title: Discovery of Dark pH-Dependent H(+) Migration in a [NiFe]-Hydrogenase and Its Mechanistic Relevance: Mobilizing the Hydrido Ligand of the Ni-C Intermediate.
abstract: Despite extensive studies on [NiFe]-hydrogenases, the mechanism by which these enzymes produce and activate H2 so efficiently remains unclear. A well-known EPR-active state produced under H2 and known as Ni-C is assigned as a Ni(III)-Fe(II) species with a hydrido ligand in the bridging position between the two metals. It has long been known that low-temperature photolysis of Ni-C yields distinctive EPR-active states, collectively termed Ni-L, that are attributed to migration of the bridging-H species as a proton; however, Ni-L has mainly been regarded as an artifact with no mechanistic relevance. It is now demonstrated, based on EPR and infrared spectroscopic studies, that the Ni-C to Ni-L interconversion in Hydrogenase-1 (Hyd-1) from Escherichia coli is a pH-dependent process that proceeds readily in the dark-proton migration from Ni-C being favored as the pH is increased. The persistence of Ni-L in Hyd-1 must relate to unassigned differences in proton affinities of metal and adjacent amino acid sites, although the unusually high reduction potentials of the adjacent Fe-S centers in this O2-tolerant hydrogenase might also be a contributory factor, impeding elementary electron transfer off the [NiFe] site after proton departure. The results provide compelling evidence that Ni-L is a true, albeit elusive, catalytic intermediate of [NiFe]-hydrogenases.
authors.affiliation: †Department of Chemistry and ‡Centre for Advanced Electron Spin Resonance, University of Oxford, Oxford OX1 3QR, United Kingdom.
authors.forename: Bonnie J
authors.initials: BJ
authors.lastname: Murphy
authors.affiliation: †Department of Chemistry and ‡Centre for Advanced Electron Spin Resonance, University of Oxford, Oxford OX1 3QR, United Kingdom.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Hidalgo
authors.affiliation: †Department of Chemistry and ‡Centre for Advanced Electron Spin Resonance, University of Oxford, Oxford OX1 3QR, United Kingdom.
authors.forename: Maxie M
authors.initials: MM
authors.lastname: Roessler
authors.affiliation: †Department of Chemistry and ‡Centre for Advanced Electron Spin Resonance, University of Oxford, Oxford OX1 3QR, United Kingdom.
authors.forename: Rhiannon M
authors.initials: RM
authors.lastname: Evans
authors.affiliation: †Department of Chemistry and ‡Centre for Advanced Electron Spin Resonance, University of Oxford, Oxford OX1 3QR, United Kingdom.
authors.forename: Philip A
authors.initials: PA
authors.lastname: Ash
authors.affiliation: †Department of Chemistry and ‡Centre for Advanced Electron Spin Resonance, University of Oxford, Oxford OX1 3QR, United Kingdom.
authors.forename: William K
authors.initials: WK
authors.lastname: Myers
authors.affiliation: †Department of Chemistry and ‡Centre for Advanced Electron Spin Resonance, University of Oxford, Oxford OX1 3QR, United Kingdom.
authors.forename: Kylie A
authors.initials: KA
authors.lastname: Vincent
authors.affiliation: †Department of Chemistry and ‡Centre for Advanced Electron Spin Resonance, University of Oxford, Oxford OX1 3QR, United Kingdom.
authors.forename: Fraser A
authors.initials: FA
authors.lastname: Armstrong
mesh_terms: D002244:Carbon / Q000737:chemistry*; D020134:Catalytic Domain; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006864:Hydrogenase / Q000737:chemistry*; D008024:Ligands; D008670:Metals / Q000737:chemistry; D009532:Nickel / Q000737:chemistry; D009584:Nitrogen / Q000737:chemistry*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D010782:Photolysis; D011522:Protons; D013696:Temperature
chemical_list: D029968:Escherichia coli Proteins; D008024:Ligands; D008670:Metals; D011522:Protons; D002244:Carbon; D009532:Nickel; D006859:Hydrogen; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; C000602631:hydrogenase 1, E coli; D009584:Nitrogen; D010100:Oxygen