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The soluble [NiFe]-hydrogenase from Ralstonia eutropha contains four cyanides in its active site, one of which is responsible for the insensitivity towards oxygen

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Abstract

Infrared spectra of 15N-enriched preparations of the soluble cytoplasmic NAD+-reducing [NiFe]-hydrogenase from Ralstonia eutropha are presented. These spectra, together with chemical analyses, show that the Ni-Fe active site contains four cyanide groups and one carbon monoxide molecule. It is proposed that the active site is a (RS)2(CN)Ni(μ-RS)2Fe(CN)3(CO) centre (R=Cys) and that H2 activation solely takes place on nickel. One of the two FMN groups (FMN-a) in the enzyme can be reversibly released upon reduction of the enzyme. It is now reported that at longer times also one of the cyanide groups, the one proposed to be bound to the nickel atom, could be removed from the enzyme. This process was irreversible and induced the inhibition of the enzyme activity by oxygen; the enzyme remained insensitive to carbon monoxide. The Ni-Fe active site was EPR undetectable under all conditions tested. It is concluded that the Ni-bound cyanide group is responsible for the oxygen insensitivity of the enzyme.

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Abbreviations

BV:

benzyl viologen

DCIP:

2,6-dichlorophenol-indophenol

EXAFS:

extended X-ray absorption fine structure

FTIR:

Fourier transform infrared

MV:

methyl viologen

SH:

soluble NAD+-reducing hydrogenase

XAS:

X-ray absorption spectroscopy

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Acknowledgements

This work was supported by the Netherlands Organization for Scientific Research (NWO), the Deutsche Forschungsgemeinschaft, EU-project BIO4-98-0280, and the European Union Cooperation in the field of Scientific and Technical Research (COST), Action-818 and Action-841. We thank Dr K. Schneider (University of Bielefeld, Germany) for the kind gift of enzyme 98% enriched in 15N, Dr R.P. Happe for the IR spectrum of the oxidized enriched enzyme, and W. Roseboom for expert technical assistance.

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Authors and Affiliations

  1. Swammerdam Institute for Life Sciences, Biochemistry, University of Amsterdam, Plantage Muidergracht 12, 1018 TV , Amsterdam, The Netherlands

    Eddy Van der Linden, Boris Bleijlevens & Simon P. J. Albracht

  2. Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, 10115 , Berlin, Germany

    Tanja Burgdorf, Michael Bernhard & Bärbel Friedrich

Authors
  1. Eddy Van der Linden
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  2. Tanja Burgdorf
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  3. Michael Bernhard
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  4. Boris Bleijlevens
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  5. Bärbel Friedrich
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  6. Simon P. J. Albracht
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Correspondence to Simon P. J. Albracht.

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Van der Linden, E., Burgdorf, T., Bernhard, M. et al. The soluble [NiFe]-hydrogenase from Ralstonia eutropha contains four cyanides in its active site, one of which is responsible for the insensitivity towards oxygen. J Biol Inorg Chem 9, 616–626 (2004). https://doi.org/10.1007/s00775-004-0555-y

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