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Title: Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O 2-tolerant NAD +-reducing [NiFe] hydrogenase

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 57Fe 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 13C labeling of the CO ligand. This strategy also revealed the molecular coordinates that dominate the individual Fe–COmore » 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.« less
 [1] ;  [2] ;  [3] ;  [4] ;  [5] ;  [6] ;  [3] ;  [3] ;  [2]
  1. Technische Univ. Berlin, Berlin (Germany); Univ. of California, Davis, CA (United States)
  2. Univ. of California, Davis, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  3. Technische Univ. Berlin, Berlin (Germany)
  4. Univ. of California, Davis, CA (United States)
  5. JARSI, Sayo-gun, Hyogo (Japan)
  6. RIKEN, Sayo-gun, Hyogo (Japan)
Publication Date:
Grant/Contract Number:
AC02-05CH11231; GM-65440
Accepted Manuscript
Journal Name:
Chemical Science
Additional Journal Information:
Journal Volume: 6; Journal Issue: 2; Journal ID: ISSN 2041-6520
Royal Society of Chemistry
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
Country of Publication:
United States
OSTI Identifier: