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

Lauterbach, Lars; Wang, Hongxin; Horch, Marius; Gee, Leland B.; Yoda, Yoshitaka; Tanaka, Yoshihito; Zebger, Ingo; Lenz, Oliver; Cramer, Stephen P.

doi:10.1039/C4SC02982H

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

Journal Article · Thu Oct 30 00:00:00 EDT 2014 · Chemical Science

DOI:https://doi.org/10.1039/C4SC02982H· OSTI ID:1214545

Lauterbach, Lars ^[1]; Wang, Hongxin ^[2]; Horch, Marius ^[3]; Gee, Leland B. ^[4]; Yoda, Yoshitaka ^[5]; Tanaka, Yoshihito ^[6]; Zebger, Ingo ^[3]; Lenz, Oliver ^[3]; Cramer, Stephen P. ^[2]

Technische Univ. Berlin, Berlin (Germany); Univ. of California, Davis, CA (United States)
Univ. of California, Davis, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Technische Univ. Berlin, Berlin (Germany)
Univ. of California, Davis, CA (United States)
JARSI, Sayo-gun, Hyogo (Japan)
RIKEN, Sayo-gun, Hyogo (Japan)

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.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1214545

Journal Information:: Chemical Science, Journal Name: Chemical Science Journal Issue: 2 Vol. 6; ISSN 2041-6520; ISSN CSHCBM

Publisher:: Royal Society of ChemistryCopyright Statement

Country of Publication:: United States

Language:: English

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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 Serrano, Pauline N.; Wang, Hongxin; Crack, Jason C. Angewandte Chemie, Vol. 128, Issue 47 https://doi.org/10.1002/ange.201607033	journal	October 2016
Nitrosylation of Nitric-Oxide-Sensing Regulatory Proteins Containing [4Fe-4S] Clusters Gives Rise to Multiple Iron-Nitrosyl Complexes Serrano, Pauline N.; Wang, Hongxin; Crack, Jason C. Angewandte Chemie International Edition, Vol. 55, Issue 47 https://doi.org/10.1002/anie.201607033	journal	October 2016
Spectroscopic and Computational Investigations of Ligand Binding to IspH: Discovery of Non-diphosphate Inhibitors O'Dowd, Bing; Williams, Sarah; Wang, Hongxin ChemBioChem, Vol. 18, Issue 10 https://doi.org/10.1002/cbic.201700052	journal	April 2017
Hydride bridge in [NiFe]-hydrogenase observed by nuclear resonance vibrational spectroscopy Ogata, Hideaki; Krämer, Tobias; Wang, Hongxin Nature Communications, Vol. 6, Issue 1 https://doi.org/10.1038/ncomms8890	journal	August 2015
Understanding the structure and dynamics of hydrogenases by ultrafast and two-dimensional infrared spectroscopy Horch, Marius; Schoknecht, Janna; Wrathall, Solomon L. D. Chemical Science, Vol. 10, Issue 39 https://doi.org/10.1039/c9sc02851j	journal	January 2019

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