title: Single-Amino Acid Modifications Reveal Additional Controls on the Proton Pathway of [FeFe]-Hydrogenase.
abstract: The proton pathway of [FeFe]-hydrogenase is essential for enzymatic H2 production and oxidation and is composed of four residues and a water molecule. A computational analysis of this pathway in the [FeFe]-hydrogenase from Clostridium pasteurianum revealed that the solvent-exposed residue of the pathway (Glu282) forms hydrogen bonds to two residues outside of the pathway (Arg286 and Ser320), implying that these residues could function in regulating proton transfer. In this study, we show that substituting Arg286 with leucine eliminates hydrogen bonding with Glu282 and results in an ∼3-fold enhancement of H2 production activity when methyl viologen is used as an electron donor, suggesting that Arg286 may help control the rate of proton delivery. In contrast, substitution of Ser320 with alanine reduces the rate ∼5-fold, implying that it either acts as a member of the pathway or influences Glu282 to permit proton transfer. Interestingly, quantum mechanics/molecular mechanics and molecular dynamics calculations indicate that Ser320 does not play a structural role or indirectly influence the barrier for proton movement at the entrance of the channel. Rather, it may act as an additional proton acceptor for the pathway or serve in a regulatory role. While further studies are needed to elucidate the role of Ser320, collectively these data provide insights into the complex proton transport process.
authors.affiliation: Department of Biochemistry & Molecular Biology, Michigan State University , East Lansing, Michigan 48824, United States.|Great Lakes Bioenergy Research Center, Michigan State University , East Lansing, Michigan 48824, United States.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Cornish
authors.affiliation: Physical Sciences Division, Pacific Northwest National Laboratory , Richland, Washington 99352, United States.
authors.forename: Bojana
authors.initials: B
authors.lastname: Ginovska
authors.affiliation: Department of Biochemistry & Molecular Biology, Michigan State University , East Lansing, Michigan 48824, United States.
authors.forename: Adam
authors.initials: A
authors.lastname: Thelen
authors.affiliation: Department of Fundamental Chemistry, Federal University of Pernambuco , Cidade Universitária,50740-560 Recife, PE, Brazil.
authors.forename: Julio C S
authors.initials: JC
authors.lastname: da Silva
authors.affiliation: Department of Fundamental Chemistry, Federal University of Pernambuco , Cidade Universitária,50740-560 Recife, PE, Brazil.
authors.forename: Thereza A
authors.initials: TA
authors.lastname: Soares
authors.affiliation: Physical Sciences Division, Pacific Northwest National Laboratory , Richland, Washington 99352, United States.
authors.forename: Simone
authors.initials: S
authors.lastname: Raugei
authors.affiliation: Physical Sciences Division, Pacific Northwest National Laboratory , Richland, Washington 99352, United States.
authors.forename: Michel
authors.initials: M
authors.lastname: Dupuis
authors.affiliation: Physical Sciences Division, Pacific Northwest National Laboratory , Richland, Washington 99352, United States.
authors.forename: Wendy J
authors.initials: WJ
authors.lastname: Shaw
authors.affiliation: Department of Biochemistry & Molecular Biology, Michigan State University , East Lansing, Michigan 48824, United States.|Great Lakes Bioenergy Research Center, Michigan State University , East Lansing, Michigan 48824, United States.
authors.forename: Eric L
authors.initials: EL
authors.lastname: Hegg
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology*; D006864:Hydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017136:Ion Transport; D007501:Iron / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D011522:Protons
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D050505:Mutant Proteins; D011522:Protons; D007501:Iron; C118830:iron hydrogenase; D006864:Hydrogenase