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Characterization of a HoxEFUYH type of [NiFe] hydrogenase from Allochromatium vinosum and some EPR and IR properties of the hydrogenase module

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Abstract

A soluble hydrogenase from Allochromatium vinosum was purified. It consisted of a large (M r = 52 kDa) and a small (M r = 23 kDa) subunit. The genes encoding for both subunits were identified. They belong to an open reading frame where they are preceded by three more genes. A DNA fragment containing all five genes was cloned and sequenced. The deduced amino acid sequences of the products characterized the complex as a member of the HoxEFUYH type of [NiFe] hydrogenases. Detailed sequence analyses revealed binding sites for eight Fe–S clusters, three [2Fe–2S] clusters and five [4Fe–4S] clusters, six of which are also present in homologous subunits of [FeFe] hydrogenases and NADH:ubiquione oxidoreductases (complex I). This makes the HoxEFUYH type of hydrogenases the one that is evolutionary closest to complex I. The relative positions of six of the potential Fe–S clusters are predicted on the basis of the X-ray structures of the Clostridium pasteurianum [FeFe] hydrogenase I and the hydrophilic domain of complex I from Thermus thermophilus. Although the HoxF subunit contains binding sites for flavin mononucleotide and NAD(H), cell-free extracts of A. vinosum did not catalyse a H2-dependent reduction of NAD+. Only the hydrogenase module (HoxYH) could be purified. Its electron paramagnetic resonance (EPR) and IR spectral properties showed the presence of a Ni–Fe active site and a [4Fe–4S] cluster. Its activity was sensitive to carbon monoxide. No EPR signals from a light-sensitive Nia–C* state could be observed. This study presents the first IR spectroscopic data on the HoxYH module of a HoxEFUYH type of [NiFe] hydrogenase.

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Abbreviations

BV:

Benzyl viologen

DCIP:

2,6-Dichlorophenol-indophenol

EPR:

Electron paramagnetic resonance

FMN:

Flavin mononucleotide

FPLC:

Fast protein liquid chromatography

KPi:

Potassium phosphate (50 mM)

MBH:

Membrane-bound hydrogenase

MV:

Methyl viologen

PAGE:

Polyacrylamide gel electrophoresis

SDS:

Sodium dodecyl sulfate

SH:

Soluble hydrogenase

Tris:

Tris(hydroxymethyl)aminomethane–HCl buffer (50 mM)

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Acknowledgements

M.L. was supported by grants from the National Natural Science Foundation of China (30470395) and the National 863 Project of China (2002AA515030). S.P.J.A. thanks the Netherlands Organization for Scientific Research (NWO), Division of Chemical Sciences, for support. We thank A.O.M. Muijsers for the N-terminal sequencing work.

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Authors and Affiliations

  1. School of Life Sciences, Bio-energy Center, Xiamen University, Xiamen, 361005, People’s Republic of China

    Minnan Long, Jingjing Liu & Zhifeng Chen

  2. Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV, Amsterdam, The Netherlands

    Boris Bleijlevens, Winfried Roseboom & Simon P. J. Albracht

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  1. Minnan Long
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  2. Jingjing Liu
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  3. Zhifeng Chen
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Correspondence to Minnan Long or Simon P. J. Albracht.

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Long, M., Liu, J., Chen, Z. et al. Characterization of a HoxEFUYH type of [NiFe] hydrogenase from Allochromatium vinosum and some EPR and IR properties of the hydrogenase module. J Biol Inorg Chem 12, 62–78 (2007). https://doi.org/10.1007/s00775-006-0162-1

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