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Spectroscopic and biochemical insight into an electron-bifurcating [FeFe] hydrogenase.

ABSTRACT: The heterotrimeric electron-bifurcating [FeFe] hydrogenase (HydABC) from Thermotoga maritima (Tm) couples the endergonic reduction of protons (H⁺) by dihydronicotinamide adenine dinucleotide (NADH) (?G⁰???18 kJ mol^-1) to the exergonic reduction of H⁺ by reduced ferredoxin (Fd_red) (?G⁰???- 16 kJ mol^-1). The specific mechanism by which HydABC functions is not understood. In the current study, we describe the biochemical and spectroscopic characterization of TmHydABC recombinantly produced in Escherichia coli and artificially maturated with a synthetic diiron cofactor. We found that TmHydABC catalyzed the hydrogen (H₂)-dependent reduction of nicotinamide adenine dinucleotide (NAD⁺) in the presence of oxidized ferredoxin (Fd_ox) at a rate of? ?17 ?mol NADH min^-1 mg^-1. Our data suggest that only one flavin is present in the enzyme and is not likely to be the site of electron bifurcation. FTIR and EPR spectroscopy, as well as FTIR spectroelectrochemistry, demonstrated that the active site for H₂ conversion, the H-cluster, in TmHydABC behaves essentially the same as in prototypical [FeFe] hydrogenases, and is most likely also not the site of electron bifurcation. The implications of these results are discussed with respect to the current hypotheses on the electron bifurcation mechanism of [FeFe] hydrogenases. Overall, the results provide insight into the electron-bifurcating mechanism and present a well-defined system for further investigations of this fascinating class of [FeFe] hydrogenases.

SUBMITTER: Chongdar N

PROVIDER: S-EPMC7064455 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Spectroscopic and biochemical insight into an electron-bifurcating [FeFe] hydrogenase.

Chongdar Nipa N Pawlak Krzysztof K Rüdiger Olaf O Reijerse Edward J EJ Rodríguez-Maciá Patricia P Lubitz Wolfgang W Birrell James A JA Ogata Hideaki H

Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 20191210 1

The heterotrimeric electron-bifurcating [FeFe] hydrogenase (HydABC) from Thermotoga maritima (Tm) couples the endergonic reduction of protons (H+) by dihydronicotinamide adenine dinucleotide (NADH) (∆G0 ≈ 18 kJ mol-1) to the exergonic reduction of H+ by reduced ferredoxin (Fdred) (∆G0 ≈ - 16 kJ mol-1). The specific mechanism by which HydABC functions is not understood. In the current study, we describe the biochemical a ...[more]

PMID: 31823008

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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 biochemical insight into an electron-bifurcating [FeFe] hydrogenase.

Publications

Spectroscopic and biochemical insight into an electron-bifurcating [FeFe] hydrogenase.

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