title: Investigations on the role of proton-coupled electron transfer in hydrogen activation by [FeFe]-hydrogenase.
abstract: Proton-coupled electron transfer (PCET) is a fundamental process at the core of oxidation-reduction reactions for energy conversion. The [FeFe]-hydrogenases catalyze the reversible activation of molecular H2 through a unique metallocofactor, the H-cluster, which is finely tuned by the surrounding protein environment to undergo fast PCET transitions. The correlation of electronic and structural transitions at the H-cluster with proton-transfer (PT) steps has not been well-resolved experimentally. Here, we explore how modification of the conserved PT network via a Cys → Ser substitution at position 169 proximal to the H-cluster of Chlamydomonas reinhardtii [FeFe]-hydrogenase (CrHydA1) affects the H-cluster using electron paramagnetic resonance (EPR) and Fourier transform infrared (FTIR) spectroscopy. Despite a substantial decrease in catalytic activity, the EPR and FTIR spectra reveal different H-cluster catalytic states under reducing and oxidizing conditions. Under H2 or sodium dithionite reductive treatments, the EPR spectra show signals that are consistent with a reduced [4Fe-4S]H(+) subcluster. The FTIR spectra showed upshifts of νCO modes to energies that are consistent with an increase in oxidation state of the [2Fe]H subcluster, which was corroborated by DFT analysis. In contrast to the case for wild-type CrHydA1, spectra associated with Hred and Hsred states are less populated in the Cys → Ser variant, demonstrating that the exchange of -SH with -OH alters how the H-cluster equilibrates among different reduced states of the catalytic cycle under steady-state conditions.
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory , Golden, Colorado 80401, United States.
authors.forename: David W
authors.initials: DW
authors.lastname: Mulder
authors.forename: Michael W
authors.initials: MW
authors.lastname: Ratzloff
authors.forename: Maurizio
authors.initials: M
authors.lastname: Bruschi
authors.forename: Claudio
authors.initials: C
authors.lastname: Greco
authors.forename: Evangeline
authors.initials: E
authors.lastname: Koonce
authors.forename: John W
authors.initials: JW
authors.lastname: Peters
authors.forename: Paul W
authors.initials: PW
authors.lastname: King
mesh_terms: D019943:Amino Acid Substitution; D002248:Carbon Monoxide / Q000494:pharmacology; D016825:Chlamydomonas reinhardtii / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D006864:Hydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D011522:Protons; D011789:Quantum Theory
chemical_list: D011522:Protons; D002248:Carbon Monoxide; D006864:Hydrogenase