Health, Safety & Environment


The Challenging in silico Description of Carbon Monoxide Oxidation as Catalyzed by Molybdenum-Copper CO Dehydrogenase

Carbon monoxide (CO) is a highly toxic gas to many living organisms. However, some microorganisms are able to use this molecule as the sole source of carbon and energy. Soil bacteria such as the aerobic Oligotropha carboxidovorans are responsible for the annual removal of about 2x10(8) tons of CO from the atmosphere. Detoxification through oxidation of CO to CO2 is enabled by the MoCu-dependent CO-dehydrogenase enzyme (MoCu-CODH) which-differently from other enzyme classes with similar function-retains its catalytic activity in the presence of atmospheric O2. In the last few years, targeted advancements have been described in the field of bioengineering and biomimetics, which is functional for future technological exploitation of the catalytic properties of MoCu-CODH and for the reproduction of its reactivity in synthetic complexes. Notably, a growing interest for the quantum chemical investigation of this enzyme has recently also emerged. This mini-review compiles the current knowledge of the MoCu-CODH catalytic cycle, with a specific focus on the outcomes of theoretical studies on this enzyme class. Rather controversial aspects from different theoretical studies will be highlighted, thus illustrating the challenges posed by this system as far as the application of density functional theory and hybrid quantum-classical methods are concerned.

A. Rovaletti, M. Bruschi, G. Moro, U. Cosentino, and C. Greco,The Challenging in silico Description of Carbon Monoxide Oxidation as Catalyzed by Molybdenum-Copper CO Dehydrogenase, Frontiers in chemistry, 2018, 6, 630.


The oxidation-reduction and electrocatalytic properties of CO dehydrogenase from Oligotropha carboxidovorans

CO dehydrogenase (CODH) from the Gram-negative bacterium Oligotropha carboxidovorans is a complex metalloenzyme from the xanthine oxidase family of molybdenum-containing enzymes, bearing a unique binuclear Mo-S-Cu active site in addition to two [2Fe-2S] clusters (FeSI and FeSII) and one equivalent of FAD. CODH catalyzes the oxidation of CO to CO2 with the concomitant introduction of reducing equivalents into the quinone pool, thus enabling the organism to utilize CO as sole source of both carbon and energy. Using a variety of EPR monitored redox titrations and spectroelectrochemistry, we report the redox potentials of CO dehydrogenase at pH7.2 namely Mo(VI/V), Mo(V/IV), FeSI(2+/+), FeSII(2+/+), FAD/FADH and FADH/FADH(-). These potentials are systematically higher than the corresponding potentials seen for other members of the xanthine oxidase family of Mo enzymes, and are in line with CODH utilising the higher potential quinone pool as an electron acceptor instead of pyridine nucleotides. CODH is also active when immobilised on a modified Au working electrode as demonstrated by cyclic voltammetry in the presence of CO.

P. Kalimuthu, M. Petitgenet, D. Niks, S. Dingwall, J. R. Harmer, R. Hille, and P. V. Bernhardt,The oxidation-reduction and electrocatalytic properties of CO dehydrogenase from Oligotropha carboxidovorans, Biochimica et biophysica acta. Bioenergetics, 2019, 148118.  


Functional Studies on Oligotropha carboxidovorans Molybdenum-Copper CO Dehydrogenase Produced in Escherichia coli

The Mo/Cu-dependent CO dehydrogenase (CODH) from Oligotropha carboxidovorans is an enzyme that is able to catalyze both the oxidation of CO to CO2 and the oxidation of H2 to protons and electrons. Despite the close to atomic resolution structure (1.1 angstrom), significant uncertainties have remained with regard to the reaction mechanism of substrate oxidation at the unique Mo/Cu center, as well as the nature of intermediates formed during the catalytic cycle. So far, the investigation of the role of amino acids at the active site was hampered by the lack of a suitable expression system that allowed for detailed site-directed mutagenesis studies at the active site. Here, we report on the establishment of a functional heterologous expression system of O. carboxidovorans CODH in Escherichia coli. We characterize the purified enzyme in detail by a combination of kinetic and spectroscopic studies and show that it was purified in a form with characteristics comparable to those of the native enzyme purified from O. carboxidovorans. With this expression system in hand, we were for the first time able to generate active-site variants of this enzyme. Our work presents the basis for more detailed studies of the reaction mechanism for CO and H2 oxidation of Mo/Cu-dependent CODHs in the future.

P. Kaufmann, B. R. Duffus, C. Teutloff, and S. Leimkuhler,Functional Studies on Oligotropha carboxidovorans Molybdenum-Copper CO Dehydrogenase Produced in Escherichia coli, Biochemistry, 2018, 57, 2889-2901.


Carbon monoxide dehydrogenase

Structure and reactivity - molybdenum-copper site

We review the xanthine oxidase (XO) family of pyranopterin molybdenum enzymes with a specific emphasis on electronic structure contributions to reactivity. In addition to xanthine and aldehyde oxidoreductases, which catalyze the two-electron oxidation of aromatic heterocycles and aldehyde substrates, this mini-review highlights recent work on the closely related carbon monoxide dehydrogenase (CODH) that catalyzes the oxidation of CO using a unique Mo-Cu heterobimetallic active site. A primary focus of this mini-review relates to how spectroscopy and computational methods have been used to develop an understanding of critical relationships between geometric structure, electronic structure, and catalytic function.

Stein, B. W. and Kirk, M. L., Electronic structure contributions to reactivity in xanthine oxidase family enzymes, Journal of Biological Inorganic Chemistry, 2015, 20, 183-194.


We review here the recent literature dealing with the molybdenum- and copper-dependent CO dehydrogenase, with particular emphasis on the structure of the enzyme and recent advances in our understanding of the reaction mechanism of the enzyme.

Hille R, Dingwall S, Wilcoxen J. The aerobic CO dehydrogenase from Oligotropha carboxidovorans J Biol Inorg Chem. 2015 Mar;20(2):243-51. doi: 10.1007/s00775-014-1188-4. Epub 2014 Aug 26.

CO dehydrogenase (EC 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.

Reversible inactivation of CO dehydrogenase with thiol compounds

Carbon monoxide dehydrogenase (CO dehydrogenase) from Oligotropha carboxidovorans is a structurally characterized member of the molybdenum hydroxylase enzyme family. It catalyzes the oxidation of CO (CO + H2O + CO2 + 2e- + 2H+) which proceeds at a unique [CuSMo(=O)OH] metal cluster. Because of changing activities of CO dehydrogenase, particularly in subcellular fractions, we speculated whether the enzyme would be subject to regulation by thiols (RSH).

Here we establish inhibition of CO dehydrogenase by thiols and report the corresponding K-1-values (mM): L-cysteine (5.2), D-cysteine (9.7), N-acetyl-L-cysteine (8.2), D,L-homocysteine (25.8), L-cysteine-glycine (2.0), dithiothreitol (4.1), coenzyme A (8.3), and 2-mercaptoethanol (9.3).

Inhibition of the enzyme was reversed by CO or upon lowering the thiol concentration.

Electron paramagnetic resonance spectroscopy (EPR) and X-ray absorption spectroscopy (XAS) of thiol-inhibited CO dehydrogenase revealed a bimetallic site in which the RSH coordinates to the Cu-ion as a third ligand {[Mo-VI(=O)OH2SCuI(SR)S-Cys]} leaving the redox state of the Cu(I) and the Mo(VI) unchanged.

Collectively, our findings establish a regulation of CO dehydrogenase activity by thiols in vitro.

They also corroborate the hypothesis that CO interacts with the Cu-ion first.

The result that thiol compounds much larger than CO can freely travel through the substrate channel leading to the bimetallic cluster challenges previous concepts involving chaperone function and is of importance for an understanding how the sulfuration step in the assembly of the bimetallic cluster might proceed. (C) 2014 The Authors. Published by Elsevier Inc.

Kress, O., Gnida, M., Pelzmann, A. M., Marx, C., Meyer-Klaucke, W., and Meyer, O., Reversible inactivation of CO dehydrogenase with thiol compounds, Biochemical and Biophysical Research Communications, 2014, 447, 413-418.