Reduction of Protein Bound Methionine Sulfoxide by a Periplasmic Dimethyl Sulfoxide Reductase
In proteins, methionine (Met) can be oxidized into Met sulfoxide (MetO). The ubiquitous methionine sulfoxide reductases (Msr) A and B are thiol-oxidoreductases reducing MetO. Reversible Met oxidation has a wide range of consequences, from protection against oxidative stress to fine-tuned regulation of protein functions. Bacteria distinguish themselves by the production of molybdenum-containing enzymes reducing MetO, such as the periplasmic MsrP which protects proteins during acute oxidative stress. The versatile dimethyl sulfoxide (DMSO) reductases were shown to reduce the free amino acid MetO, but their ability to reduce MetO within proteins was never evaluated. Here, using model oxidized proteins and peptides, enzymatic and mass spectrometry approaches, we showed that the Rhodobacter sphaeroides periplasmic DorA-type DMSO reductase reduces protein bound MetO as efficiently as the free amino acid L-MetO and with catalytic values in the range of those described for the canonical Msrs. The identification of this fourth type of enzyme able to reduce MetO in proteins, conserved across proteobacteria and actinobacteria, suggests that organisms employ enzymatic systems yet undiscovered to regulate protein oxidation states.
L. Tarrago, S. Grosse, D. Lemaire, L. Faure, M. Tribout, M. I. Siponen, M. Kojadinovic-Sirinelli, D. Pignol, P. Arnoux, and M. Sabaty,Reduction of Protein Bound Methionine Sulfoxide by a Periplasmic Dimethyl Sulfoxide Reductase, Antioxidants (Basel, Switzerland), 2020, 9.
New insights into the molecular physiology of sulfoxide reduction in bacteria [Review]
Sulfoxides occur in biology as products of the S-oxygenation of small molecules as well
as in peptides and proteins and their formation is often associated with oxidative stress
and can affect biological function. In bacteria, sulfoxide damage can be reverse by
different types of enzymes. Thioredoxin-dependent peptide methionine sulfoxide
reductases ( MSR proteins) repair oxidized methionine residues and are found in all Domains
of life. In bacteria MSR proteins are often found in the cytoplasm but in some
bacteria, including Neisseria, Streptococci, and Haemophilus they a e extracytoplasmic.
Mutants lacking MSR proteins are often sensitive to oxidative stress and in
pathogens exhibit decreased virulence as indicated by reduced suuvival in host cell or
animal model systems. Molybdenum enzymes are also known to reduce S-oxides and
traditionally their physiological role was considered to be in anaerobic respiration using
dimethylsulfoxide (DMSO) as an electron acceptor. However, it now appears that some
enzymes (MtsZ) of the DMSO reductase family of Mo enzymes use methione sulfoxide
as preferred physiological substrate and thus may be involved in scavenging/
recycling of this amino acid. Similarly, an enzyme (MsrP/YedY) of the sulfite oxidase
family of Mo enzymes has been shown to be involved in repair of methionine sulfoxides
sin periplasmic proteins. Again, some mutants deficient in Mo-dependent sulfoxide
reductases exhibit reduced virulence, and there is evidence that these Mo enzymes and
some MSR systems are induced by hypochlorite produced by the innate immune system.
This review describes recent advances in the understanding of the molecular
microbiology of MSR systems and the broadening of the role of Mo-dependent sulfoxide
reductase to encompass functions beyond anaerobic respiration.
U. Kappler, M. Nasreen, and A. McEwan, New insights into the molecular physiology of sulfoxide reduction in bacteria. In Advances in microbial physiology, 2019, 75, 1-51.
Addressing ligand-based redox in molybdenum-dependent methionine sulfoxide reductase [enzyme]
A combination of pulsed EPR, CW EPR, and X-ray absorption spectroscopies has been employed to probe the geometric and electronic structure of the E. coli periplasmic molybdenum-dependent methionine sulfoxide reductase (MsrP). O-17 and H-1 pulsed EPR spectra show that the as-isolated Mo(V) enzyme form does not possess an exchangeable H2O/OH- ligand bound to Mo as found in the sulfite oxidizing enzymes of the same family. The nature of the unusual CW EPR spectrum has been reevaluated in light of new data on the MsrP-N45R variant and related small-molecule analogues of the active site. These data point to a novel "thiol-blocked" [(PDT)(MoO)-O-v(S-Cys)(thiolate)](-) structure, which is supported by new EXAFS data. We discuss these new results in the context of ligand-based and metal-based redox chemistry in the enzymatic oxygen atom transfer reaction.
L. J. Ingersol, J. Yang, K. C. Khadanand, A. Pokhrel, A. V. Astashkin, J. H. Weiner, C. A. Johnston, and M. L. Kirk, Addressing ligand-based redox in molybdenum-dependent methionine sulfoxide reductase, Journal of the American Chemical Society, 2020, 142, 2721-2725.
A novel dimethylsulfoxide reductase family of molybdenum enzyme, Idr, is involved in iodate respiration by pseudomonas sp. SCT
Pseudomonas sp. strain SCT is capable of using iodate (IO3‑) as a terminal electron acceptor for anaerobic respiration. A possible key enzyme, periplasmic iodate reductase (Idr), was visualized by active staining on non-denaturing gel electrophoresis. Liquid chromatography-tandem mass spectrometry analysis revealed that at least four proteins, designated as IdrA, IdrB, IdrP1 , and IdrP2 , were involved in Idr. IdrA and IdrB were homologues of catalytic and electron transfer subunits of respiratory arsenite oxidase (Aio); however, IdrA defined a novel clade within the dimethylsulfoxide (DMSO) reductase family. IdrP1 and IdrP2 were closely related to each other and distantly related to cytochrome c peroxidase. The idr genes (idrABP 1 P 2 ) formed an operon-like structure, and their transcription was upregulated under iodate-respiring conditions. Comparative proteomic analysis also revealed that Idr proteins and high affinity terminal oxidases (Cbb3 and Cyd), various H2 O2 scavengers, and chlorite (ClO2 (-) ) dismutase-like proteins were expressed specifically or abundantly under iodate-respiring conditions. These results suggest that Idr is a respiratory iodate reductase, and that both O2 and H2 O2 are formed as by-products of iodate respiration. We propose an electron transport chain model of strain SCT, in which iodate, H2 O2 , and O2 are used as terminal electron acceptors.
C. Yamazaki, S. Kashiwa, A. Horiuchi, Y. Kasahara, S. Yamamura, and S. Amachi, A novel dimethylsulfoxide reductase family of molybdenum enzyme, idr, is involved in iodate respiration by pseudomonas sp. Sct, Environ Microbiol, 2020.
Effect of the protein ligand in DMSO reductase studied by computational methods
The DMSO reductase family is the largest and most diverse family of mononuclear molybdenum oxygen-atom-transfer proteins. Their active sites contain a Mo ion coordinated to two molybdopterin ligands, one oxo group in the oxidised state, and one additional, often protein-derived ligand. We have used density-functional theory to evaluate how the fourth ligand (serine, cysteine, selenocysteine, OH-, O2-, SH-, or S2-) affects the geometries, reaction mechanism, reaction energies, and reduction potentials of intermediates in the DMSO reductase reaction. Our results show that there are only small changes in the geometries of the reactant and product states, except from the elongation of the MoX bond as the ionic radius of XO, S, Se increases. The five ligands with a single negative charge gave an identical two-step reaction mechanism, in which DMSO first binds to the reduced active site, after which the SO bond is cleaved, concomitantly with the transfer of two electrons from Mo in a rate-determining second transition state. The five models gave similar activation energies of 69-85kJ/mol, with SH- giving the lowest barrier. In contrast, the O2- and S2- ligands gave much higher activation energies (212 and 168kJ/mol) and differing mechanisms (a more symmetric intermediate for O2- and a one-step reaction without any intermediate for S2-). The high activation energies are caused by a less exothermic reaction energy, 13-25kJ/mol, and by a more stable reactant state owing to the strong MoO2- or MoS2- bonds.
Dong, G., and Ryde, U.,Effect of the protein ligand in DMSO reductase studied by computational methods, Journal of inorganic biochemistry, 2017, 171, 45-51.
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.