A model system for [NiFe] hydrogenase maturation studies: Purification of an active site-containing hydrogenase large subunit without small subunit

Gordon Winter; Thorsten Buhrke; Oliver Lenz; Anne Katherine Jones; Michael Forgber; Bärbel Friedrich

doi:10.1016/j.febslet.2005.06.064

A model system for [NiFe] hydrogenase maturation studies: Purification of an active site-containing hydrogenase large subunit without small subunit

Gordon Winter, Thorsten Buhrke, Oliver Lenz, Anne Katherine Jones, Michael Forgber, Bärbel Friedrich

Arizona State University

Research output: Contribution to journal › Article › peer-review

33 Citations (Scopus)

Abstract

The large subunit HoxC of the H₂-sensing [NiFe] hydrogenase from Ralstonia eutropha was purified without its small subunit. Two forms of HoxC were identified. Both forms contained iron but only substoichiometric amounts of nickel. One form was a homodimer of HoxC whereas the second also contained the Ni-Fe site maturation proteins HypC and HypB. Despite the presence of the Ni-Fe active site in some of the proteins, both forms, which lack the Fe-S clusters normally present in hydrogenases, cannot activate hydrogen. The incomplete insertion of nickel into the Ni-Fe site provides direct evidence that Fe precedes Ni in the course of metal center assembly.

Original language	English
Pages (from-to)	4292-4296
Number of pages	5
Journal	FEBS Letters
Volume	579
Issue number	20
DOIs	https://doi.org/10.1016/j.febslet.2005.06.064
Publication status	Published - Aug 15 2005

Keywords

Maturation
Metal center assembly
Ralstonia eutropha
[NiFe] hydrogenase

ASJC Scopus subject areas

Biophysics
Structural Biology
Biochemistry
Molecular Biology
Genetics
Cell Biology

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A model system for [NiFe] hydrogenase maturation studies : Purification of an active site-containing hydrogenase large subunit without small subunit. / Winter, Gordon; Buhrke, Thorsten; Lenz, Oliver; Jones, Anne Katherine; Forgber, Michael; Friedrich, Bärbel.

In: FEBS Letters, Vol. 579, No. 20, 15.08.2005, p. 4292-4296.

Research output: Contribution to journal › Article › peer-review

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title = "A model system for [NiFe] hydrogenase maturation studies: Purification of an active site-containing hydrogenase large subunit without small subunit",

abstract = "The large subunit HoxC of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha was purified without its small subunit. Two forms of HoxC were identified. Both forms contained iron but only substoichiometric amounts of nickel. One form was a homodimer of HoxC whereas the second also contained the Ni-Fe site maturation proteins HypC and HypB. Despite the presence of the Ni-Fe active site in some of the proteins, both forms, which lack the Fe-S clusters normally present in hydrogenases, cannot activate hydrogen. The incomplete insertion of nickel into the Ni-Fe site provides direct evidence that Fe precedes Ni in the course of metal center assembly.",

keywords = "Maturation, Metal center assembly, Ralstonia eutropha, [NiFe] hydrogenase",

author = "Gordon Winter and Thorsten Buhrke and Oliver Lenz and Jones, {Anne Katherine} and Michael Forgber and B{\"a}rbel Friedrich",

note = "Funding Information: We thank S. L{\"o}scher and M. Haumann (FU Berlin) for the AAS measurements, I. Zebger (TU Berlin), H. Stosnach and U. Waldschl{\"a}ger (R{\"o}ntec AG, Berlin) for TXRF analysis, A.L. DeLacey (Madrid, Spain) for the H + /D 2 exchange measurements and J. St{\"o}hr for excellent technical assistance. This work was funded by the Deutsche Forschungsgemeinschaft (SFB498, TP C1) and by the Fonds der Chemischen Industrie. A.K. Jones thanks the Humboldt foundation for a fellowship. ",

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AU - Lenz, Oliver

AU - Jones, Anne Katherine

AU - Forgber, Michael

AU - Friedrich, Bärbel

N1 - Funding Information: We thank S. Löscher and M. Haumann (FU Berlin) for the AAS measurements, I. Zebger (TU Berlin), H. Stosnach and U. Waldschläger (Röntec AG, Berlin) for TXRF analysis, A.L. DeLacey (Madrid, Spain) for the H + /D 2 exchange measurements and J. Stöhr for excellent technical assistance. This work was funded by the Deutsche Forschungsgemeinschaft (SFB498, TP C1) and by the Fonds der Chemischen Industrie. A.K. Jones thanks the Humboldt foundation for a fellowship.

PY - 2005/8/15

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N2 - The large subunit HoxC of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha was purified without its small subunit. Two forms of HoxC were identified. Both forms contained iron but only substoichiometric amounts of nickel. One form was a homodimer of HoxC whereas the second also contained the Ni-Fe site maturation proteins HypC and HypB. Despite the presence of the Ni-Fe active site in some of the proteins, both forms, which lack the Fe-S clusters normally present in hydrogenases, cannot activate hydrogen. The incomplete insertion of nickel into the Ni-Fe site provides direct evidence that Fe precedes Ni in the course of metal center assembly.

AB - The large subunit HoxC of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha was purified without its small subunit. Two forms of HoxC were identified. Both forms contained iron but only substoichiometric amounts of nickel. One form was a homodimer of HoxC whereas the second also contained the Ni-Fe site maturation proteins HypC and HypB. Despite the presence of the Ni-Fe active site in some of the proteins, both forms, which lack the Fe-S clusters normally present in hydrogenases, cannot activate hydrogen. The incomplete insertion of nickel into the Ni-Fe site provides direct evidence that Fe precedes Ni in the course of metal center assembly.

KW - Maturation

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KW - Ralstonia eutropha

KW - [NiFe] hydrogenase

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