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Spectroscopic and Computational Evidence that [FeFe] Hydrogenases Operate Exclusively with CO-Bridged Intermediates.

ABSTRACT: [FeFe] hydrogenases are extremely active H₂-converting enzymes. Their mechanism remains highly controversial, in particular, the nature of the one-electron and two-electron reduced intermediates called H_redH⁺ and H_sredH⁺. In one model, the H_redH⁺ and H_sredH⁺ states contain a semibridging CO, while in the other model, the bridging CO is replaced by a bridging hydride. Using low-temperature IR spectroscopy and nuclear resonance vibrational spectroscopy, together with density functional theory calculations, we show that the bridging CO is retained in the H_sredH⁺ and H_redH⁺ states in the [FeFe] hydrogenases from Chlamydomonas reinhardtii and Desulfovibrio desulfuricans, respectively. Furthermore, there is no evidence for a bridging hydride in either state. These results agree with a model of the catalytic cycle in which the H_redH⁺ and H_sredH⁺ states are integral, catalytically competent components. We conclude that proton-coupled electron transfer between the two subclusters is crucial to catalysis and allows these enzymes to operate in a highly efficient and reversible manner.

SUBMITTER: Birrell JA

PROVIDER: S-EPMC6956316 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Spectroscopic and Computational Evidence that [FeFe] Hydrogenases Operate Exclusively with CO-Bridged Intermediates.

Birrell James A JA Pelmenschikov Vladimir V Mishra Nakul N Wang Hongxin H Yoda Yoshitaka Y Tamasaku Kenji K Rauchfuss Thomas B TB Cramer Stephen P SP Lubitz Wolfgang W DeBeer Serena S

Journal of the American Chemical Society 20191230 1

[FeFe] hydrogenases are extremely active H2-converting enzymes. Their mechanism remains highly controversial, in particular, the nature of the one-electron and two-electron reduced intermediates called HredH+ and HsredH+. In one model, the HredH+ and HsredH+ states contain a semibridging CO, while in the other model, the bridging CO is replaced by a bridging hydride. Using low-temperature IR spectr ...[more]

PMID: 31820961

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Project description:A non-bifurcating NADH-dependent, dimeric [FeFe]-hydrogenase (HydAB) from Syntrophus aciditrophicus was heterologously produced in Escherichia coli, purified and characterized. Purified recombinant HydAB catalyzed NAD+ reduction coupled to hydrogen oxidation and produced hydrogen from NADH without the involvement of ferredoxin. Hydrogen partial pressures (2.2-40.2 Pa) produced by the purified recombinant HydAB at NADH to NAD+ ratios of 1-5 were similar to the hydrogen partial pressures generated by pure and cocultures of S. aciditrophicus (5.9-36.6 Pa). Thus, the hydrogen partial pressures observed in metabolizing cultures and cocultures of S. aciditrophicus can be generated by HydAB if S. aciditrophicus maintains NADH to NAD+ ratios greater than one. The flavin-containing beta subunits from S. aciditrophicus HydAB and the non-bifurcating NADH-dependent S. wolfei Hyd1ABC share a number of conserved residues with the flavin-containing beta subunits from non-bifurcating NADH-dependent enzymes such as NADH:quinone oxidoreductases and formate dehydrogenases. A number of differences were observed between sequences of these non-bifurcating NADH-dependent enzymes and [FeFe]-hydrogenases and formate dehydrogenases known to catalyze electron bifurcation including differences in the number of [Fe-S] centers and in conserved residues near predicted cofactor binding sites. These differences can be used to distinguish members of these two groups of enzymes and may be relevant to the differences in ferredoxin-dependence and ability to mediate electron-bifurcation. These results show that two phylogenetically distinct syntrophic fatty acid-oxidizing bacteria, Syntrophomonas wolfei a member of the phylum Firmicutes, and S. aciditrophicus, a member of the class Deltaproteobacteria, possess functionally similar [FeFe]-hydrogenases that produce hydrogen from NADH during syntrophic fatty acid oxidation without the involvement of reduced ferredoxin. The reliance on a non-bifurcating NADH-dependent [FeFe]-hydrogenases may explain the obligate requirement that many syntrophic metabolizers have for a hydrogen-using partner microorganism when grown on fatty, aromatic and alicyclic acids.

Dataset Information

Spectroscopic and Computational Evidence that [FeFe] Hydrogenases Operate Exclusively with CO-Bridged Intermediates.

Publications

Spectroscopic and Computational Evidence that [FeFe] Hydrogenases Operate Exclusively with CO-Bridged Intermediates.

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