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Mechanism of proton transfer in [FeFe]-hydrogenase from Clostridium pasteurianum.

ABSTRACT: [FeFe]-Hydrogenases are complex metalloproteins that catalyze the reversible reduction of protons to molecular hydrogen utilizing a unique diiron subcluster bridged to a [4Fe4S] subcluster. Extensive studies have concentrated on the nature and catalytic activity of the active site, yet relatively little information is available concerning the mechanism of proton transport that is required for this activity. Previously, structural characterization of [FeFe]-hydrogenase from Clostridium pasteurianum indicated a potential proton transport pathway involving four residues (Cys-299, Glu-279, Ser-319, and Glu-282) that connect the active site to the enzyme surface. Here, we demonstrate that substitution of any of these residues resulted in a drastic reduction in hydrogenase activity relative to the native enzyme, supporting the importance of these residues in catalysis. Inhibition studies of native and amino acid-substituted enzymes revealed that Zn(2+) specifically blocked proton transfer by binding to Glu-282, confirming the role of this residue in the identified pathway. In addition, all four of these residues are strictly conserved, suggesting that they may form a proton transport pathway that is common to all [FeFe]-hydrogenases.

SUBMITTER: Cornish AJ

PROVIDER: S-EPMC3207428 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Mechanism of proton transfer in [FeFe]-hydrogenase from Clostridium pasteurianum.

Cornish Adam J AJ Gärtner Katrin K Yang Hui H Peters John W JW Hegg Eric L EL

The Journal of biological chemistry 20110907 44

[FeFe]-Hydrogenases are complex metalloproteins that catalyze the reversible reduction of protons to molecular hydrogen utilizing a unique diiron subcluster bridged to a [4Fe4S] subcluster. Extensive studies have concentrated on the nature and catalytic activity of the active site, yet relatively little information is available concerning the mechanism of proton transport that is required for this activity. Previously, structural characterization of [FeFe]-hydrogenase from Clostridium pasteurian ...[more]

PMID: 21900241

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Project description:[FeFe] hydrogenases, metalloenzymes catalyzing proton/dihydrogen interconversion, have attracted intense attention due to their remarkable catalytic properties and (bio-)technological potential for a future hydrogen economy. In order to unravel the factors enabling their efficient catalysis, both their unique organometallic cofactors and protein structural features, i.e., "outer-coordination sphere" effects have been intensively studied. These structurally diverse enzymes are divided into distinct phylogenetic groups, denoted as Group A-D. Prototypical Group A hydrogenases display high turnover rates (104-105 s-1). Conversely, the sole characterized Group D representative, Thermoanaerobacter mathranii HydS (TamHydS), shows relatively low catalytic activity (specific activity 10-1 μmol H2 mg-1 min-1) and has been proposed to serve a H2-sensory function. The various groups of [FeFe] hydrogenase share the same catalytic cofactor, the H-cluster, and the structural factors causing the diverging reactivities of Group A and D remain to be elucidated. In the case of the highly active Group A enzymes, a well-defined proton transfer pathway (PTP) has been identified, which shuttles H+ between the enzyme surface and the active site. In Group D hydrogenases, this conserved pathway is absent. Here, we report on the identification of highly conserved amino acid residues in Group D hydrogenases that constitute a possible alternative PTP. We varied two proposed key amino acid residues of this pathway (E252 and E289, TamHydS numbering) via site-directed mutagenesis and analyzed the resulting variants via biochemical and spectroscopic methods. All variants displayed significantly decreased H2-evolution and -oxidation activities. Additionally, the variants showed two redox states that were not characterized previously. These findings provide initial evidence that these amino acid residues are central to the putative PTP of Group D [FeFe] hydrogenase. Since the identified residues are highly conserved in Group D exclusively, our results support the notion that the PTP is not universal for different phylogenetic groups in [FeFe] hydrogenases.

Dataset Information

Mechanism of proton transfer in [FeFe]-hydrogenase from Clostridium pasteurianum.

Publications

Mechanism of proton transfer in [FeFe]-hydrogenase from Clostridium pasteurianum.

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