Molybdenum in Biology - An Essential Trace Element

Molybdoenzymes

A volume in the series Metal Ions in Biological Systems is devoted to molybdenum and tungsten:

A. Sigel and H. Sigel (eds.), Metal Ions in Biological Systems, Vol. 39: Molybdenum and Tungsten: Their Roles in Biological Processes, Marcel Dekker, New York, 2002.

See especially:

Stiefel, E.L., The biogeochemistry of molybdenum and tungsten, Molybdenum and Tungsten: Their Roles in Biological Processes, 2002, 39, 1-29.
Lowe, D.J., Enzymes of the xanthine oxidase family: The role of molybdenum, Molybdenum and Tungsten: Their Roles in Biological Processes, 2002, 39, 455-479.
Turnlund, J.R., Molybdenum metabolism and requirements in humans, Molybdenum and Tungsten: Their Roles in Biological Processes, 2002, 39, 727-739.
Lagarde, F. and Leroy, M., Metabolism and toxicity of tungsten in humans and animals, Molybdenum and Tungsten: Their Roles in Biological Processes , 2002, 39, 741-759.

Schindelin, H., Kisker, C., Rees , D.C. , The molybdenum-cofactor: a crystallographic perspective, Journal Of Biological Inorganic Chemistry , 1997, 2 , 773-781.

Mendel, R.R., The role of the molybdenum cofactor in humans, Biofactors, 2000, 11 , 147-148.

Vorholt, J.A. and Thauer, R. K., Molybdenum and tungsten enzymes in C1 metabolism, Molybdenum and Tungsten: Their Roles in Biological Processes, 2002, 39, 571-619.

L'vov, N.P., Nosikov, A. N., and Antipov, A. N., Tungsten-containing enzymes, Biochemistry-Moscow, 2002, 67, 196-200.

Noodleman, L., Lovell, T., Liu, T. Q., Himo, F., and Torres, R. A., Insights into properties and energetics of iron-sulfur proteins from simple clusters to nitrogenase, Current Opinion in Chemical Biology , 2002, 6, 259-273.

Williams, R.J.P. and da Silva, J. J. R. F., The involvement of molybdenum in life, Biochemical and Biophysical Research Communications, 2002, 292, 293-299.

Hille, R., Molybdenum and tungsten in biology, Trends in Biochemical Sciences , 2002, 27, 360-367.

Molybdenum-containing hydroxylases catalyze the hydroxylation of carbon centers using oxygen derived ultimately from water, rather than O₂, as the source of the oxygen atom incorporated into the product, and do not require an external source of reducing equivalents. The mechanism by which this interesting chemistry takes place has been the subject of investigation for some time, and in the last several years the chemical course of the reaction has become increasingly well understood. This review summarizes recent mechanistic and structure/function studies of the molybdenum-containing hydroxylases.

Hille, R., Molybdenum-containing hydroxylases, Archives of Biochemistry and Biophysics, 2005, 433, 107-116.

Review of molybdenum uptake into the cell, via formation of the molybdenum cofactor and its storage, to the final modification of molybdenum cofactor and its insertion into apo-metalloenzymes

Mendel, R.R., Molybdenum: biological activity and metabolism, Dalton Transactions, 2005, 3404-3409.

Schwarz, G., Molybdenum cofactor biosynthesis and deficiency, Cellular and Molecular Life Sciences, 2005, 62, 2792-2810.

Noriega, C., Hassett, D. J., and Rowe, J. J., The mobA gene is required for assimilatory and respiratory nitrate reduction but not xanthine dehydrogenase activity in Pseudomonas aeruginosa, Current Microbiology, 2005, 51, 419-424.

Molybdenum is essential for most biological systems as it is required by enzymes catalyzing reactions in carbon, sulfur and nitrogen metabolism. Molybdenum is biologically inactive unless it is complexed by a cofactor. Mo is bound to a pterin forming the molybdenum cofactor (Moco) which is the active compound at the catalytic site of Mo-enzymes except nitrogenase. In eukaryotes, the most prominent Mo-enzymes are (1) sulfite oxidase, which catalyzes the final step in the degradation of sulfur-containing amino acids and is involved in detoxifying excess sulfite, (2) xanthine dehydrogenase, which is involved in purine catabolism and reactive oxygen production, (3) aldehyde oxidase, which oxidizes a variety of aldehydes and is essential for the biosynthesis of the phytohormone abscisic acid, and in autotrophic organisms and (4) nitrate reductase, which catalyzes the key step in inorganic nitrogen assimilation. Mo-enzymes, except plant sulfite oxidase, need at least one more redox active center, many of them involving iron in electron transfer. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also includes iron as well as copper in an indespensable way. Moco as released after synthesis is likely to be distributed to the apoproteins of Mo-enzymes by putative Moco-carrier proteins. Xanthine dehydrogenase and aldehyde oxidase, but not sulfite oxidase and nitrate reductase, require the postranslational sulfuration of their Mo-site for becoming active. This final maturation step is catalyzed by a Moco-sulfurase enzyme, which mobilizes sulfur from L-cysteine in a pyridoxal phosphate-dependent manner as typical for cysteine desulfurases.

Mendel, R. R. and Bittner, F., Cell biology of molybdenum, Biochimica et Biophysica Acta-Molecular Cell Research, 2006, 1763, 621-635.

Review of molybdenum uptake into the cell, via formation of the molybdenum cofactor and its storage, to the final modification of molybdenum cofactor and its insertion into apo-metalloenzymes

Mendel, R.R., Molybdenum: biological activity and metabolism, Dalton Transactions, 2005, 3404-3409.

Schwarz, G. and Mendel, R. R., Molybdenum cofactor biosynthesis and molybdenum enzymes, Annual Review of Plant Biology, 2006, 57, 623-647

Harrison, R., Milk xanthine oxidase: Properties and physiological roles, International Dairy Journal, 2006, 16, 546-554.

An issue of Journal Inorganic Biochemistry dedicated to Dr Ed Stiefel is devoted largely to molybdenum biology and enzymes:

CONTENTS

Preface for Stiefel Issue1543

John H Dawson and Harry B Gray

Edward I Stiefel – Catalysis by Enthusiasm1544

Jay Groves and Tom Spiro

Edward I Stiefel – A Personal Retrospective1546

Stephen J Lippard

Personal Remembrance

Ferrocentric memories of Edward I Stiefel1548

Elizabeth C Theil

General Review

Life, the environment and our ecosystem1550

R J P Williams

The chemistry and biochemistry of molybdenum

Facets of early transition metal–sulfur chemistry: Metal–sulfur ligand redox, induced internal electron transfer, and the reactions of metal–sulfur complexes with alkynes 1562

Charles G Young

Reactivity of potential anti-diabetic molybdenum(VI) complexes in biological media: A XANES spectroscopic study1586

Aviva Levina, Andrew IMcLeod, Jan Seuring and Peter ALay

Sulfur K-edge XAS of WV@O vs MoV@O bis(dithiolene) complexes: Contributions of relativistic effects to electronic structure and reactivity of tungsten enzymes1594

Adam L Tenderholt, Robert K Szilagyi, Richard H Holm, Keith O Hodgson, Britt Hedman and Edward I Solomon

Synthesis, characterization, and spectroscopy of model molybdopterin complexes 601

Sharon J Nieter Burgmayer, Mary Kim, Rebecca Petit, Amy Rothkopf, Alison Kim, Shadia BelHamdounia, Ying Hou,

Arpad Somogyi, Diana Habel-Rodriguez, Antonio Williams and Martin L Kirk

EPR characterization of the molybdenum(V) forms of formate dehydrogenase from Desulfovibrio desulfuricans ATCC 27774 upon formate reduction1617

Marý´a G Rivas, Pablo J Gonza´lez, Carlos D Brondino, Jose´ J G Moura and Isabel Moura

Toward modeling the high chloride, low pH form of sulfite oxidase: Ka-band ESEEM of equatorial chloro ligands in oxomolybdenum(V) complexes1623

Andrei V Astashkin, Eric L Klein and John H Enemark

Molecular insights into nitrogenase FeMoco insertion – The role of His 274 and His 451 of MoFe protein a subunit 1630

Aaron W Fay, Yilin Hu, Benedikt Schmid and Markus W Ribbe

Alkyne substrate interaction within the nitrogenase MoFe protein1642

Patricia C Dos Santos, Suzanne M Mayer, Brett M Barney, Lance C Seefeldt and Dennis R Dean

Conformations generated during turnover of the Azotobacter vinelandii nitrogenase MoFe protein and their relationship to physiological function1649

Karl Fisher, David J Lowe, Pedro Tavares, Alice S Pereira, Boi Hanh Huynh, Dale Edmondson and William E Newton

Model molybdopterin complexes

New model pterin-substituted dithiolene complexes of molybdenum(V) and molybdenum(IV) for the molybdenum cofactor are reported: Tp*MoX(pterin-R-dithiolene) (Tp* = tris(3,5,-dimethylpyrazolyl)borate), X = O, S, R = aryl.

Burgmayer, S. J. N., Kim, M., Petit, R., Rothkopf, A., Kim, A., BelHamdounia, S., Hou, Y., Somogyi, A., Habel-Rodriguez, D., Williams, A., and Kirk, M. L., Synthesis, characterization, and spectroscopy of model molybdopterin complexes, Journal of Inorganic Biochemistry, 2007, 101, 1601-1616.

Active sites of molybdoenzymes

Molybdenum enzymes can be grouped on the basis of the structure of the metal centre:

molybdenum hydroxylases	(pyranopterin)MoOS(OH)	Xanthine oxidase and xanthine dehydrogenase
eukaryotic oxotransferases	(pyranopterin)MoO₂(S- Cys)	sulfite oxidases and plant nitrate reductases.
bacterial oxotransferases	(pyranopterin)₂MoOX	pyranopterin, cofactor; X, ligated serine, cysteine or selenocysteine

The active sites possess a catalytically labile Mo-OH (or possibly Mo-OH₂) group that is transferred to substrate in the course of the hydroxylation reaction. Water rather than molecular oxygen is the ultimate source of the oxygen atom incorporated into product.

Hille, R., Molybdenum enzymes, Essays Biochem., 1999, 34, 125-137.

Review: active centres

Recent characterisation of molybdenum and tungsten enzymes revealed novel structural types of reaction centres, as well as providing new subjects of interest as synthetic chemical analogues. This tutorial review highlights the structure/reactivity relationships of the enzyme reaction centres and chemical analogues. Chemical analogues for the oxygen atom transfer enzymes have been well expanded in structure and reactivity. Other types of chemical analogues that exhibit different coordination chemistry have recently been presented for reaction centres of the hydroxylation and dehydrogenation enzymes and others

Sugimoto, H. and Tsukube, H., Chemical analogues relevant to molybdenum and tungsten enzyme reaction centres toward structural dynamics and reaction diversity, Chemical Society Reviews, 2008, 37, 2609-2619.

Review: molybdenum and tungsten enzymes

Tungsten is widely distributed in biology; the majority of the tungsten-containing enzymes so far purified are from anaerobic archaea and bacteria. Tungsten is taken up by cells as tungstate, and then coordinated by sulfur in a cofactor, tungstopterin. equivalent to molybdopterin, the active center in several molybdenum-containing enzymes.

In biology tungsten is different from molybdenum. This review describes the (bio)molecular basis of this differential cellular use of W to Mo in terms of their active transport, cofactor synthesis, and functioning as catalytically active sites.

Bevers, L. E., Hagedoorn, P. L., and Hagen, W. R., The bioinorganic chemistry of tungsten, Coordination Chemistry Reviews, 2009, 253, 269-290

X-ray absorption spectra of molybdoenzymes

The X-ray absorption spectra at the molybdenum and selenium K- edges and the tungsten L-2,L-3-edges are reported for fourteen Mo(IV) and W(IV,VI) bis(dithiolene) complexes related to the active sites of molybdo- and tungstoenzymes. These and previous XAS results should prove useful in characterizing and refining metric features and structures of enzyme sites.

Musgrave, K.B., Lim, B. S., Sung, K. M., Holm, R. H., Hedman, B., and Hodgson, K. O., X-ray spectroscopy of enzyme active site analogues and related molecules: Bis(dithiolene)molybdenum(IV) and -tungsten(IV,VI) complexes with variant terminal ligands, Inorganic Chemistry, 2000, 39 , 5238-5247.

Aldehyde oxidase

The aldehyde oxidoreductase, isolated from the sulfate reducer Desulfovibrio desulfuricans (ATCC 27774), and the homologous enzyme from Desulfovibrio gigas are members of the xanthine oxidase family of molybdenum-containing enzymes. They have similar substrate specificity. The primary sequences from both enzymes show 68 % identity. Both enzymes are very closely related in their sequences and 3D structures. The comparison allowed confirmation and establishment of features that are essential for their function: conserved residues in the active site, catalytically relevant water molecules and recognition of the physiological electron acceptor docking site.

Rebelo, J., Macieira, S., Dias, J. M., Huber, R., Ascenso, C. S., Rusnak, F., Moura, J. J. G., Moura, I., and Romao, M. J., Gene sequence and crystal structure of the aldehyde oxidoreductase from Desulfovibrio desulfuricans ATCC 27774, Journal of Molecular Biology, 2000, 297 , 135-146.

The current knowledge of genetics, evolution, structure, enzymology, tissue distribution and regulation of mammalian aldehyde oxidases is reviewed.

Garattini, E., Fratelli, M., and Terao, M., Mammalian aldehyde oxidases: genetics, evolution and biochemistry, Cellular and Molecular Life Sciences, 2008, 65, 1019-1048.

Oxidative removal of glutaraldehyde

Maeda, Y., Yagyu, A., Sakurai, A., Fujii, Y., and Uchida, H., Characterization of aldehyde oxidase from Brevibacillus sp MEY43 and its application to oxidative removal of glutaraldehyde, World Journal of Microbiology & Biotechnology, 2008, 24, 797-804.

Molybdenum iron-sulfur flavin hydroxylases in the pathogenesis of

liver injuries injuries

The role of molybdenum iron-sulfur flavin hydroxylases in the pathogenesis of

liver injuries injuries in rats induced

(a) by carbon tetrachloride (CCl₄), thioacetamide (TAA) and chloroform (CHCl₃), which produce free radicals were associated with elevated activity levels of hepatic Mo-Fe-S flavin hydroxylases. Inhibition of these hydroxylases by sodium tungstate suppressed biochemical and oxidative stress markers of hepatic tissue damage.

(b) by acetaminophen (AAP) and bromobenzene (BB), which cause severe glutathione depletion. Mo-Fe-S flavin hydroxylases did not with these toxicants show any change. Mo-Fe-S hydroxylases contribute to the hepatic injury inflicted by free radical generating agents. and does not play any role in hepatic injury produced by glutathione depleting agents. The study has implication for understanding human liver diseases caused by liver toxicants and for inhibitors of Mo-Fe-S flavin hydroxylases as potential therapeutic agents.

Ali, S., Pawa, S., Naime, M., Prasad, R., Ahmad, T., Farooqui, H., and Zafar, H., Role of mammalian cytosolic molybdenum Fe-S flavin hydroxylases in hepatic injury, Life Sciences, 2008, 82, 780-788.

Cytosol = the non-particulate components of the cytoplasm

Cytoplasm = that part of the cell outside the nucleus but inside the cell wall if it exists

Molybdenum hydroxylases drug-metabolizing ability

Drug metabolizing ability of molybdenum hydroxylases, which include aldehyde oxidase and xanthine oxidoreductase, and the variation of the activity amongst humans, with the highest activity, rats and mice and dogs are described in this review. Molybdenum hydroxylases, are involved in the metabolism of some medicines in humans. Interindividual variation of aldehyde oxidase activity is present in humans. Drug-drug interactions associated with aldehyde oxidase and xanthine oxidoreductase are of potential clinical significance.

Kitamura, S., Sugihara, K., and Ohta, S., Drug-metabolizing ability of molybdenum hydroxylases, Drug Metabolism and Pharmacokinetics, 2006, 21, 83-98.

Reduction of N-hydroxylated prodrugs by a molybdenum enzyme

The recently discovered mammalian molybdoprotein mARC1 is capable of reducing N-hydroxylated compounds. Upon reconstitution with cytochrome b(5) and b5 reductase, benzamidoxime, pentamidine, and diminazene amidoximes, N-hydroxymelagatran, guanoxabenz, and N-hydroxydebrisoquine are efficiently reduced. These substances are amidoxime/N-hydroxyguanidine prodrugs, leading to improved bioavailability compared to the active amidines/guanidines. Thus, the recombinant enzyme allows prediction about in vivo reduction of N-hydroxylated prodrugs. Furthermore, the prodrug principle is not dependent on cytochrome P450 enzymes

Gruenewald, S., Wahl, B., Bittner, F., Hungeling, H., Kanzow, S., Kotthaus, J., Schwering, U., Mendel, R. R., and Clement, B., The Fourth Molybdenum Containing Enzyme mARC: Cloning and Involvement in the Activation of N-Hydroxylated Prodrugs, Journal of Medicinal Chemistry, 2008, 51, 8173-8177.

Selenium-dependent molybdenum hydroxylases

Haft, D.H. and Self, W. T., Orphan SeID proteins and selenium-dependent molybdenum hydroxylases, Biology Direct, 2008, 3,

Aldehyde oxidase

Aldehyde oxidase (AO) is a homodimer with a subunit molecular mass of approximately 150 kDa. Each subunit consists of about 20 kDa 2Fe-2S cluster domain storing reducing equivalents, about 40 kDa flavine adenine dinucleotide (FAD) domain and about 85 kDa molybdenum cofactor (MoCo) domain containing a substrate binding site. In order to clarify the properties of each domain, especially substrate binding domain, chimeric cDNAs were constructed by mutual exchange of 2Fe-2S/FAD and MoCo domains between monkey and rat. Chimeric monkey/rat AO was referred to one with monkey type 2Fe-2S/FAD domains and a rat type MoCo domain. Rat/monkey AO was vice versa. AO-catalyzed 2-oxidation activities of (S)-RS-8359 were measured using the expressed enzyme in Escherichia coli. Substrate inhibition was seen in rat AO and chimeric monkey/rat AO, but not in monkey AO and chimeric rat/monkey AO, suggesting that the phenomenon might be dependent on the natures of MoCo domain of rat. A biphasic Eadie-Hofstee profile was observed in monkey AO and chimeric rat/monkey AO, but not rat AO and chimeric monkey/rat AO, indicating that the biphasic profile might be related to the properties of MoCo domain of monkey. Two-fold greater V-max, values were observed in monkey AO than in chimeric rat/monkey AO, and in chimeric monkey/rat AO than in rat AO, suggesting that monkey has the more effective electron transfer system than rat. Thus, the use of chimeric enzymes revealed that 2Fe-2S/FAD and MoCo domains affect the velocity and the quantitative profiles of AO-catalyzed (S)-RS-8359 2-oxidation, respectively

Itoh, K., Asakawa, T., Hoshino, K., Adachi, M., Fukiya, K., Watanabe, N., and Tanaka, Y., Functional Analysis of Aldehyde Oxidase Using Expressed Chimeric Enzyme between Monkey and Rat, Biological & Pharmaceutical Bulletin, 2009, 32, 31-35.

Arsenate and selenate reductases

In arsenate and selenate respiring bacteria, the oxidation of organic substrates acetate, lactate, pyruvate, glycerol, ethanol or hydrogen can be coupled to the reduction of arsenate and selenate. Arsenate and selenate reductases have been characterized; they all contain molybdenum.

Stolz, J.F., Oremland, R.S., Bacterial respiration of arsenic and selenium, Fems Microbiology Reviews , 1999, 23 , 5, 615-627.

Arsenite Oxidase

Arsenite oxidase from Alcaligenes faecalis NCIB 8687 is a molybdenum/iron protein involved in the detoxification of arsenic. It oxidizes arsenite [(AsO₂^-)-O-III], which binds to essential sulfhydryl groups of proteins and dithiols, to the relatively less toxic arsenate [(AsO₄^3-)-O-V]. Arsenite oxidase consists of a large subunit of 825 residues and a small subunit of approximately 134 residues. The large subunit contains a Mo site, consisting of a Mo atom bound to two pterin cofactors, and a [3Fe-4S] cluster. The large subunit of arsenite oxidase is similar to other members of the dimethylsulfoxide (DMSO) reductase family of molybdenum enzymes, particularly the dissimilatory periplasmic nitrate reductase from Desulfovibrio desulfuricans , but is unique in having no covalent bond between the polypeptide and the Mo atom. The small subunit has no counterpart among known Mo protein structures but is homologous to the Rieske [2Fe-2S] protein domain of the cytochrome be, and cytochrome b(6)f complexes and to the Rieske domain of naphthalene 1,2-dioxygenase

Ellis, P.J., Conrads, T., Hille, R., and Kuhn, P., Crystal structure of the 100 kDa arsenite oxidase from Alcaligenes faecalis in two crystal forms at 1.64 angstrom and 2.03 angstrom, Structure, 2001, 9, 125-132.

Lebrun, E., Brugna, M., Baymann, F., Muller, D., Lievremont, D., Lett, M. C., and Nitschke, W., Arsenite oxidase, an ancient bioenergetic enzyme, Molecular Biology and Evolution, 2003, 20, 686-693.

Biotin sulfoxide reductase

Vibrational modes associated with a terminal oxo ligand and the two molybdopterin dithiolene ligands have been assigned. The enzyme cycles between mono-oxo- Mo(VI) and des-oxo-Mo(IV) forms with both molybdopterin dithiolene ligands remaining co-ordinated. In both redox states the terminal oxo group at the molybdenum centre is exchangeable with water during redox cycling and originates from the substrate in substrate-oxidized samples. Product-induced changes in the Mo=O stretching frequency provide direct evidence for a product- associated mono-oxo-Mo(VI) catalytic intermediate.

Garton, S.D., Temple, C. A., Dhawan, I. K., Barber, M. J., Rajagopalan, K. V., and Johnson, M. K., Resonance Raman characterization of biotin sulfoxide reductase - Comparing oxomolybdenum enzymes in the Me2SO reductase family, Journal of Biological Chemistry , 2000, 275 , 6798-6805.

Carbon monoxide dehydrogenase

CO dehydrogenase (EC 1.2.99.2) catalyses the oxidation of CO
CO + H2O --> CO2 + 2 e- + 2 H+
It is a selenium-containing molybdo-iron-sulfur- flavoenzyme.It has been crystallized from the aerobic CO utilizing bacteria Oligotropha carboxidovorans and Hydrogenophaga pseudoflava. It has been structurally characterized in its oxidized state. The enzymes are dimers of two heterotrimers. Each heterotrimer is composed of a molybdoprotein, a flavoprotein, and an iron-sulfur protein. The substituents in the first co-ordination sphere of the Mo-ion are the enedithiolate sulfur atoms of the molybdopterin-cytosine dinucleotide, two oxo- and a sulfide-group. Extended X-ray absorption fine structure spectroscopy , along with the crystal structure of CO dehydrogenase at 1.85 Angstrom resolution, have identified a sulfur atom at 2.3 Angstrom from the Mo-ion. The sulfur reacts with cyanide yielding thiocyanate. Both CO dehydrogenase structures show only minor differences. CO oxidation takes place at the molybdoprotein which contains a 1:1 mononuclear complex of molybdopterin-cytosine dinucleotide and a Mo-ion, along with a catalytically essential S- selanylcysteine. The corresponding inactive desulfo-CO dehydrogenase shows a typical desulfo inhibited-type of Mo-electron paramagnetic resonance spectrum. Structural changes at the SeMo-site during catalysis are suggested by the Mo to Se distance of 3.7 A and the Mo-S-Se angle of 113 degrees in the oxidized enzyme which increase to 4.1 Angstrom, and 121", respectively, in the reduced enzyme. The intramolecular electron transport chain in CO dehydrogenase involves the following prosthetic groups and minimal distances: CO --> [Mo of the molybdenum cofactor] - 14.6 Angstrom - [2Fe-2S] I - 12.4 Angstrom - [2Fe-2S] II - 8.7 Angstrom - [FAD]

Meyer, O., Gremer, L., Ferner, R., Ferner, M., Dobbek, H., Gnida, M., Meyer-Klaucke, W., and Huber, R., The role of Se, Mo and Fe in the structure and function of carbon monoxide dehydrogenase, Biological Chemistry , 2000, 381 , 865-876.

See also

Hanzelmann, P., Dobbek, H., Gremer, L., Huber, R., and Meyer, O., The effect of intracellular molybdenum in Hydrogenophaga pseudoflava on the crystallographic structure of the seleno- molybdo-iron-sulfur flavoenzyme carbon monoxide dehydrogenase, Journal of Molecular Biology , 2000, 301 , 1221-1235.

The molybdoenzyme carbon monoxide dehydrogenase (CODH) catalyzes the oxidation Of CO to CO2 in the aerobic bacterium Oligotropha carboxidovorans. The active site in oxidized CODH contains a bimetallic [(CuSMOVI)- S-I(=O)(2)] cluster which was converted into a [(CuSMoIV)-S-I(=O)- OH(2)] cluster upon reduction. The Cu...Mo distance is 3.70 Angstrom in the oxidized form and is increased to 4.23 Angstrom upon reduction.

Gnida, M., Ferner, R., Gremer, L., Meyer, O., and Meyer-Klaucke, W., A novel binuclear [CuSMo] cluster at the active site of carbon monoxide dehydrogenase: Characterization by X-ray absorption spectroscopy, Biochemistry, 2003, 42, 222-230.

Dimethylsulfide dehydrogenase

Hanson, G.R., McDevitt, C. A., and McEwan, A. G., Dimethylsulfide dehydrogenase from rhodovulum sulfidophilum : EPR spectroscopy and biochemical analysis reveal its place in the DMSO reductase family of molybdenum enzymes, Journal of Inorganic Biochemistry, 2001, 86, 248.

Dimethyl sulfoxide (DMSO) reductase

The molybdenum site of the oxidized Mo(VI) dimethyl sulfoxide (DMSO) reductase enzyme possesses one terminal oxygen ligand (Mo=O) at 1.68 Angstrom, four thiolate ligands at 2.44 Angstrom, and one oxygen at 1.92 Angstrom. The dithionite-reduced Mo(IV) enzyme possesses a desoxo species with three or four Mo-S at 2.33 Angstrom and two different Mo-O ligands at 2.16 and 1.92 Angstrom. One exchangeable oxygen ligand, most likely an Mo- OH, in the ESR-active signal-giving species originates from the Mo=O of the oxidized enzyme. The enzyme reduced by dimethylsulfide contains a desoxo active site with four Mo-S at 2.36 and two different Mo-O ligands at 1.94 and 2.14 Angstrom. Recombinant wild-type R. sphaeroides DMSO reductase expressed in Escherichia coli. initially has a dioxo structure (two Mo=O at 1.72 Angstrom and four Mo-S at 2.48 Angstrom) but assumes the wild-type Mo(VI) structure after a cycle of reduction and reoxidation. The site-directed Ser147-->Cys mutant possesses a monooxo active site in the oxidized state (Mo=O at 1.70 Angstrom) with five sulfur ligands (at 2.40 Angstrom), consistent with cysteine 147 coordination to Mo. The dithionite reduced form of the mutant possesses a desoxo site also with five. Mo-S ligands (at 2.37 Angstrom) and one Mo-O at 2.12 Angstrom.

George, G.N., Hilton, J., Temple, C., Prince, R.C., Rajagopalan, K.V., Structure of the molybdenum site of dimethyl sulfoxide reductase, Journal Of The American Chemical Society , 1999, 121 , 1256-1266.

Dimethyl sulfoxide (DMSO) reductase from purple bacteria contains a mononuclear Mo co-ordinated by two molybdopterin guanine dinucleotides as its single cofactor. Crystallographic studies on the enzyme from Rhodobacter sphaeroides and Rhodobacter capsulatus revealed substantial differences in the Mo co-ordination environment in the oxidized Mo(VI) state, despite a close structural similarity in the overall fold of the protein. The crystal structure of DMSO reductase from R. sphaeroides identified a Mo environment with a mono-oxo ligation and an asymmetric co-ordination by the two molybdopterins, with three short and one very long Mo-S bond.
In contrast, two independent crystallographic studies of the enzyme from R. capsulatus revealed two additional Mo co-ordination environments: a pentacoordinated dioxo metal ligation sphere in which one molybdopterin is completely dissociated from the Mo and a heptacoordinated environment with symmetrical metal co-ordination by both molybdopterins and two oxo ligands. In all three structures the side chain of a serine was a ligand to the Mo. EXAFS studies on the R. sphaeroides enzyme suggested a hexacoordinated active site geometry, whereas for the R. capsulatus enzyme EXAFS indicated seven ligands.

Baugh, P.E., Garner, C.D., Charnock, J.M., Collison, D., Davies, E.S., McAlpine, A.S., Bailey, S., Lane, I., Hanson, G.R., McEwan, A.G, X-ray absorption spectroscopy of dimethylsulfoxide reductase from Rhodobacter capsulatus, Journal Of Biological Inorganic Chemistry , 1997, 2 , 634-643.

The 1.3 Angstrom resolution crystal structure of oxidized DMSOR from R. sphaeroides reveals plasticity at the active site. The Mo is discretely disordered and exists in a hexacoordinated and a pentacoordinated ligation sphere. The hexacoordinated model reconciles the existing differences in active site co-ordination of R. sphaeroides DMSO reductase as studied by crystallographic and EXAFS techniques. In addition, the pentacoordinated structure closely resembles one of the reported R. capsulatus crystal structures.

Trieber, C.A., Rothery, R.A., Weiner, J.H., Consequences Of Removal Of A Molybdenum Ligand (Dmsa-Ser-176) Of Escherichia-Coli Dimethyl-Sulfoxide Reductase, Journal Of Biological Chemistry, 1996, 271 , 27339-27345.

The active site geometry in the previously reported 2.2 Angstrom crystal structure of R, sphaeroides DMSO reductase appears to represent an average of the two conformations described here. Thus, structural flexibility at the active site appears to give rise to the observed differences in the Mo co-ordination environment

Li, H.K., Temple , C., Rajagopalan, K. V., and Schindelin, H., The 1.3 angstrom crystal structure of Rhodobacter sphaeroides dimethyl sulfoxide reductase reveals two distinct molybdenum co-ordination environments, Journal of the American Chemical Society , 2000, 122 , 7673-7680.

Dimethylsulfoxide reductase ― molybdenum coordination in the reduced enzyme

Reduced dimethylsulfoxide reductase (DMSOR) enzymes have an active-site which (a) lacks a terminal oxo ligand (unlike the reduced active sites of other pyranopterin Mo enzymes); (b) has two pyranopterin-ene-1,2-dithiolate ligands (unlike other pyranopterin Mo enzymes but analogous to all of the currently known tungsten-containing enzymes). The Mo-bisdithiolene electronic structure and bonding in the absence of a strong-field oxo ligand is discussed in relation to the electronic and Raman spectra of model complexes.

McNaughton, R.L., Lim, B. S., Knottenbelt, S. Z., Holm, R. H., and Kirk, M. L., Spectroscopic and electronic structure studies of symmetrized models for reduced members of the dimethylsulfoxide reductase enzyme family, Journal of the American Chemical Society, 2008, 130, 4628-4636.

Ethylbenzene dehydrogenase

The first step in anaerobic ethylbenzene mineralization in denitrifying Azoarcus so. strain EB1 is the oxidation of ethylbenzene to (S)-(-)-1-phenylethanol. Ethylbenzene dehydrogenase, which catalyzes this reaction, is a unique enzyme in that it mediates the stereoselective hydroxylation of an aromatic hydrocarbon in the absence of molecular oxygen. Purified ethylbenzene dehydrogenase contains approximately 0.5 mol of molybdenum, 16 mol of iron, and 15 mol of acid-labile sulfur per mol of holoenzyme, and a molydopterin cofactor. According to sequence analysis and biochemical data ethylbenzene dehydrogenase is a novel member of the dimethyl sulfoxide reductase family of molybdopterin-containing enzymes

Johnson, H.A., Pelletier, D. A., and Spormann, A. M., Isolation and characterization of anaerobic ethylbenzene dehydrogenase, a novel Mo-Fe-S enzyme, Journal of Bacteriology, 2001, 183, 4536-4542.

Formate dehydrogenase

Escherichia coli can perform two modes of formate metabolism. Under respiratory conditions, two periplasmically-located formate dehydrogenase isoenzymes couple formate oxidation to the generation of a transmembrane electrochemical gradient; and under fermentative conditions a third cytoplasmic isoenzyme is involved in the disproportionation of formate to CO₂and H₂. The respiratory formate dehydrogenases are redox enzymes that comprise three subunits: a molybdenum cofactor- and FeS cluster-containing catalytic subunit; an electron-transferring ferredoxin; and a membrane-integral cytochrome b. The catalytic subunit and its ferredoxin partner are targeted to the periplasm as a complex by the twin-arginine transport (Tat) pathway. Biosynthesis of these enzymes is under control of an accessory protein, FdhE. E. coli FdhE interacts with the catalytic subunits of the respiratory formate dehydrogenases. Purification of recombinant FdhE demonstrates the protein is an iron-binding rubredoxin that can adopt monomeric and homodimeric forms. Bacterial two-hybrid analysis suggests the homodimer form of FdhE is stabilized by anaerobiosis. Site-directed mutagenesis shows that conserved cysteine motifs are essential for the physiological activity of the FdhE protein and are also involved in iron ligation

Luke, I., Butland, G., Moore, K., Buchanan, G., Lyall, V., Fairhurst, S., Greenblatt, J., Emili, A., Palmer, T., and Sargent, F., Biosynthesis of the respiratory formate dehydrogenases from Escherichia coli: characterization of the FdhE protein, Archives of Microbiology, 2008, 190, 685-696.

Molybdopterin cofactor

Molybdenum cofactor

The molybdopterin cofactor is required for the activity of a variety of oxidoreductases. The xanthine oxidase class of molybdoenzymes requires the molybdopterin cofactor to have a terminal, cyanolysable sulfur ligand. In the sulfite oxidase/nitrate reductase class, an oxygen is present in the same position.

Mutations in both the ma-l gene of Drosophila melanogaster and the hxB gene of Aspergillus nidulans cause loss of activities of those molybdoenzymes that require a cyanolysable sulfur in the active centre. The ma-l and hxB genes encode highly similar proteins containing domains common to pyridoxal phosphate-dependent cysteine transulphurases, including the cofactor binding site and a conserved cysteine, which is the putative sulfur donor.
Key similarities were found with NifS, the enzyme involved in the generation of the iron-sulphur centres in nitrogenase. These similarities suggest an analogous mechanism for the generation of the terminal molybdenum-bound sulfur ligand. Putative homologues have been identified of these genes in organisms, including humans.

Amrani, L., Primus, J., Glatigny, A., Arcangeli, L., Scazzocchio, C., and Finnerty, V., Comparison of the sequences of the aspergillus nidulans hxB and drosophila melanogaster ma-l genes with nifS from azotobacter vinelandii suggests a mechanism for the insertion of the terminal sulfur atom in the molybdopterin cofactor, Mol.Microbiol. , 2000, 38 , 114-125.

All mononuclear molybdoenzymes bind molybdenum in a complex with an organic cofactor termed molybdopterin. In many bacteria, including Escherichia coli, molybdopterin can be further modified by attachment of a guanine monophosphate group to the terminal phosphate of molybdopterin to form molybdopterin guanine dinucleotide. This modification reaction is required for the functioning of many bacterial molybdoenzymes, including the nitrate reductases, dimethylsulfoxide and trimethylamine-N-oxide reductases, and formate dehydrogenases. The guanine monophosphate attachment step is catalyzed by the cellular enzyme MobA. The crystal structure of the 21.6 kDa E. coli MobA has been determined.

Stevenson, C.E.M., Sargent, F., Buchanan, G., Palmer, T., and Lawson, D. M., Crystal structure of the molybdenum cofactor biosynthesis protein MobA from Escherichia coli at near-atomic resolution, Structure, 2000, 8 , 1115-1125.

Molybdopterin synthase

Molybdopterin is inserted into xanthine dehydrogenase only after molybdenum chelation, and both metal chelation and molybdopterin insertion can occur only under high molybdate concentrations. In the biosynthesis of the molybdenum cofactor the biosynthesis of molybdopterin and molybdopterin guanine dinucleotide are split at a stage when the molybdenun atom is added to molybdopterin.

Leimkuhler, S., Angermuller, S., Schwarz, G., Mendel, R.R., Klipp,W., Activity of the molybdopterin-containing xanthine dehydrogenase of Rhodobacter capsulatus can be restored by high molybdenum concentrations in a moeA mutant defective in molybdenum cofactor biosynthesis, Journal Of Bacteriology , 1999, 181 , 19, 5930-5939.

Molybdenum cofactor (Moco) biosynthesis is an evolutionarily conserved pathway present in eubacteria, archaea and eukaryotes, including humans. Genetic deficiencies of enzymes involved in Moco biosynthesis in humans lead to a severe and usually fatal disease. Moco contains a tricyclic pyranopterin, termed molybdopterin, that bears the cis-dithiolene group responsible for molybdenum ligation. The dithiolene group of molybdopterin is generated by molybdopterin synthase, which consists of a large and small subunits. The 1.45 Angstrom resolution crystal structure of molybdopterin synthase reveals a heterotetrameric protein in which the C-terminus of each small subunit is inserted into a large subunit to form the active site. In the activated form of the enzyme this C-terminus is present as a thiocarboxylate. In the structure of a covalent complex of molybdopterin synthase, an isopeptide bond is present between the C-terminus of the small subunit and a Lys side chain in the large subunit. The strong structural similarity between the small subunit of molybdopterin synthase and ubiquitin provides evidence for the evolutionary antecedence of the Moco biosynthetic pathway to the ubiquitin dependent protein degradation pathway.

Rudolph, M.J., Wuebbens, M. M., Rajagopalan, K. V., and Schindelin, H., Crystal structure of molybdopterin synthase and its evolutionary relationship to ubiquitin activation, Nature Structural Biology, 2001, 8, 42-46.

See also

Schrag, J.D., Huang, W. J., Sivaraman, J., Smith, C., Plamondon, J., Larocque, R., Matte, A., and Cygler, M., The crystal structure of Escherichia coli MoeA, a protein from the molybdopterin synthesis pathway, Journal of Molecular Biology , 2001, 310, 419-431.

Molybdopterin is a pyranopterin with a unique dithiolene group coordinating molybdenum or tungsten in all molybdenum- and tungsten-enzymes except nitrogenase. In Escherichia coli, molybdopterin is formed by incorporation of two sulfur atoms into a precursor, which is catalyzed by the molybdopterin synthase enzyme. A two-step reaction of molybdopterin synthesis is proposed where the dithiolene is generated by two thiocarboxylates derived from a single tetrameric molybdopterin synthase.

Gutzke, G., Fischer, B., Mendel, R. R., and Schwarz, G., Thiocarboxylation of molybdopterin synthase provides evidence for the mechanism of dithiolene formation in metal-binding pterins, Journal of Biological Chemistry, 2001, 276, 36268-36274.

See also

Leimkuhler, S., Wuebbens, M. M., and Rajagopalan, K. V., Characterization of Escherichia coli MoeB and its involvement in the activation of molybdopterin synthase for the biosynthesis of the molybdenum cofactor, Journal of Biological Chemistry, 2001, 276, 34695-34701.

Krepinsky, K. and Leimkuhler, S., Site-directed mutagenesis of the active site loop of the rhodanese-like domain of the human molybdopterin synthase sulfurase MOCS3 - Major differences in substrate specificity between eukaryotic and bacterial homologs, Febs Journal, 2007, 274, 2778-2787.

Regulski, E.E., Moy, R. H., Weinberg, Z., Barrick, J. E., Yao, Z., Ruzzo, W. L., and Breaker, R. R., A widespread riboswitch candidate that controls bacterial genes involved in molybdenum cofactor and tungsten cofactor metabolism, Molecular Microbiology, 2008, 68, 918-932.

Biosynthesis of molybdenum and tungsten enzymes

See Mo transport.

Bevers, L. E., Hagedoorn, P.L., Santamaria-Araujo, J.A., Magalon, A., Hagen, W.R., Schwarz, G., BIOCHEMISTRY, 47, 949-956, 2008.

Molybdenum cofactor sulfuration

In almost all biological life forms, molybdenum and tungsten are coordinated by molybdopterin (MPT), a tricyclic pyranopterin containing a cis-dithiolene group. Molybdenum and the pterin moiety form the redox reactive molybdenum cofactor (Moco). Mutations in patients with deficiencies in Moco biosynthesis usually occur in the enzymes catalyzing the first and second steps of biosynthesis, leading to the formation of: precursor Z and MPT, respectively. The conversion of the sulfur- and metal-free precursor Z to MPT by MPT synthase involves sulfur atom transfer from a C-terminal MoaD thiocarboxylate to the C-1' and C-2' positions of precursor Z. The crystal structures of non-thiocarboxylated MPT synthase from Staphylococcus aureus in its apo form and in complex with precursor Z. A is reported.

Daniels, J. N., Wuebbens, M. M., Rajagopalan, K. V., and Schindelin, H., Crystal structure of a molybdopterin synthase-precursor Z complex: Insight into its sulfur transfer mechanism and its role in molybdenum cofactor deficiency, Biochemistry, 2008, 47,615-626, 3315.

Wollers, S., Heidenreich, T., Zarepour, M., Zachmann, D., Kraft, C., Zhao, Y. D., Mendel, R. R., and Bittner, F., Binding of sulfurated molybdenum cofactor to the C-terminal domain of ABA3 from Arabidopsis thaliana provides insight into the mechanism of molybdenum cofactor sulfuration, Journal of Biological Chemistry, 2008, 283, 9642-9650.

Molybdopterin co-factor coordination chemistry review

Dithiolenes in natural systems are ligands that bind molybdenum or tungsten at the catalytic centre of enzymes which catalyse the transfer of an oxygen atom to or from the substrate: e.g. the sulfite oxidases, sulfite to sulfate, and the nitrate reductases, nitrate to nitrite. The catalytic centres have one or two molybdopterin (MPT) cofactors bound to a mononuclear metal centre via their dithiolene group. The review covers the biosynthesis of MPT, its role in the function of the oxotransferase enzymes and the coordination chemistry that has been stimulated by the present knowledge of the nature and function of the catalytic centres of these enzymes.

Hine, F. J_., Taylor, A. J_., and Garner, C. D_., Dithiolene complexes and the nature of molybdopterin, Coordination Chemistry Reviews, 2010, 254, 1570-1579.

FeMo-co precursor

George, S.J., Igarashi, R. Y., Xiao, Y., Hernandez, J. A., Demuez, M., Zhao, D., Yoda, Y., Ludden, P. W., Rubio, L. M., and Cramer, S. P., Extended X-ray absorption fine structure and nuclear resonance vibrational Spectroscopy reveal that NifB-co, a FeMo-co precursor, comprises a 6Fe core with an interstitial light atom, Journal of the American Chemical Society, 2008, 130, 5673-5680.

Inhibitor

Johannes, J., Unciuleac, M. C., Friedrich, T., Warkentin, E., Ermler, U., and Boll, M., Inhibitors of the molybdenum cofactor containing 4-Hydroxybenzoyl-CoA reductase, Biochemistry, 2008, 47, 4964-4972.

Sulfuration

The Moco (molybdenum cofactor) sulfurase ABA3 from Arabidopsis thaliana catalyses the sulfuration of the Moco of aldehyde oxidase and xanthine oxidoreductase, which represents the final activation step of these enzymes.

ABA3 consists of an N-terminal NifS-like domain that exhibits L-cysteine desulfurase activity and a C-terminal domain that binds sulfurated Moco.

The strictly conserved Cys(430) in the NifS-like domain binds a persulfide intermediate, which is abstracted from the substrate L-cysteine and finally needs to be transferred to the Moco of aldehyde oxidase and xanthine oxidoreductase.

In addition to Cys(430), another eight cysteine residues are located in the NifS-like domain, with two of them being highly conserved among Moco sulfurase proteins and, at the same time, being in close proximity to Cys(430).

By determination of the number of surface-exposed cysteine residues and the number of persulfide-binding cysteine residues in combination with the sequential substitution of each of the nine cysteine residues, a second persulfide-binding cysteine residue, Cys(206), was identified.

Furthermore, the active-site Cys(430) was found to be located on top of a loop structure, formed by the two flanking residues Cys(428) and Cys(435), which are likely to form an intramolecular disulfide bridge.

These findings are confirmed by a structural model of the NifS-like domain, which indicates that Cys(428) and Cys(435) are within disulfide bond distance and that a persulfide transfer from Cys(430) to Cys(206) is indeed possible.

[‘NifS-like proteins are ubiquitous, homodimeric, proteins which belong to the α-family of pyridoxal-5’-phosphate dependent enzymes. They are proposed to donate elementary sulphur, generated from cysteine, via a cysteine persulphide intermediate during iron sulphur cluster biosynthesis.’ Kaiser, J.T., Clausen, T. Gleb P. Bourenkow, G.P., Bartunik, H.D., Steinbacher,S., and Huber, R.,J. Mol. Biol.,2000, 297, 451-464. Crystal Structure of a NifS-like Protein fromThermotoga maritima: Implications for Iron Sulphur Cluster Assembly.]

Lehrke, M., Rump, S., Heidenreich, T., Wissing, J., Mendel, R., Bittner, F., biochemical Journal, 2012, 441, 823-832. Identification of persulfide-binding and disulfide-forming cysteine residues in the NifS-like domain of the molybdenum cofactor sulfurase ABA3 by cysteine-scanning mutagenesis.

Biosynthesis reactions from Caspi et al, Nucleic Acids Research 38:D473-D479 2010 Page generated by SRI InternationalPathway Tools version 16.0 http://www.metacyc.org/META/NEW-IMAGE?type=PATHWAY&object=PWY-5963&detail-level=3 ©2011 SRI International.

Molybdenum and enzymes molybdenum cofactor

Cyclic pyranopterin monophosphate

Hydrogenated pterins are found in all living organisms, where they are involved in key metabolic processes. Molybdenum in its biologically active form is bound to a fully reduced tetrahydropyranopterin, a metal-binding pterin (MPT), forming the molybdenum cofactor (Moco).

Cyclic pyranopterin monophosphate (cPMP) is the first isolatable intermediate in molybdenum cofactor biosynthesis. The (13)C NMR data for cPMP confirm the tetrahydropyranopterin nature of cPMP and the presence of a gem-diol in the C1' position of the side chain. The gem-diol is not a chemical artifact, but is chemically stable and not in equilibrium with the keto form.

The kinetics of cPMP oxidation in the presence of metal centers, chelating agents, and different buffers and pH values were studied. Oxidation is metal-dependent and can be retarded by EDTA.

Santamaria-Araujo, J.A., WrayV., Schwarz, G., Journal of Biological Inorganic Chemistry, 2012, 17, 1,113-122 Structure and stability of the molybdenum cofactor intermediate cyclic pyranopterin monophosphate.

Cyclic pyranopterin monophosphate (cPMP)

Nitrate reductase

In plants and some animals the first stage in the reduction of nitrate is to nitrite. The reduction is catalysed by nitrate reductase, a flavoprotein enzyme which has molybdenum as the only metal requirement. Molybdenum acts as an electron acceptor from reduced FAD in the enzyme. The molybdenum cofactor is an oxomolybdenum sulfur species with a pterin ligand.

Berks, B. C., Ferguson, S. J., Moir, J. W. B. and Richardson, D. J., Biochim. Biophys. Acta - Bioenergetics , 1995, 1232, 97.

Campbell, W. H., Plant Physiology , 1996, 111 , 355.

Collison, D., Garner, C. D. and Joule, J. A., Chem. Soc. Rev. , 1996, 25 , 25.

Although NR always catalyzes the conversion of nitrate to nitrite, its location in the cell, structure, and function are organism-dependent. Protein sequencing is used to determine phylogenetic relationships and to examine similarities in structure and function. Conserved binding sites for molybdenum and pterin cofactors are found.

Stolz, J.F. and Basu, P., Evolution of nitrate reductase: Molecular and structural variations on a common function, Chembiochem, 2002, 3, 198-206.

Gonzalez, P. J., Correia, C., Moura, I., Brondino, C. D., and Moura, J. J. G., Bacterial nitrate reductases: Molecular and biological aspects of nitrate reduction, Journal of Inorganic Biochemistry, 2006, 100, 1015-1023.

Nitrate reductase

Enzymes of the DMSO reductase family use a mononuclear Mo-bis(molybdopterin) cofactor (MoCo) to catalyze a variety of oxo-transfer reactions. Site-directed mutagenesis, EPR and protein film voltammetry were used to demonstrate that the MoCo in R. sphaeroides periplasmic nitrate reductase (NapAB) is subject to an irreversible reductive activation process. This activation quantitatively correlates with the disappearance of the so-called "Mo(V) high-g" EPR signal, but this reductive process is too slow to be part of the normal catalytic cycle. Therefore, in NapAB, this most intense and most commonly observed signature of the MoCo arises from a dead-end, inactive state that gives a catalytically competent species only after reduction. This activation proceeds, even without substrate, according to a reduction followed by an irreversible nonredox step, both of which are pH independent. An apparently similar process occurs in other nitrate reductases (both assimilatory and membrane bound), and this also recalls the redox cycling procedure, which activates periplasmic DMSO reductases and simplifies their spectroscopic signatures.

It is proposed that heterogeneity at the active site and reductive activation are common properties of enzymes from the DMSO reductase family.

Regarding NapAB, that no Mo EPR signal could be detected upon reoxidizing the fully reduced enzyme suggests that the catalytically active form of the Mo(V) is thermodynamically unstable, as for other enzymes of the DMSO reductase family.

Fourmond, V., Burlat, B., Dementin, S., Arnoux, P., Sabaty, M., Boiry, S., Guigliarelli, B., Bertrand, P., Pignol, D., and Leger, C., Major Mo(V) EPR Signature of Rhodobacter sphaeroides Periplasmic Nitrate Reductase Arising from a Dead-End Species That Activates upon Reduction. Relation to Other Molybdoenzymes from the DMSO Reductase Family, Journal of Physical Chemistry B, 2008, 112, 15478-15486.

Nitrate reductase in winter wheat enhanced by molybdenum

The objective was to study whether the accumulation and utilization of plant nitrogen are controlled by molybdenum status in winter wheat cultivars. Mo-efficient cultivar 97003 and Mo-inefficient cultivar 97014 were grown in severely Mo-deficient acidic soil (0.112 mg Mo kg^-1) with and without the application of 0.13 mg Mo kg^-1. The accumulation and use efficiency of plant total nitrogen were higher in the molybdenum-treated soil. The overall activity of nitrate reductase was higher in the molybdenum-treated soil and the activity of glutamine synthetase was lower. Concentration of nitrate and glutamate were also lower in the molybdenum-treated soil; evidences for enhanced nitgrogen use efficiency due to added molybdenum. Molybdenumd promote nitrogen accumulation and utilization in winter wheat which was directly related to nitrate reductase and feedback regulated by glutamine synthetase. Higher molybdenum status also results in higher accumulation and utilization of plant nitrogen in the Mo-efficient cultivar.

Yu, M., Hu, C. X., Sun, X. C., and Wang, Y. H., Influences of Mo on Nitrate Reductase, Glutamine Synthetase and Nitrogen Accumulation and Utilization in Mo-Efficient and Mo-Inefficient Winter Wheat Cultivars, Agricultural Sciences in China, 2010, 9, 355-361.

Nitrate reductase mechanism

Calculations show that the reduction of nitrate to nitrite can occur through an association of nitrate to the Mo center, followed by rupture of the Mo-O-NO₂- bond. the direct coordination of nitrate to Mo is an almost barrier-free process, and the barrier for the rate-determining step of the Mo-O-NO₂- bond cleavage is about 11.7 kcal mol^-1, significantly lower than those in other plausible mechanisms. The presence of a disulfide bond in the active site can influence the interconversion of Mo(IV) to Mo(VI).

Xie, H. J. and Cao, Z. X., Enzymatic Reduction of Nitrate to Nitrite: Insight from Density Functional Calculations, Organometallics, 2010, 29, 436-441.

Nitrogenase

Nitrogenase is the complex metalloenzyme responsible for biological dinitrogen reduction. The mechanism of nitrogen fixation in enzymes and in model systems in vitro has been extensively investigated.

Burris, R. H. and Roberts, G. P., Ann. Rev. Nutrition , 1993, 13 , 317.

Sellman, D., Agnew. Chem. Int. Ed. , 1993, 105 , 64.

The microorganisms which fix molecular nitrogen fall into two classes: (a) the symbiotic microorganisms which fix nitrogen in association with plants, e.g., Rhizobium; (b) asymbiotic microorganisms which are free-living and include Azotobacter vinelandii and Clostridium Pasteurianum. From cell-free extracts of C. Pasteurianum, two metalloproteins have been obtained. The hydrogen donating system, azoferredoxin, which contains iron and sulfide and the nitrogen activating system, molybdoferredoxin, which contains molybdenum, iron, and sulfide. The structure of the active centre has been shown by X-ray crystallography to be a Fe-Mo-S cluster.

Kim, J., Woo, D. and Rees, D. C., Biochemistry ,1993 , 32 ,7104.

Chen, J., Christiansen, J., Campbasso, N., Bolin, J. T., Tittsworth, B. C., Hales, B. J., Rehr, J. J. and Cramer, S. P., Angew. Chem. Int. Edn. ,1993, 32,1592.

The mechanism of the N₂ fixing enzyme nitrogenase has been reviewed including a discussion of why low molecular weight complexes have not yet been able to copy nitrogenase catalyzed reactions or to act as competitive catalysts with nitrogenase-like activity.

Sellmann, D., Utz, J., Blum, N., Heinemann, F.W., On the function of nitrogenase FeMo cofactors and competitive catalysts: chemical principles, structural blue-prints, and the relevance of iron sulfur complexes for N-2 fixation, Coordination Chemistry Reviews , 1999, 192 , 607-627.

Recent developments in understanding the mechanism of the Mo-based nitrogenase are reviewed: how nucleotide binding and hydrolysis are coupled to electron transfer and substrate reduction, how electrons are accumulated and transferred within the MoFe-protein, and how substrates bind and are reduced at the active site metal cluster.

Christiansen, J., Dean, D. R., and Seefeldt, L. C., Mechanistic features of the Mo-containing nitrogenase, Annual Review of Plant Physiology and Plant Molecular Biology, 2001, 52, 269.

The Mo-site and its ligand environment of the FeMo-cofactor (FeMo-co) were studied using the hybrid density functional method B3LYP.

Szilagyi, R.K., Musaev, D. G., and Morokuma, K., Theoretical studies of biological nitrogen fixation. I. Density functional modeling of the Mo-site of the FeMo-cofactor, Inorganic Chemistry , 2001, 40, 766-775

The MoFe protein of the nitrogenase enzyme from Azotobacter vinelandii was altered by substituting the alpha Gly(69) residue by serine. Then both CO and acetylene become competitive inhibitors of dinitrogen reduction. CO is also converted from a non-competitive inhibitor of the wild type enzyme to a competitive inhibitor of acetylene, nitrous oxide, and azide reduction. These results are interpreted in terms of a two-site model with both sites located in close proximity within a specific 4Fe-4S face of FeMo cofactor. Site 1 is a high affinity acetylene-binding site to which CO also binds; but dinitrogen, azide, and nitrous oxide do not bind. Site 2 is a low affinity acetylene-binding site to which CO, dinitrogen, azide, and nitrous oxide also bind.

Christiansen, J., Seefeldt, L. C., and Dean, D. R., Competitive substrate and inhibitor interactions at the physiologically relevant active site of nitrogenase, Journal of Biological Chemistry , 2000, 275 , 36104-36107.

The role of molybdenum in nitrogenase has been reviewed with particular reference to theoretical modeling of the molybdenum centre.

Barriere, F., Modeling of the molybdenum center in the nitrogenase FeMo- cofactor, Coordination Chemistry Reviews, 2003, 236, 71-89.

See also

Bell, J., Dunford, A. J., Hollis, E., and Henderson, R. A., The role of Mo atoms in nitrogen fixation: Balancing substrate reduction and dihydrogen production, Angewandte Chemie-International Edition, 2003, 42 , 1149-1152.
Benton, P.M.C., Laryukhin, M., Mayer, S. M., Hoffman, B. M., Dean, D. R., and Seefeldt, L. C., Localization of a substrate binding site on the FeMo-cofactor in nitrogenase: Trapping propargyl alcohol with an alpha-70- substituted MoFe protein, Biochemistry, 2003, 42, 9102-9109.
Hinnemann, B. and Norskov, J. K., Modeling a central ligand in the nitrogenase FeMo cofactor, Journal of the American Chemical Society, 2003, 125, 1466-1467.

Barney, B. M., Lee, H. I., Dos Santos, P. C., Hoffmann, B. M., Dean, D. R., and Seefeldt, L. C., Breaking the N-2 triple bond: insights into the nitrogenase mechanism, Dalton Transactions, 2006, 2277-2284.

Nitrogenase mechanism possible involvement of molybdenum in substrate interactions during catalytic turnover

Molybdenum does not participate in binding a hydride of the catalytically central E-4 intermediate and only Fe ions are involved. Nonetheless, the response of the molybdenum coupling to subtle conformational changes in E-0 and to the formation of E-4 suggests that molybdenum is intimately involved in tuning the geometric and electronic properties of FeMo-co in these states

Lukoyanov, D., Yang, Z. Y., Dean, D. R., Seefeldt, L. C., and Hoffman, B. M., Is Mo Involved in Hydride Binding by the Four-Electron Reduced (E-4) Intermediate of the Nitrogenase MoFe Protein, Journal of the American Chemical Society, 2010, 132, 2526.

Nitrogenase ― tungstate toxicity and effect of catechol

Tungsten is toxic to N₂-fixing bacteria, partly through substituting for Mo in nitrogenase. Catechol siderophores produced by A. vinelandii (essential for iron acquisition) modulate the relative uptake of molybdenum and tungsten. At high tungsten, concentrations of these catechol siderophores (particularly protochelin) in the growth medium increase sharply; they complex all the tungstate along with molybdate and some of the iron. The molybdenum-catechol complex is taken up much more rapidly than the tungsten complex, allowing A. vinelandii to satisfy its molybdenum requirement and avoid tungsten toxicity. Mutants deficient in the production of catechol siderophores are more sensitive to tungstate and have higher cellular tungsten quotas than the wild type. The binding of metals by excreted catechol siderophores allows A. vinelandii to discriminate in its uptake of essential metals, iron and molybdenum, over that of the toxic metal, tungsten, and to sustain high growth rates under adverse environmental conditions.

Wichard, T., Bellenger, J. P., Loison, A., and Kraepiel, A. M. L., Catechol siderophores control tungsten uptake and toxicity in the nitrogen-fixing bacterium Azotobacter vinelandii, Environmental Science & Technology , 2008, 42, 2408-2413.

Nitrogenase ― Assembly of MoFe protein

Recent advances in elucidation of the mechanism of FeMoco biosynthesis in four aspects: (1) the ex situ assembly of FeMoco on NifEN, (2) the incorporation of FeMoco into MoFe protein, (3) the in situ assembly of P-cluster on MoFe protein, and (4) the stepwise assembly of MoFe protein are reviewed.

Hu, Y.L., Fay, A. W., Lee, C. C., Yoshizawa, J., and Ribbe, M. W., Assembly of nitrogenase MoFe protein, Biochemistry, 2008, 47, 3973-3981.

Nitrogenase biosynthesis

Recent developments in the understanding of the biosynthesis of the active site of the nitrogenase enzyme, the structure of the iron centre of [Fe]-hydrogenase and the structure and biomimetic chemistry of the [FeFe] hydrogenase H-cluster as deduced by application of X-ray spectroscopy are reviewed.

Best, S. P. and Cheah, M. H., Applications of X-ray absorption spectroscopy to biologically relevant metal-based chemistry, Radiation Physics and Chemistry, 2010, 79, 185-194.

Vanadium nitrogenase

Vanadium is a cofactor in the alternative V-nitrogenase that is expressed by some N₂-fixing bacteria when Mo is not available. The V requirements, the kinetics of V uptake, and the production of catechol compounds across a range of concentrations of vanadium in diazotrophic cultures of the soil bacterium Azotobacter vinelandii were investigated. In strain CA11.70, a mutant that expresses only the V-nitrogenase, V concentrations in the medium between 10^-8 and 10^-6 M sustain maximum growth rates; they are limiting below this range and toxic above. A. vinelandii excretes in its growth medium micromolar concentrations of the catechol siderophores azotochelin and protochelin, which bind the vanadate oxoanion. The production of catechols increases when V concentrations become toxic. Short-term uptake experiments with the radioactive isotope V-49 show that bacteria take up the V-catechol complexes through a regulated transport system, which shuts down at high V concentrations. The modulation of the excretion of catechols and of the uptake of the V-catechol complexes allows A. vinelandii to precisely manage its V homeostasis over a range of V concentrations, from limiting to toxic.

Bellenger, J. P., Wichard, T., and Kraepiel, A. M. L., Vanadium requirements and uptake kinetics in the dinitrogen-fixing bacterium Azotobacter vinelandii, Applied and Environmental Microbiology, 2008, 74, 1478-1484.

Nitrogenfixation: nitrogenase

Molecular nitrogen is the source of all of the nitrogen necessary to sustain life on earth. Biological nitrogen fixation takes N₂ and converts it into ammonia using nitrogenase enzymes, whereas industrial nitrogen fixation converts N₂ and H₂ to NH₃ using heterogeneous iron or ruthenium surfaces. In both cases, the processes are energy-intensive. Is it possible to discover a homogeneous catalyst that can convert molecular nitrogen into higher-value organonitrogen compounds using a less energy-intensive pathway? If this could be achieved, it would be considered a major breakthrough.

In contrast to carbon monoxide, which is reactive and an important feedstock in many homogeneous catalytic reactions, the ischelectronic but inert N₂ molecule is a very poor ligand and not a common industrial feedstock, except for the industrial production of NH₃ .

Because N₂ is available from the atmosphere and because nitrogen is an essential element for the biosphere, attempts to discover new processes involving this simple small molecule have occupied chemists for over a century. Since the first discovery of a dinitrogen complex in 1965, inorganic chemists have been key players in this area and have contributed much fundamental knowledge on structures, binding modes, and reactivity patterns. For the most part, the synthesis of dinitrogen complexes relies on the use of reducing agents to generate an electron-rich intermediate that can interact with this rather inert molecule. In this Account, a facile reaction of dinitrogen with a ditantalum tetrahydride species to generate the unusual side-on end-on bound N₂ moiety is described. This particular process is one of a growing number of new, milder ways to generate dinitrogen complexes. Furthermore, the resulting dinitrogen complex undergoes a number of reactions that expand the known patterns of reactivity for coordinated N₂. This Account reviews the reactions of ([NPN]Ta)(2)(mu-H)(2)(mu-eta(1):eta(2)-N-2), 2 (where NPN = PhP(CH2SiMe2NPh)(2)), with a variety of simple hydride reagents, E-H (where E-H = R2BH, R2AlH, RSiH3, and Cp2ZrCl(H)), each of which results in the cleavage of the N-N bond to form various functionalized imide and nitride moieties. This work is described in the context of a possible catalytic cycle that in principle could generate higher-value nitrogen-containing materials and regenerate the starting ditantalum tetrahydride. How this fails for each particular reagent is discussed and evaluated

Fryzuk, M. D., Side-on End-on Bound Dinitrogen: An Activated Bonding Mode That Facilitates Functionalizing Molecular Nitrogen, Accounts of Chemical Research, 2009, 42, 127-133.

Nitrogenase mechanism - homologue approach

The (Mo)-nitrogenase is a complex metalloenzyme that catalyzes the key step in the global nitrogen cycle, the reduction of atmospheric dinitrogen (N-2) to bioavailable ammonia (NH3), at the iron-molybdenum cofactor (FeMoco) site of its molybdenum-iron (MoFe) protein component. One major challenge for the mechanistic study of nitrogenase is the redox versatility of its FeMoco center. The ability of FeMoco to shuttle between oxidation states in a rapid and unsynchronized manner results in a mixed oxidation state of the cofactor population during turnover. The substrate and the various intermediates can only interact with the FeMoco site in a transient manner, so it is extremely difficult to capture any substrate- or intermediate-bound form of nitrogenase for the direct examination of substrate-enzyme interactions during catalysis.

In this Account, we describe the approach of identifying a partially "defective" nitrogenase homologue, one with a slower turnover rate, as a means of overcoming this problem.

The NifEN protein complex serves as an ideal candidate for this purpose. It is an alpha(2)beta(2)-heterotetramer that contains cluster-binding sites homologous to those found in the MoFe protein: the "P-cluster site" at the interface of the alpha beta-subunit dimer, which accommodates a [Fe4S4]-type cluster; and the "FeMoco site" within the alpha-subunit, which houses an all-iron homologue to the FeMoco. Moreover, NifEN mimics the MoFe protein in catalysis: it is capable of reducing acetylene (C2H2) and azide (N-3(-)) in an ATP- and iron (Fe) protein-dependent manner. However, NifEN is unable to reduce proton (H+) and N-2, and it is an inefficient enzyme with a restricted electron flux during the turnover.

The extremely slow turnover rate of NifEN and the possible "synchronization" of its FeMoco homologue at a certain oxidation level permit the observation of a new S = 1/2 EPR signal upon turnover of C2H2 by NifEN, which is analogous to the signal reported for a MoFe protein variant upon turnover of the same substrate.

This result is exciting, because it suggests the possibility of naturally enriching a C₂H₂-bound form of NifEN for the successful crystallization of the first intermediate-bound nitrogenase homologue.

On the other hand, the fact that NifEN represents a partially "defective" homologue of the MoFe protein makes it a promising mutational platform on which a functional MoFe protein equivalent may be reconstructed by introducing the missing features of MoFe protein step-by-step into NifEN.

Such a strategy allows us to define the function of each feature and address questions such as the following: What is the function of P-cluster in catalysis? Are Mo and homocitrate the essential constituents of the cofactor in N-2 reduction? How does substrate accessibility affect the reactivity of the enzyme? This homologue approach could complement the mechanistic analysis of the nitrogenase MoFe protein, and information derived from both approaches will help achieve the ultimate goal of solving the riddle of biological nitrogen fixation

Hu, Y. L. and Ribbe, M. W., Decoding the Nitrogenase Mechanism: The Homologue Approach, Accounts of Chemical Research, 2010, 43, 475-484.

Nitrogen fixation pre-biotic

Nitrogen reduction by iron(II) has been suggested as an important mechanism in the formation of ammonia on pre-biotic Earth. This paper examines the effects of adsorption of Fe²⁺ onto a goethite (alpha-FeOOH) substrate on the thermodynamic driving force and rate of a Fe²⁺-mediated reduction of N₂ as compared with the homogeneous aqueous reaction. The key to reduction in both cases is N₂adsorption to multiple transition metal centers with competitive H₂ production

Wander, M.C.F., Kubicki, J. D., and Schoonen, M. A. A., Reduction of N₂by Fe²⁺ via homogeneous and heterogeneous reactions Part 2: The role of metal binding in activating N₂ for reduction; a requirement for both pre-biotic and biological mechanisms, Origins of Life and Evolution of Biospheres, 2008, 38, 195-209.

Selenate and nitrate

Some of the common themes and variations between the different classes of nitrate and selenate reductases are reviewed.

Watts, C.A., Ridley, H., Dridge, E. J., Leaver, J. T., Reilly, A. J., Richardson, D. J., and Butler, C. S., Microbial reduction of selenate and nitrate: common themes and variations, Biochemical Society Transactions, 2005, 33, 173-175.

Sulfite oxidase

See also below: Molybdenum cofactor deficiency in humans: neurological consequences of sulfite oxidase deficiency.

Sulfite is the main intermediate in the oxidation of sulfur compounds to sulfate, the major product of most dissimilatory sulfur-oxidizing prokaryotes. Two pathways of sulfite oxidation are known: (1) direct oxidation to sulfate catalyzed by a sulfite: acceptor oxidoreductase, which is thought to be a molybdenum-containing enzyme; (2) indirect oxidation under the involvement of the enzymes adenylylsulfate (APS) reductase and ATP sulfurylase and/or adenylylsulfate phosphate adenylyltransferase with APS as an intermediate. Direct oxidation appears to have a wider distribution. In many pro- and also eukaryotes sulfite is formed as a degradative product from molecules containing sulfur as a heteroatom. In these organisms detoxification of sulfite is generally achieved by direct oxidation to sulfate.

Kappler, U. and Dahl, C., Enzymology and molecular biology of prokaryotic sulfite oxidation, Fems Microbiology Letters, 2001, 203 , 1-9.

Sulfite oxidase which was deficient in molybdopterin was reconstituted in vitro with the molybdenum cofactor (Moco) synthesized de novo from precursor and molybdate. In vitro reconstitution of the purified apoprotein was achieved using an incubation mixture containing purified precursor, purified molybdopterin synthase, and sodium molybdate.

Leimkuhler, S. and Rajagopalan, K. V., In vitro incorporation of nascent molybdenum cofactor into human sulfite oxidase, Journal of Biological Chemistry, 2001, 276, 1837-1844.

The electronic (charge-transfer) spectrum of the enzyme sulfite oxidase has been probed by temperature-dependent magnetic circular dichroism (MCD) spectroscopy. The enzyme was poised in the catalytically relevant [ Mo(V):Fe(II)] state by anaerobic reduction with sulfite in the absence of cytochrome c. A feature at 22 250 cm-1 in the MCD is assigned as the cysteine S(sigma)-->Mo d(xy) charge transfer. The primary role of the co-ordinated cysteine is to decrease the effective nuclear charge on Mo by charge donation to the metal, statically poising the active site at more negative reduction potentials during electron transfer regeneration.

Helton, M.E., Pacheco, A., McMaster, J., Enemark, J. H., and Kirk, M. L., An MCD spectroscopic study of the molybdenum active site in sulfite oxidase: insight into the role of co-ordinated cysteine, Journal of Inorganic Biochemistry , 2000, 80 , 227-233.

Molybdenum is an essential constituent of the enzyme hepatic sulfite oxidase. The reduced enzyme, like xanthine oxidase, gives an electron paramagnetic resonance signal of molybdenum(V).

Sheep and cows, develop adverse reactions to feed containing 2-30 ppm molybdenum; horses and pigs tolerate feed with concentrations > 1000 ppm molybdenum [Smyth, 1956].

Cohen, H. J., Fridovich, I. and Rajagopalan, K. V., J. Biol. Chem., 1971, 246 , 374.

Smyth, H.E., Hygienic standard for daily inhalation. Ind Hyg Q, 1956, 17 ,129-185.

Molybdenum K-edge X-ray absorption studies of the oxidized and reduced active sites of the sulfite dehydrogenase from Starkeya novella showed that the molybdenum atom of the oxidized enzyme is bound by two Mo=O ligands at 1.73 angstrom and three thiolate Mo-S ligands at 2.42 angstrom, whereas the reduced enzyme has one oxo at 1.74 angstrom, one long oxygen at 2.19 angstrom (characteristic of Mo-OH₂), and three Mo-S ligands at 2.40 angstrom

Doonan, C. J., Kappler, U., and George, G. N., Structure of the active site of sulfite dehydrogenase from Starkeya novella, Inorganic Chemistry, 2006, 45, 7488-7492.

Enemark, J. H., Astashkin, A. V., and Raitsimring, A. M., Investigation of the coordination structures of the molybdenum(v) sites of sulfite oxidizing enzymes by pulsed EPR spectroscopy, Dalton Transactions, 2006, 3501-3514.

Hemann, C., Hood, B. L., Fulton, M., Hansch, R., Schwarz, G., Mendel, R. R., Kirk, M. L., and Hille, R., Spectroscopic and kinetic studies of Arabidopsis thaliana sulfite oxidase: Nature of the redox-active orbital and electronic structure contributions to catalysis, Journal of the American Chemical Society, 2005, 127, 16567-16577.

The recent developments in our understanding of sulfite oxidizing enzyme mechanisms that are driven by a combination of molecular biology, rapid kinetics, pulsed electron paramagnetic resonance (EPR), and computational techniques are the subject of this review.

Feng, C. J., Tollin, G., and Enernark, J. H., Sulfite oxidizing enzymes, Biochimica et Biophysica Acta-Proteins and Proteomics, 2007, 1774, 527-539.

Chloride and sulfite oxidase

Chloro ligands in model oxomolybdenum(V) chloro complexes in an electron spin echo envelope modulation (ESEEM) spectroscopy study had greater ESEEM amplitude than chloride near the oxomolybdenum active site in the high chloride, low-pH form of sulfite oxidase so ruling out equatorial coordination of chloride in the enzyme.

Astashkin, A. V., Klein, E. L., and Enemark, J. H., Toward modeling the high chloride, low pH form of sulfite oxidase: K-a-band ESEEM of equatorial chloro ligands in oxomolybdenum(V) complexes, Journal of Inorganic Biochemistry, 2007, 101, 1623-1629.

Sulfite oxidase coordinated sulfate

Astashkin, A. V., Johnson-Winters, K., Klein, E. L., Byrne, R. S., Hille, R., Raitsimring, A. M., and Enemark, J. H., Direct demonstration of the presence of coordinated sulfate in the reaction pathway of Arabidopsis thaliana sulfite oxidase using S-33 Labeling and ESEEM Spectroscopy, Journal of the American Chemical Society, 2007, 129, 14800-14810

Sulfite oxidase vasorelaxation

The effect of dietary sulphite supplementation on vascular responsiveness in sulphite oxidase (SO)-deficient rats was studied. Increased production of reactive oxygen species and the resultant increment in L-arginine/nitric oxide consumption may play a role in the reduced endothelium-dependent vasorelaxation in sulphite-treated SO-deficient rats

Nacitarban, C., Kucukatay, V., Sadan, G., Ozturkl, O. H., and Agao, A., Effects of sulphite supplementation on vascular responsiveness in sulphite oxidase-deficient rats, Clinical and Experimental Pharmacology and Physiology, 2008, 35, 268-272.

Mo(V) center of the Y343F mutant of human sulfite oxidase by variable frequency pulsed EPR spectroscopy

Raitsimring, A.M., Astashkin, A. V., Feng, C., Wilson, H. L., Rajagopalan, K. V., and Enemark, J. H., Studies of the Mo(V) center of the Y343F mutant of human sulfite oxidase by variable frequency pulsed EPR spectroscopy, Inorganica Chimica Acta, 2008, 361, 941-946.

Sulfite oxidase

Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased K-m(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (similar to 60 and 200 s^-1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH

Bailey, S., Rapson, T., Johnson-Winters, K., Astashkin, A. V., Enemark, J. H., and Kappler, U., Molecular Basis for Enzymatic Sulfite Oxidation HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY, Journal of Biological Chemistry, 2009, 284, 2053-2063.

Sulfite oxidase in plants

The occurrence of sulfite oxidase in plants has been established by identification of a cDNA from Arabidopsis thaliana encoding a functional sulfite oxidase. The aim was to identify herbaceous and woody plants (Azardirachta indica L., Cassia fistula L., Saraca indica L., Spinacea oleracea L., and S Syzyzium cumini L.) with sulfite oxidase activity and to characterize some of its immuno-biochemical properties. The Syzyzium cumini was chosen to characterize sulfite oxidase as it showed maximum enzyme activity in the crude extract as compared to other plants. Absorption spectra of sulfite oxidase revealed two peaks at 235 and 277 nm, but no distinct peak in the visible region. Crude extracts of the plants were studied for immuno-biochemical studies. Despite protein structure-functional similarities between plant and animal sulfite oxidase, no cross-reactivity could be established between the two sources of sulfite oxidase. These data suggested that plants sulfite oxidase, however, differed with regards to their immuno-biochemical properties

Ahmad, A. and Ahmad, S., Screening and partial immunochemical characterization of sulfite oxidase from plant source, Indian Journal of Experimental Biology, 2010, 48, 83-86.

Sulfite oxidase mechanism molecular dynamics simulations to understand the large-scale domain motions of the enzyme

Molecular dynamics simulations were undertaken to understand the large-scale domain motions of the enzyme. Motion of the N-terminal domain into an orientation similar to that postulated for rapid electron transfer was observed. Simulations also probe the dynamics of the active site and surrounding residues, adding a further level of structural and thermodynamic detail in understanding sulfite oxidase function

Pushie, M. J. and George, G. N., Active-Site Dynamics and Large-Scale Domain Motions of Sulfite Oxidase: A Molecular Dynamics Study, Journal of Physical Chemistry B, 2010, 114, 3266-3275

Essential molybdenum - Molybdenum-dependent sulfite-oxidizing enzymes

Sulfite-oxidizing enzymes (SOEs) are molybdenum-dependent and are found in vertebrates, plants and bacteria. They catalyze the oxidation of sulfite to sulfate :.

SO₃²⁻+ H₂O = SO₄^{2 −} + 2H⁺ + 2e ⁻

The oxygen atom that is incorporated into sulfite comes from water (rather than from dioxygen). In the fully oxidized resting state of the enzymes, the catalytic site is a nearly square-pyramidal dioxo-molybdenum centre that has three equatorial sulfur ligands (one from the conserved cysteinyl side chain and two from the molybdopterin (MPT) cofactor) and two oxo ligands—one equatorial and one axial.The mechanism for SOEs involves attack of sulfite on the electrophilic equatorial oxo ligand that is exposed to solvent, followed by hydrolysis of sulfate and two sequential one-electron oxidations to return the enzyme to the fully oxidized resting state:

Mo^VI=O + SO₃^{2 −} à Mo^IV-OSO₃, + H₂O à Mo^V-OH, − SO₄^{2 −}àMo^VI=O, − H⁺, − e ⁻

In chloride-depleted samples at low pH a blocked form of the enzyme is obtained having molybdenum(V) binding sulfite.

Enemark, J. H., Raitsimring, A. M., Astashkin, A. V., and Klein, E. L., Implications for the mechanism of sulfite oxidizing enzymes from pulsed EPR spectroscopy and DFT calculations for "difficult'' nuclei, Faraday Discussions, 2011, 148, 249-267.

Sulfite oxidase variants having nitrate reductase activity.

Eukaryotic sulfite oxidase is a dimeric protein that contains molybdenum cofactor and catalyzes the metabolically essential conversion of sulfite to sulfate as the terminal step in the metabolism of cysteine and methionine.

Nitrate reductase is an evolutionarily related molybdoprotein in lower organisms that is essential for growth on nitrate

Human and chicken sulfite oxidase variants in which the active site has been modified to alter substrate specificity and activity from sulfite oxidation to nitrate reduction are described.

The crystal structures of the Mo domains of the double and triple mutants were determined to and 2.1 angstrom resolution.

Qiu, J.A., Wilson, H.L., Rajagopalan, K. V. BIOCHEMISTRY, 2012, 51,1134-1147. Structure-Based Alteration of Substrate Specificity and Catalytic Activity of Sulfite Oxidase from Sulfite Oxidation to Nitrate Reduction

Xanthine dehydrogenase and xanthine oxidase

Xanthine oxidase (EC 1.1.3.22) and xanthine dehydrogenase (EC 1.1.1.204) belong to the molybdenum hydroxylase flavoprotein family; they represent different forms of the same gene product. Xanthine oxidoreductase activity is rate-limiting in purine catabolism. The enzymes can metabolize xenobiotics, including a number of anticancer compounds, to their active metabolites.
Xanthine oxidoreductase is implicated in the pathophysiology of inflammatory diseases and atherosclerosis and in ischemia- reperfusion injury.

Pritsos , C.A. , Cellular distribution, metabolism and regulation of the xanthine oxidoreductase enzyme system, Chem.Biol.Interact. , 12-1-2000 , 129 , 195-208.

The role of molybdenum in xanthine oxidase has been extensively studied [Collison et al., 1996; Peive, 1968]. During the enzyme-catalysed reaction the oxidation state of molybdenum changes and so molybdenum is involved in the electron-transfer pathway. The substrate interacts directly with molybdenum. The molybdenum cofactor is an oxomolybdenum pterin complex similar to the cofactor of nitrate reductase [Berks et al., 1995; Campbell , 1996; Collison et al., 1996; Peive, 1968].

Collison, D., Garner, C. D. and Joule, J. A., Chem. Soc. Rev. , 1996, 25 , 25.

Peive, Ya. V.,(ed.), Biol. Rol Molibdena, Sb. Tr. Simp. 1968 (Publ. 1972), Nauka, Moscow , U.S.S.R., 207, 235.

Berks, B. C., Ferguson, S. J., Moir, J. W. B. and Richardson, D. J., Biochim. Biophys. Acta - Bioenergetics , 1995, 1232, 97.

Campbell, W. H., Plant Physiology , 1996, 111 , 355.

Collison, D., Garner, C. D. and Joule, J. A., Chem. Soc. Rev. , 1996, 25 , 25.

Xanthine oxidizing enzymes isolated from leaves of leguminous plants did not react with molecular oxygen at a significant rate, indicating that all of them are xanthine dehydrogenase . The visible absorption spectrum of the pure protein was assigned: 312 - 390 nm, related to the molybdopterin component of the enzyme; 420 - 510 nm, flavin chromophores; 550 nm, iron-sulphur centres. The fluorescence excitation spectrum showed peaks at 274 and 368 nm, similar to pterin and xanthopterin. The fluorescence emission spectrum was characterized by two maxima at about 400 and 460 nm, typical of flavin chromophores.

Montalbini, P., Xanthine dehydrogenase from leaves of leguminous plants: Purification, characterization and properties of the enzyme, Journal of Plant Physiology , 2000, 156 , 3-16.

Xanthine dehydrogenase (xanthine dehydrogenase ) is a member of the molybdenum hydroxylase family of enzymes catalyzing the oxidation of hypoxanthine and xanthine to uric acid. The enzyme is also required for the production of one of the major Drosophila eye pigments, drosopterin. The xanthine dehydrogenase gene has been isolated.

Pitts, R.J. and Zwiebel, L. J., Isolation and characterization of the xanthine dehydrogenase gene of the Mediterranean fruit fly, Ceratitis capitata, Genetics , 2001, 158, 1645-1655.

The compound 1-(3, 4- dimethoxy-2-chlorobenzylideneamino)-3-hydroxyguanidine (PR5) is a member of a novel class of compounds, xanthine oxidase electron acceptor-inhibitor drugs. They have potential use in the prevention of free radical mediated tissue damage in organ ischemia- reperfusion diseases.
PR5 acts as an alternative electron acceptor in xanthine oxidase catalysed oxidation of xanthine. The action of PR5 is associated with the inhibition of superoxide radical formation. It is suggested that PR5 binds and becomes reduced at the molybdenum centre of the xanthine oxidase.

Dambrova, M., Baumane, L., Kiuru, A., Kalvinsh, I., and Wikberg, J. E., N-Hydroxyguanidine compound 1-(3,4-dimethoxy- 2-chlorobenzylideneamino)- 3-hydroxyguanidine inhibits the xanthine oxidase mediated generation of superoxide radical, Arch.Biochem.Biophys. , 5-1-2000, 377 , 101-108.

The drug allopurinol is used to treat xanthine dehydrogenase -catalyzed uric acid build-up occurring in gout or during cancer chemotherapy. As a hypoxanthine analog, it is oxidized to alloxanthine, which cannot be further oxidized but acts as a tight binding inhibitor of xanthine dehydrogenase . The 3.0 Angstrom resolution structure of the xanthine dehydrogenase -alloxanthine complex shows direct coordination of alloxanthine to the molybdenum via a nitrogen atom. These results provide a starting point for the rational design of new xanthine dehydrogenase inhibitors

Truglio, J.J., Theis, K., Leimkuhler, S., Rappa, R., Rajagopalan, K. V., and Kisker, C., Crystal structures of the active and alloxanthine-inhibited forms of xanthine dehydrogenase from Rhodobacter capsulatus, Structure , 2002, 10, 115-125.

Reactive oxygen species, either superoxide anion radical or hydrogen peroxide, are generated when xanthine oxidase catalyses the hydroxylation of purines. Xanthine oxidase serum levels are increased in various pathological states: hepatitis, inflammation, ischemia-reperfusion, carcinogenesis and aging. The reactive oxygen species are involved in oxidative damage. The inhibition of this enzymatic pathway might be beneficial. Excess of uric acid, the metabolic product of xanthine oxidase, can lead to gout. Allopurinol is a clinically useful inhibitor used in the treatment of gout.The structure and mechanism of the enzyme, associated pathological states, and the development of new xanthine oxidase inhibitors are reviewed.

Xanthine oxidase inhibition

Coumarin derivatives competitively inhibited xanthine oxidase. Esculetin (6,7-dihydroxycoumarin) had the highest affinity toward the binding site of xanthine oxidase due to the interaction of 6-hydroxyl with the E802 residue of xanthine oxidase. Esculetin was the most potent suppressor of reactive oxygen species. Esculetin was the most potent agent at protecting living cells against A beta-damage mediated by reactive oxygen species.

Lin, H.C., Tsai, S. H., Chen, C. S., Chang, Y. C., Lee, C. M., Lai, Z. Y., and Lin, C. M., Structure-activity relationship of coumarin derivatives on xanthine oxidase-inhibiting and free radical-scavenging activities, Biochemical Pharmacology, 2008, 75, 1416-1425.

Borges, F., Fernandes, E., and Roleira, F. , Progress towards the discovery of xanthine oxidase inhibitors, Current Medicinal Chemistry, 2002, 9, 195-217.

An overview of the current state of our understanding of the reaction mechanism of the molybdenum-containing enzyme xanthine oxidoreductase is presented, with an emphasis on work done in the past five years. Recent studies of the biosynthesis of the pterin cofactor bound to the metal in the active site are also reviewed, as is crystallographic work that has clarified the coordination geometry of the molybdenum center. This structural work provides the context in which to understand recent mechanistic studies of the enzyme, in particular those aimed at elucidating the role of specific amino acid residues in the active site of the enzyme

Hille, R., Structure and function of xanthine oxidoreductase, European Journal of Inorganic Chemistry, 2006, 1913-1926.

Acute lung injury represents a wide spectrum of pathologic processes, the most severe being the acute respiratory distress syndrome. Reactive oxygen intermediates have been implicated in the pathobiochemistry of acute lung injury. The endogenous sources that contribute to the generation of reactive oxygen intermediates in acute lung injury probably include, inter alia, the molybdenum hydroxylases. Gene expression of xanthine dehydrogenase /XO is regulated in a cell-specific manner and is markedly affected by inflammatory cytokines, steroids, and physiologic events such as hypoxia. Posttranslational processing is also important in regulating xanthine dehydrogenase /XO activity.

Hoidal, JR, Xu, P, Huecksteadt, T, Sanders, KA, Pfeffer, K, Sturrock, AB, Lung injury and oxidoreductases, Environmental Health Perspectives , 1998, 106 , 1235-1239.

Xanthine Oxidase Mechanism of Inhibition by Flavonoids and Gallic Acid Derivatives

The main active center of the molybdoenzyme xanthine oxidase is a molybdopterin buried in a cavity. One possible mechanism of inhibition is the attraction of an inhibitor molecule inside the cavity. It is important to understand the mechanisms of the enzyme inhibition to help in the search of new inhibitors. In this work the attraction was studied computationally by ab initio (DFT) calculations. Two properties were shown to be important in the design of new inhibitors of medium size derived from flavonoids: the molecule must be polar, with a longitudinal dipole moment, and must be weakly dissociated at physiological pH.

Xanthine oxidoreductase (XOR) is a molybdoflavoenzyme, which is abundant in cow’s milk. It appears in two interconvertible forms xanthine dehydrogenase (XDH), and xanthine oxidase (XO).

Xanthine oxidoreductase catalyzes the hydroxylation of hypoxanthine to xanthine and of xanthine to urate. Oxidative hydroxylation occurs at the molybdenum center. With XDH NAD⁺ is reduced; with XO molecular oxygen is reduced at the flavin center.

Reduction of molecular oxygen produces free radicals which can damage tissues. XO is a potentiallydestructive agent in the vasculature.

Xanthine oxidase is a well-established target of drugs against gout and hyperuricemia.

Some pathological conditions show enhanced plasma XO levels: hepatic, acute viral infection, hemorrhagic shock, thermal stress,and hypercholesterolemia.

Allopurinol is applied as an inhibitor of xanthine oxidase but may induce hypersensivity reactions in patients with renal insufficiency and concomitant administration of thiazide diuretics. These undesirable effects have prompted efforts to isolate other types of XO inhibitors. Ethnobotanical research has provided XO inhibitors of natural origin.

Two main classes of molecules have been selected.

Flavonoids are natural compounds in plants, fruits, and vegetables. They are anti-oxidants. They are able to inhibit xanthine oxidase.

Gallic acid derivatives extracted from a Neo-Caledonian plant, Cunonia macrophylla, have been tested in vitro toward xanthine oxidase: gallic acid, ellagic acid, and ellagic acid-4-O-β-D-xylopyranoside.The last is the most active toward xanthine oxidase.

To evaluate the attraction inside the cavity, the electrostatic potential between the charged molybdopterin molecule and two series of inhibitors, flavonoids and some gallic acid derivatives, have been calculated using the multipolar development supplied by the Gaussian package. The good concordance between the electrostatic force and IC50 (the half maximal inhibitory concentration) shows that the attraction is an important factor in the inhibition and must be taken into account in the designing of new drugs

Lespade, L. and Bercion, S., Theoretical Study of the Mechanism of Inhibition of Xanthine Oxidase by Flavonoids and Gallic Acid Derivatives, Journal of Physical Chemistry B, 2010, 114, 921-928.

Flavonoids interaction with molybdenum hydroxylases review

Molybdenum hydroxylases, aldehyde oxidase and xanthine oxidase, are metalloflavoproteins that catalyze both oxidation and reduction of a broad range of drugs and other xenobiotics indicating the importance of these enzymes in drug oxidation, detoxification and activation. Both enzymes are also involved in some physiological processes and also the metabolism of some endogenous compounds which may indicate their important roles in in vivo conditions. Superoxide radical and hydrogen peroxide produced during molybdenum hydroxylases-catalyzed reactions may be relevant in various disease conditions. Therefore, the interference with the function of molybdenum hydroxylases could be of great importance. Flavonoids are a large group of polyphenolic compounds that are able to interfere with xanthine oxidase and aldehyde oxidase function. As flavonoids are consumed in high content in our daily life, their potential to interfere with molybdenum hydroxylases could be a serious concern for consumer safety. However, the subject has not received enough attention and has usually been overshadowed by that of cytochrome P450 as the most important drug metabolizing enzyme system. The present review focuses on the different aspects of flavonoids interaction with molybdenum hydroxylases considering literature published mainly in the last 2 decades. The review also provides insight into some research areas that may offer a great potential for future studies

Rashidi, M. R. and Nazemiyeh, H., Inhibitory effects of flavonoids on molybdenum hydroxylases activity, Expert Opinion on Drug Metabolism & Toxicology, 2010, 6, 133-152.

Substrate binding to molybdenum in the enzyme xanthine oxidoreductase rate limiting step

X-ray crystal structures of the urate complexes of the demolybdo-form of the D428A mutant of rat xanthine oxidoreductase at 1.7 angstrom and of the reduced bovine milk xanthine oxidoreductase2.1 angstrom, representing a reaction intermediate, are reported. The urate molecule is near the Mo ion, and high electron density connects them: hencea covalent link between molybdenum and urate via a bridging oxygen from urate. Thebridging electron density is bent, spanning a total of 3.5 Å, and

connects to the C8 atom of urate, the position where xanthine becomes hydroxylated during the reaction.

The structure presents a picture of arrested catalysis at the step of the intermediate formed in the course of oxygen atom transfer from the molybdenum coordination sphere to the substrate carbon to be hydroxylated.

The rate-limiting step in the enzymatic catalysis is breaking of the Mo-O bond accompanied by electron transfer and followed by release of the product.

Okamoto, K., Kawaguchi, Y., Eger, B. T., Pai, E. F., and Nishino, T., Crystal Structures of Urate Bound Form of Xanthine Oxidoreductase: Substrate Orientation and Structure of the Key Reaction Intermediate, Journal of the American Chemical Society, 2010, 132, 17080-17083.

Copper inhibition of xanthine oxidase

Milk xanthine oxidase forms optically observable complexes with Cu²⁺ ion. Cu²⁺ ion binds to milk xanthine oxidase with sulfur and nitrogenous ligands. Two Cu²⁺ bound milk xanthine oxidase complexes are formed at two different time scales of the interaction, earlier than 5 ms and at around 20 s. The second complex may be responsible for the inhibition of the enzyme activity.

Sau, A.K., Mondal, M. S., and Mitra, S., Interaction of Cu2+ ion with milk xanthine oxidase, Biochimica Et Biophysica Acta-Protein Structure and Molecular Enzymology, 2001, 1544, 89-95.

Xanthine oxidase (XO)-catalyses nitrite reduction to nitric oxide under anaerobic conditions. The XO reducing substrates xanthine, NADH, and 2,3 -dihydroxybenzaldehyde triggered nitrite reduction to NO, and the molybdenum-binding XO inhibitor oxypurinol inhibited this NO formation, indicating that nitrite reduction occurs at the molybdenum site. However, at higher xanthine concentrations, partial inhibition was seen, suggesting the formation of a substrate-bound reduced enzyme complex with xanthine blocking the molybdenum site. Studies of the pH dependence of NO formation indicated that XO-mediated nitrite reduction occurred via an acid-catalyzed mechanism, Nitrite and reducing substrate concentrations were important regulators of XO-catalyzed NO generation. XO-catalyzed nitrite reduction can be an important source of NO generation under ischemic conditions

Li, H.T., Samouilov, A., Liu, X. P., and Zweier, J. L., Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrite reduction - Evaluation of its role in nitric oxide generation in anoxic tissues, Journal of Biological Chemistry, 2001, 276, 24482-24489.

Xanthine oxidase and purine metabolism

Xanthine oxidase is a molybdoflavoprotein enzyme with the composition 2FAD:8Fe:2Mo [Bray et al., 1996; Collison et al., 1996; Bray, 1963; Bray and Swann, 1972; Peive,1968]. The enzyme has a low specificity and will catalyse oxidation of many purines and aldehydes with very different rates of reaction. The oxidation of xanthine to uric acid is an essential stage in the catabolism of purine bases in some animals. For example, in parts of New Zealand where the concentration of molybdenum in the soil was low (0.03 ppm compared with 0.4 ppm in other areas) serious losses of sheep occurred owing to the formation of xanthine calculi in the kidneys of the sheep [Underwood, 1962]. Because of the low molybdenum concentration in the pasture xanthine oxidase activity was low and the conversion of xanthine to uric acid was hindered with the consequence that xanthine was precipitated in the kidneys. Raising the molybdenum level in the pasture prevented the development of renal calculi. The relationship between molybdenum concentration and xanthine oxidase activity is not, however, quite straightforward. At low concentrations molybdenum may stimulate the activity of xanthine oxidase; but at high concentrations molybdenum may actually reduce the activity of the enzyme. The activity of xanthine oxidase in cows' milk is proportional to the molybdenum content and both depend on the quantity of ingested molybdenum [Kiermeir and Capellani, 1957]. Addition of molybdenum to the diet causes an increase in the milk molybdenum but does not affect xanthine oxidase activity if this is high before feeding additional molybdenum.

The effect of molybdenum on xanthine oxidase activity in rats has been studied but the results are conflicting. Thus in one study increased dietary molybdenum (0-800 ppm), although causing an increase in liver molybdenum, caused a decrease in liver xanthine oxidase activity and in the concentration of uric acid in the blood [Cox et al., 1960]. With rats fed 1.0 mg Mo/kg body weight daily there were no changes in purine metabolism but 20 mg Mo daily caused increased xanthine oxidase activity in the liver and kidneys and in the blood urate and urea levels [Grigoryan et al., 1969]. According to another report rats receiving added dietary molybdenum (50-500 ug) showed increased xanthine oxidase activity and increased uric acid concentrations in the blood and urine, but 5 ug/day decreased xanthine oxidase activity [Peive, 1968; Gusev, 1969]. Xanthine oxidase activity rises markedly during virus multiplication and it is possible that the enzyme has a controlling effect on the pattern of nucleic acid synthesis in vivo [Bray, 1963].

Bray, R. C., in The Enzymes , eds Boyer, P. D., Hardy, L. and Myrback, K., Academic Press, New York , 2nd Edn., 1963, 7, 533.

Bray, R. C., Knowles, P. F. and Meriwether, L. S., in Magnetic Resonance in Biological Systems , ed. Ehrenberg, A., Malstrom, B. G. and Vanngard, T., Pergamon, 1967, 249.

Bray, R. C. and Swann, J. C., Structure and Bonding, 1972, 11 , 107.

Bray, R. C., in Proceedings of the Climax International Conference on the Chemistry and Uses of Molybdenum, ed. Mitchell, P. C. H., Climax Molybdenum Co. Ltd, London and Ann Arbor , 1973, 216.

Bray, R. C., Bennett, B.m Burke, J. F., Chovnick, A., Doyle, W. A., Howes, B. D., Lowe, D. J., Richards, R. L., Turner, N. A., Ventom, A. and Whittle, J. R-S, Biochem. Soc. Trans. , 1996, 24, 99.

Collison, D., Garner, C. D. and Joule, J. A., Chem. Soc. Rev. , 1996, 25 , 25.

Peive, Ya. V.,(ed.), Biol. Rol Molibdena, Sb. Tr. Simp. 1968 (Publ. 1972), Nauka, Moscow , U.S.S.R., 207, 235.

Underwood, J. E., Trace Elements in Human and Animal Nutrition, Academic Press, London , 2nd Ed., 1962, 100.

Kiermeir, F. and Capellani, K., Naturwiss., 1957, 44 , 69.

Peive, Ya. V.,(ed.), Biol. Rol Molibdena, Sb. Tr. Simp. 1968 (Publ. 1972), Nauka, Moscow , U.S.S.R., 207, 235.

Cox, D. H., Davis, G. K., Shirley, R. L.and Jack, F. H., J. Nutr., 1960, 70 , 63.

Grigoryan, M. S. and Brutyan, A. S., Tr. Erevan. Zootekh.-Vet. Inst., 1968, 29 , 57, 61.

Grigoryan, M. S., Tatevosyan-Markaryan, L. G. and Asmangulyan,M. S. Grigoryan, L. G. Tatevosyan-Markaryan, and Asmangulyan, T. A., Biol. Zh. Arm., 1969, 22 , 102.

The function in health and disease of the Mo-based enzymes sulfite oxidase, xanthine oxidase and aldehyde oxidase has been discussed. Xanthine oxidase, which occurs in the liver of humans and catalyses the formation of uric acid, may be anticarcinogenic through the development of protective systems against oxygen radicals.

Moriwaki, Y., Yamamoto, T., Higashino, K., Distribution And Pathophysiologic Role Of Molybdenum-Containing Enzymes, Histology And Histopathology , 1997,12, 513-524.

According to a molecular modelling study of the interaction of xanthine and hypoxanthine with xanthine oxidase correct positioning of the carbonyl group in the active site cavity is essential for a productive interaction with XO. The dimensions of the active site are mapped starting from the superimposition of the physiological substrates.

Rastelli, G., Costantino, L., Albasini, A., Model of the interaction of substrates and inhibitors with xanthine oxidase, Journal of the American Chemical Society , 1997, 119 , 13, 3007-3016.

Doonan, C. J., Nielsen, D. J., Smith, P. D., White, J. M., George, G. N., and Young, C. G., Models for the molybdenum hydroxylases: Synthesis, characterization and reactivity of cis-oxosulfido-Mo(VI) complexes, Journal of the American Chemical Society, 2006, 128, 305-316.

A model system for molybdenum oxotransferases provides evidence for all biologically realistic intermediates, namely mononuclear Mo-VI, Mo-V and Mo-IV species. Dinucleation to EPR-silent [(Mo₂O₃)-O-V] species prevailing in homogeneous solution is suppressed by immobilising the active species to a polymeric support by two-point attachment.

Heinze, K. and Fischer, A., Polymer-supported dioxido-Mo-VI complexes as truly functional molybdenum oxotransferase model systems, European Journal of Inorganic Chemistry, 2007, 1020-1026.

Selenium-dependent molybdenum hydroxylases

Haft, D.H. and Self, W. T., Orphan SeID proteins and selenium-dependent molybdenum hydroxylases, Biology Direct, 2008, 3

Molybdenum hydroxylases drug-metabolizing ability

Kitamura, S., Sugihara, K., and Ohta, S., Drug-metabolizing ability of molybdenum hydroxylases, Drug Metabolism and Pharmacokinetics, 2006, 21, 83-98.

Nitro-oleic acid - a novel and irreversible inhibitor of xanthine oxidoreductase

Xanthine oxidoreductase (XOR) generates proinflammatory oxidants and secondary nitrating species, with inhibition of XOR proving beneficial in a variety of disorders. Electrophilic nitrated fatty acid derivatives, such as nitro-oleic acid (OA-NO₂), display anti-inflammatory effects with pleiotropic properties. Nitro-oleic acid inhibits XOR activity in a concentration-dependent manner with an IC50 of 0.6 mu M, limiting both purine oxidation and formation of superoxide (O₂^-). Enzyme inhibition by OA-NO₂ is not reversed by thiol reagents, including glutathione, beta-mercaptoethanol, and dithiothreitol. Structure-function studies indicate that the carboxylic acid moiety, nitration at the 9 or 10 olefinic carbon, and unsaturation is required for XOR inhibition. Enzyme turnover and competitive reactivation studies reveal inhibition of electron transfer reactions at the molybdenum cofactor accounts for OA-NO₂-induced inhibition. Importantly, OA-NO₂ more potently inhibits cell-associated XOR-dependent O₂^-. production than does allopurinol. Combined, these data establish a novel role for OA-NO₂ in the inhibition of XOR-derived oxidant formation

Kelley, E. E., Batthyany, C. I., Hundley, N. J., Woodcock, S. R., Bonacci, G., Del Rio, J. M., Schopfer, F. J., Lancaster, J. R., Freeman, B. A., and Tarpey, M. M., Nitro-oleic Acid, a Novel and Irreversible Inhibitor of Xanthine Oxidoreductase, Journal of Biological Chemistry, 2008, 283, 36176-36184.

Density-functional theory models of xanthine oxidoreductase activity

The hydroxylation mechanism of the molybdoprotein xanthine oxidoreductase (XOR) has been modelled using density-functional theory. High activation barriers are often obtained for models of this enzyme due to the absence of factors that stabilize the accumulation of charge on the substrate at the transition state. Xanthine provides much lower barriers than small model substrates such as formamide or imidazole due to charge delocalization to centers which appear to interact with key residues in the protein. Of the two mechanisms of stabilization discussed in the literature-tautomerization and protonation of xanthine-density-functional theory calculations suggest that proton transfer from Glu1261 to N9 reduces the activation barrier by similar to 30 kcal mol^-1 and leads to an intuitive product complex. Further, similar values for the activation barriers of methyl xanthine isomers lead to the conclusion that the wide variation in rates for substituted purines is due to interactions with key residues in the active site. In addition, the transition state for oxidation of xanthine can be superimposed over the X-ray structure of inhibitor-bound XO with high correlation suggesting that the enzyme active site orients the substrate into the ideal position for reaction. The activation barriers for models of a hypothetical tungsten-substituted XO are predicted to be similar to 10 kcal mol^-1 higher in energy due to the higher reduction potential of the metal and unfavourable electrostatic interactions for the hydride transfer

Bayse, C. A., Density-functional theory models of xanthine oxidoreductase activity: comparison of substrate tautomerization and protonation, Dalton Transactions, 2009, 2306-2314.

X-ray crystal structure of complex with xanthine and lumazine

Xanthine oxidoreductase is a ubiquitous cytoplasmic protein that catalyzes the final two steps in purine catabolism. We have previously investigated the catalytic mechanism of the enzyme by rapid reaction kinetics and x-ray crystallography using the poor substrate 2-hydroxy-6-methylpurine, focusing our attention on the orientation of substrate in the active site and the role of Arg-880 in catalysis. Here we report additional crystal of as-isolated, functional xanthine oxidase in the course of reaction with the pterin substrate lumazine at 2.2 angstrom resolution and of the nonfunctional desulfo form of the enzyme in complex with xanthine at 2.6 angstrom resolution. In both cases the orientation of substrate is such that the pyrimidine subnucleus is oriented opposite to that seen with the slow substrate 2-hydroxy-6-methylpurine. The mechanistic implications as to how the ensemble of active site functional groups in the active site work to accelerate reaction rate are discussed

Pauff, J. M., Cao, H., and Hille, R., Substrate Orientation and Catalysis at the Molybdenum Site in Xanthine Oxidase CRYSTAL STRUCTURES IN COMPLEX WITH XANTHINE AND LUMAZINE, Journal of Biological Chemistry, 2009, 284, 8751-8758.

Interaction of Molybdenum With Trypsin and Pepsin

Molybdate binds to cationic centres of trypsin and pepsin enzymes.

Singh, R. P., Chaudhary, R., Rani, R., Kumar, S., and Arora, S. K., Interaction of Molybdenum With Trypsin and Pepsin by Dialysis Equilibrium Method, Asian Journal of Chemistry, 2010, 22, 1029.

Enzyme inhibition by molybdate

Both arsenate and sulfate reduction were inhibited by molybdate. Arsenate-reducing bacterium, strain OREX-4, grew on lactate, with either arsenate or sulfate serving as the electron acceptor

Newman, D.K., Kennedy, E.K., Coates, J.D., Ahmann, D., Ellis, D.J., Lovley, D.R., Morel, F.M.M. ,Dissimilatory arsenate and sulfate reduction in Desulfotomaculum auripigmentum sp. nov. Archives Of Microbiology, 1997, 168 ,.380-388.

Reduction of dimethylsulfoxide was not inhibited by molybdate. In Desulfovibrio desulfuricans strain PA2805, DMSO reduction occurred simultaneously-with sulfate reduction and was not effectively inhibited by molybdate, a specific inhibitor of sulfate reduction.

Jonkers, H.M., Vandermaarel, M.J.E.C., Vangemerden, H., Hansen, T.A. ,Dimethylsulfoxide Reduction By Marine Sulfate-Reducing Bacteria, Fems Microbiology Letters, 1996, 136 , 283-287.

Townsend G.T., Ramanand K., Suflita J.M., Reductive dehalogenation and mineralization of 3-chlorobenzoate in the presence of sulfate by microorganisms from a methanogenic aquifer, Applied And Environmental Microbiology , 1997, 63 , 2785-2791.

Polycyclic aromatic hydrocarbons, naphthalene and phenanthrene, were oxidized to carbon dioxide under sulfate-reducing conditions. Hydrocarbon oxidation was sulfate dependent. Molybdate, a specific inhibitor of sulfate reduction, inhibited hexadecane oxidation.

Coates, J.D., Woodward, J., Allen, J., Philp, P., Lovley, D.R., Anaerobic degradation of polycyclic aromatic hydrocarbons and alkanes in petroleum-contaminated marine harbor sediments, Applied And Environmental Microbiology ,1997, 63 , 3589-3593.

In anaerobic treatment of wastewater molybdate inhibited both sulfidogenesis and benzoate degradation. Molybdate inhibition increased progressively with concentration. Biogranules lost 50% of their methanogenic activity when treating waste water containing 48 mg/l of molybdate. In continuous experiments the molybdate toxicity had a threshold level. Below 0.5-0.8 mM molybdate bacterial activities were unaffected. The molybdate toxicity was not permanent. Biogranules were able gradually to regain their bioactivities once molybdate was removed from the waste water.

Liu, Y., Fang, H.H.P. Effects of molybdate on sulfate reduction and benzoate degradation, Journal Of Environmental Engineering-Asce , 1997,123 ,.949-953.

Liu, S.M., Kuo, C.L. Anaerobic biotransformation of pyridine in estuarine sediments. Chemosphere , 1997, 35 , 2255-2268.

Molybdate through inhibition of sulfate-reducing bacteria inhibits mercury methylation . Group VI anions, MO₄^2-,added to sediment slurries inhibited mercury methylation: tellurate (> 50 nM )> selenate ((> 270 nM )> molybdate (100 mu M)> tungstate (700 mu M). The inhibition of mercury methylation is due to the inhibition of sulfate-reducing bacteria, which are responsible for Hg methylation. In the concentration ranges encountered in most natural aquatic environments, the inhibition of MeHg production by Group VI anions is unlikely.

Barkay, T. 1995, Methylmercury Oxidative-Degradation Potentials In Contaminated And Pristine Sediments Of The Carson River , Nevada , Applied And Environmental Microbiology, 61 , 2745-2753.

Chen Y., Bonzongo J.C.J., Lyons W.B., Miller G.C., Inhibition of mercury methylation in anoxic freshwater sediment by Group VI anions, Environmental Toxicology And Chemistry , 1997, 16 , 1568-1574 0730-7268.

Oremland, R.S., Miller, L.G., Dowdle, P., Connell, T., Barkay, T. 1995, Methylmercury Oxidative-Degradation Potentials In Contaminated And Pristine Sediments Of The Carson River, Nevada, Applied And Environmental Microbiology , 61 , 2745-2753.

The iron oxidising bacterium T. ferrooxidans AP19-3, representative of Mo sensitive strains, could not grow on a Fe²⁺-medium with 1.0 mM of sodium molybdate. Mo(V), formed by reduction of Mo(VI) by Fe(II), rather than Mo(VI), is the actual inhibitor for the iron oxidation enzyme system, especially for cytochrome c oxidase. Molybdenum(V) binds to the plasma membrane and inhibits iron oxidase; as a result, growth of the bacterium is stopped [Yong et al.,1997].

Yong N.K., Oshima M., Blake R.C., Sugio T ., Isolation and some properties of an iron-oxidizing bacterium Thiobacillus ferrooxidans resistant to molybdenum ion, Bioscience Biotechnology And Biochemistry , 1997, 61 , 1523-1526.

Molybdate inhibited biotransformation of pyridine in sediments from the Tsengwen River
Liu, S.M., Kuo, C.L. Anaerobic biotransformation of pyridine in estuarine sediments. Chemosphere , 1997, 35 , 2255-2268.

Molybdenum enters bacteria by the active transport systems for phosphate and sulfate. Mo(V) binds erythrocytic membrane proteins (Lener and Bibr 1984)and is not mutagenic like Mo(VI). Molybdate causes irreversible cleavage of ATP to AMP by bakers yeast ATP-sulfurylase. Molybdate inhibits the activities of certain enzymes including alkaline phosphatase and NADP⁺ -2’ nucleotidase (Wetterhahn-Jennette 1981).

Lener, J., and Bibr, B., Effects of molybdenum on the organism, J. Hygene, Epidemiol., Microbiol., Immunol ., 1984, 28 , 409 – 19.

Wetterhahn-Jennette, K., The role of metals in carcinogenesis, Environ. Health Perspect .,1981, 40 , 233-52.

Phosphatase inhibition by molybdate

Common bean (Phaseolus vulgaris) seedlings accumulate ureides derived from purines after germination. The first step in the conversion of purines to ureides is the removal of the 5'-phosphate group by a phosphatase that has not yes been established.

Two main phosphatase activities were detected in the embryonic axes of common bean using inosine monophosphate as substrate in an in-gel assay. Both activities differed in their sensitivity to the common phosphatase inhibitor molybdate, with the molybdate-resistant as the first enzyme induced after radicalprotrusion.

The molybdate-resistant phosphatase is the first enzyme which shows resistance to molybdate.

Cabello-Diaz, J.M., Quiles, F.A., Lambert, R., Pineda, M., Piedras, P. Plant physiology and biochemistry, 2012, 53, 54-60. Identification of a novel phosphatase with high affinity for nucleotides monophosphate from common bean (Phaseolus vulgaris).

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