The role of the active site-coordinating cysteine residues in the maturation of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha H16

Gordon Winter, Thorsten Buhrke, Anne Katherine Jones, Bärbel Friedrich

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

The H2-splitting active site of [NiFe] hydrogenases is tightly bound to the protein matrix via four conserved cysteine residues. In this study, the nickel-binding cysteine residues of HoxC, the large subunit of the H 2-sensing regulatory hydrogenase (RH) from Ralstonia eutropha, were replaced by serine. All four mutant proteins, C60S, C63S, C479S, and C482S, were inactive both in H2 sensing and H2 oxidation and did not adopt the native oligomeric structure of the RH. Nickel was bound only to the C482S derivative. The assembly of the [NiFe] active site is a complex process that requires the function of at least six accessory proteins. Among these proteins, HypC has been shown to act as a chaperone for the large subunit during the maturation process. Immunoblot analysis revealed the presence of a strong RH-dependent HypC-specific complex in extracts containing the C60S, C63S, and C482S derivatives, pointing to a block in maturation for these mutant proteins. The lack of this complex in the extract containing C479S indicates that this specific cysteine residue might be crucial for the interaction between HoxC and HypC.

Original languageEnglish
Pages (from-to)138-146
Number of pages9
JournalArchives of Microbiology
Volume182
Issue number2-3
DOIs
Publication statusPublished - Oct 2004

Fingerprint

Cupriavidus necator
Hydrogenase
Cysteine
Catalytic Domain
Mutant Proteins
Nickel
Complex Mixtures
Derivatives
Proteins
Accessories
Serine
Oxidation
asoxime chloride
nickel-iron hydrogenase

Keywords

  • [NiFe] hydrogenase
  • H sensor
  • Metal center assembly
  • Nickel metabolism
  • Ralstonia eutropha

ASJC Scopus subject areas

  • Microbiology

Cite this

The role of the active site-coordinating cysteine residues in the maturation of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha H16. / Winter, Gordon; Buhrke, Thorsten; Jones, Anne Katherine; Friedrich, Bärbel.

In: Archives of Microbiology, Vol. 182, No. 2-3, 10.2004, p. 138-146.

Research output: Contribution to journalArticle

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