NADH oxidation by the Na+-translocating NADH:quinone oxidoreductase from Vibrio cholerae: functional role of the NqrF subunit.
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.
Türk
Karin
K
Mikrobiologisches Institut der Eidgenössischen Technischen Hochschule, ETH-Zentrum, CH-8092 Zürich, Switzerland.
Puhar
Andrea
A
Neese
Frank
F
Bill
Eckhard
E
Fritz
Günter
G
Steuber
Julia
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005822:Genetic Vectors; D051379:Mice; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction / Q000379:methods; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011808:Quinone Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012964:Sodium / Q000378:metabolism*; D014734:Vibrio cholerae / Q000201:enzymology*
D017931:DNA Primers; D021122:Protein Subunits; D011994:Recombinant Proteins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D012964:Sodium; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases