NADH oxidation by the Na+-translocating NADH:quinone oxidoreductase from Vibrio cholerae: functional role of the NqrF subunit

Karin Türk, Andrea Puhar, Frank Neese, Eckhard Bill, Günter Fritz, Julia Steuber*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

49 Citations (Scopus)
14 Downloads (Pure)

Abstract

The Na(+)-translocating NADH:quinone oxidoreductase from Vibrio cholerae is a six subunit enzyme containing four flavins and a single motif for the binding of a Fe-S cluster on its NqrF subunit. This study reports the production of a soluble variant of NqrF (NqrF') and its individual flavin and Fe-S-carrying domains using V. cholerae or Escherichia coli as expression hosts. NqrF' and the flavin domain each contain 1 mol of FAD/mol of enzyme and exhibit high NADH oxidation activity (20,000 micromol min(-1) mg(-1)). EPR, visible absorption, and circular dichroism spectroscopy indicate that the Fe-S cluster in NqrF' and its Fe-S domain is related to 2Fe ferredoxins of the vertebrate-type. The addition of NADH to NqrF' results in the formation of a neutral flavosemiquinone and a partial reduction of the Fe-S cluster. The NqrF subunit harbors the active site of NADH oxidation and acts as a converter between the hydride donor NADH and subsequent one-electron reaction steps in the Na(+)-translocating NADH:quinone oxidoreductase complex. The observed electron transfer NADH --> FAD --> [2Fe-2S] in NqrF requires positioning of the FAD and the Fe-S cluster in close proximity in accordance with a structural model of the subunit.

Original languageEnglish
Pages (from-to)21349-21355
Number of pages7
JournalThe Journal of Biological Chemistry
Volume279
Issue number20
DOIs
Publication statusPublished - 14 May 2004
Externally publishedYes

Keywords

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • DNA Primers
  • Flavin-Adenine Dinucleotide/metabolism
  • Genetic Vectors
  • Mice
  • NAD/metabolism
  • Oxidation-Reduction
  • Polymerase Chain Reaction/methods
  • Protein Structure, Secondary
  • Protein Subunits/chemistry
  • Quinone Reductases/chemistry
  • Recombinant Proteins/chemistry
  • Restriction Mapping
  • Sodium/metabolism
  • Vibrio cholerae/enzymology

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