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

• Published in: The Journal of biological chemistry Vol. 279; no. 20; pp. 21349 - 21355
• Main Authors: Türk, Karin, Puhar, Andrea, Neese, Frank, Bill, Eckhard, Fritz, Günter, Steuber, Julia
• Format: Journal Article
• Language: English
• Published: United States 14-05-2004
• Subjects: 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; Protein Subunits - genetics; Protein Subunits - metabolism; Quinone Reductases - chemistry; Quinone Reductases - genetics; Quinone Reductases - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Restriction Mapping; Sodium - metabolism; Vibrio cholerae - enzymology
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M311692200

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.