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Aerobic damage to [FeFe]-hydrogenases: activation barriers for the chemical attachment of O2.

ABSTRACT: [FeFe]-hydrogenases are the best natural hydrogen-producing enzymes but their biotechnological exploitation is hampered by their extreme oxygen sensitivity. The free energy profile for the chemical attachment of O2 to the enzyme active site was investigated by using a range-separated density functional re-parametrized to reproduce high-level ab initio data. An activation free-energy barrier of 13 kcal mol(-1) was obtained for chemical bond formation between the di-iron active site and O2, a value in good agreement with experimental inactivation rates. The oxygen binding can be viewed as an inner-sphere electron-transfer process that is strongly influenced by Coulombic interactions with the proximal cubane cluster and the protein environment. The implications of these results for future mutation studies with the aim of increasing the oxygen tolerance of this enzyme are discussed.

SUBMITTER: Kubas A

PROVIDER: S-EPMC4143129 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Aerobic damage to [FeFe]-hydrogenases: activation barriers for the chemical attachment of O2.

Kubas Adam A De Sancho David D Best Robert B RB Blumberger Jochen J

Angewandte Chemie (International ed. in English) 20140311 16

[FeFe]-hydrogenases are the best natural hydrogen-producing enzymes but their biotechnological exploitation is hampered by their extreme oxygen sensitivity. The free energy profile for the chemical attachment of O2 to the enzyme active site was investigated by using a range-separated density functional re-parametrized to reproduce high-level ab initio data. An activation free-energy barrier of 13 kcal mol(-1) was obtained for chemical bond formation between the di-iron active site and O2, a valu ...[more]

PMID: 24615978

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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

Aerobic damage to [FeFe]-hydrogenases: activation barriers for the chemical attachment of O2.

Publications

Aerobic damage to [FeFe]-hydrogenases: activation barriers for the chemical attachment of O2.

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