title: 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.
authors: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319.
authors: Adam J
authors: AJ
authors: Cornish
authors: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319.
authors: Katrin
authors: K
authors: Gärtner
authors: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319.
authors: Hui
authors: H
authors: Yang
authors: Department of Chemistry and Biochemistry and Astrobiology Biogeocatalysis Research Center, Montana State University, Bozeman, Montana 59717.
authors: John W
authors: JW
authors: Peters
authors: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319. Electronic address: erichegg@msu.edu.
authors: Eric L
authors: EL
authors: Hegg
mesh_terms: D000595:Amino Acid Sequence; D001692:Biological Transport; D002384:Catalysis; D003013:Clostridium / Q000201:enzymology*; D018698:Glutamic Acid / Q000737:chemistry; D006864:Hydrogenase / Q000737:chemistry*; D007501:Iron / Q000737:chemistry*; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D007700:Kinetics; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011522:Protons; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000737:chemistry
chemical_list: D007506:Iron-Sulfur Proteins; D011522:Protons; D018698:Glutamic Acid; D007501:Iron; D006864:Hydrogenase; D015032:Zinc