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Title: Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O2-tolerant NAD+-reducing [NiFe] hydrogenase

Journal Article · · Chemical Science
DOI:https://doi.org/10.1039/C4SC02982H· OSTI ID:1214545
 [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)
+ Show Author Affiliations

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 H2 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–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.

Research Organization:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Organization:
USDOE
Grant/Contract Number:
AC02-05CH11231; GM-65440
OSTI ID:
1214545
Journal Information:
Chemical Science, Vol. 6, Issue 2; ISSN 2041-6520
Publisher:
Royal Society of ChemistryCopyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 23 works
Citation information provided by
Web of Science

References (21)

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Characterization of [4Fe-4S] Cluster Vibrations and Structure in Nitrogenase Fe Protein at Three Oxidation Levels via Combined NRVS, EXAFS, and DFT Analyses journal February 2013
Dynamics of Rhodobacter capsulatus [2Fe-2S] Ferredoxin VI and Aquifex aeolicus Ferredoxin 5 via Nuclear Resonance Vibrational Spectroscopy (NRVS) and Resonance Raman Spectroscopy † journal June 2008
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Unusual FTIR and EPR properties of the H 2 -activating site of the cytoplasmic NAD-reducing hydrogenase from Ralstonia eutropha journal January 2000
Observation of the FeCN and FeCO Vibrations in the Active Site of [NiFe] Hydrogenase by Nuclear Resonance Vibrational Spectroscopy journal November 2012
Mössbauer Spectroscopy on Respiratory Complex I: The Iron–Sulfur Cluster Ensemble in the NADH-Reduced Enzyme Is Partially Oxidized journal December 2011
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Content and localization of FMN, Fe-S clusters and nickel in the NAD-linked hydrogenase of Nocardia opaca 1b journal July 1984
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Catalytic Production of Hydrogen Peroxide and Water by Oxygen-Tolerant [NiFe]-Hydrogenase during H 2 Cycling in the Presence of O 2 journal November 2013
H 2 -driven cofactor regeneration with NAD(P) + -reducing hydrogenases journal April 2013
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Resonance Raman Spectroscopy on [NiFe] Hydrogenase Provides Structural Insights into Catalytic Intermediates and Reactions journal July 2014
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Cited By (5)

Nitrosylation of Nitric-Oxide-Sensing Regulatory Proteins Containing [4Fe-4S] Clusters Gives Rise to Multiple Iron-Nitrosyl Complexes journal October 2016
Spectroscopic and Computational Investigations of Ligand Binding to IspH: Discovery of Non-diphosphate Inhibitors journal April 2017
Hydride bridge in [NiFe]-hydrogenase observed by nuclear resonance vibrational spectroscopy journal August 2015
Understanding the structure and dynamics of hydrogenases by ultrafast and two-dimensional infrared spectroscopy journal January 2019
Nitrosylation of Nitric-Oxide-Sensing Regulatory Proteins Containing [4Fe-4S] Clusters Gives Rise to Multiple Iron-Nitrosyl Complexes journal October 2016

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