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Main Title: Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O-2-tolerant NAD(+)-reducing [NiFe] hydrogenase
Author(s): Lauterbach, Lars
Wang, Hongxin
Horch, Marius
Gee, Leland B.
Yoda, Yoshitaka
Tanaka, Yoshihito
Zebger, Ingo
Lenz, Oliver
Cramer, Stephen P.
Type: Article
Language Code: en
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) from Ralstonia eutropha is capable of H-2 conversion 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 of the various metal cofactors present in the enzyme. Here, all iron-containing cofactors of the SH were investigated by Fe-57 specific nuclear resonance vibrational spectroscopy (NRVS). Our data provide experimental evidence for one [2Fe2S] center and four [4Fe4S] clusters, which is consistent with the 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 selective C-13 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.
Issue Date: 2015
Date Available: 25-Oct-2017
DDC Class: 540 Chemie und zugeordnete Wissenschaften
Sponsor/Funder: DFG, EXC 314, Unifying Concepts in Catalysis
Journal Title: Chemical Science
Publisher: Royal Society of Chemistry
Publisher Place: Cambridge
Volume: 6
Issue: 2
Publisher DOI: 10.1039/c4sc02982h
Page Start: 1055
Page End: 1060
EISSN: 2041-6539
ISSN: 2041-6520
Appears in Collections:FG Physikalische Chemie / Biophysikalische Chemie » Publications

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