title: Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O2-tolerant NAD+-reducing [NiFe] hydrogenase.
abstract: Hydrogenases are complex metalloenzymes that catalyze the reversible splitting of molecular hydrogen into protons and electrons essentially without overpotential. The NAD+-reducing soluble hydrogenase (SH) fromRalstonia eutrophais capable of H2conversion even in the presence of usually toxic dioxygen. The molecular details of the underlying reactions are largely unknown, mainly because of limited knowledge of the structure and function the various metal cofactors present in the enzyme. Here all iron-containing cofactors of the SH were investigated by57Fe specific nuclear resonance vibrational spectroscopy (NRVS). Our data provide experimental evidence for one [2Fe2S] center and four [4Fe4S] clusters, which is consistent with amino acid sequence composition. Only the [2Fe2S] cluster and one of the four [4Fe4S] clusters were reduced upon incubation of the SH with NADH. This finding explains the discrepancy between the large number of FeS clusters and the small amount of FeS cluster-related signals as detected by electron paramagnetic resonance spectroscopic analysis of several NAD+-reducing hydrogenases. For the first time, Fe-CO and Fe-CN modes derived from the [NiFe] active site could be distinguished by NRVS through selective13C labeling of the CO ligand. This strategy also revealed the molecular coordinates that dominate the individual Fe-CO modes. The present approach explores the complex vibrational signature of the Fe-S clusters and the hydrogenase active site, thereby showing that NRVS represents a powerful tool for the elucidation of complex biocatalysts containing multiple cofactors.
authors: Institute of Chemistry, Technische Universität Berlin, Straße des 17, Juni 135, 10623 Berlin, Germany ; Department of Chemistry, University of California, One Shields Ave, Davis CA 95616, USA.
authors: Lars
authors: L
authors: Lauterbach
authors: Department of Chemistry, University of California, One Shields Ave, Davis CA 95616, USA ; Physical Biosciences Division, Lawrence Berkeley National Laboratory, One Cyclotron Road, Berkeley CA 94720, USA.
authors: Hongxin
authors: H
authors: Wang
authors: Institute of Chemistry, Technische Universität Berlin, Straße des 17, Juni 135, 10623 Berlin, Germany.
authors: Marius
authors: M
authors: Horch
authors: Department of Chemistry, University of California, One Shields Ave, Davis CA 95616, USA.
authors: Leland B
authors: LB
authors: Gee
authors: JASRI, SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5198, Japan.
authors: Yoshitaka
authors: Y
authors: Yoda
authors: RIKEN, SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5198, Japan.
authors: Yoshihito
authors: Y
authors: Tanaka
authors: Institute of Chemistry, Technische Universität Berlin, Straße des 17, Juni 135, 10623 Berlin, Germany.
authors: Ingo
authors: I
authors: Zebger
authors: Institute of Chemistry, Technische Universität Berlin, Straße des 17, Juni 135, 10623 Berlin, Germany.
authors: Oliver
authors: O
authors: Lenz
authors: Department of Chemistry, University of California, One Shields Ave, Davis CA 95616, USA ; Physical Biosciences Division, Lawrence Berkeley National Laboratory, One Cyclotron Road, Berkeley CA 94720, USA.
authors: Stephen P
authors: SP
authors: Cramer