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

doi:10.1074/jbc.m311692200

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.

Highlights

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
Assuming that the Fe-S cluster on NqrFЈ is of the 2Fe-2S type, our results indicate that 18% of NqrFЈ overproduced in V. cholerae contained an Fe-S cluster

Summary

Introduction

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. 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. Ogous to complex I of the mitochondrial respiratory chain [3,4,5] Another type of NADH-driven redox pump (called Naϩ-NQR) is found in marine bacteria like Vibrio alginolyticus or the human pathogen V. cholerae (6 –11). We demonstrate that the NqrF subunit catalyzes the initial electron transfer reactions NADH 3 FAD 3 [2Fe-2S] in the Naϩ-NQR complex. Enterobacteria like Escherichia coli possess a Naϩ-dependent NADH dehydrogenase that is homol-

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