Site-Directed Mutagenesis of Conserved Cysteine Residues in NqrD and NqrE Subunits of Na⁺-Translocating NADH:quinone Oxidoreductase

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M. S. Fadeeva¹, Y. V. Bertsova², M. I. Verkhovsky³, and A. V. Bogachev^2*

¹Department of Bioinformatics and Bioengineering, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 939-0338; E-mail: masha@genebee.msu.ru

²Department of Molecular Energetics of Microorganisms, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 939-0338; E-mail: bogachev@genebee.msu.su

³Helsinki Bioenergetics Group, Institute of Biotechnology, P. O. Box 65, (Viikinkaari 1), 00014 University of Helsinki, Helsinki, Finland; fax: (3589) 191-58001; E-mail: michael.verkhovsky@helsinki.fi

^* To whom correspondence should be addressed.

Received September 6, 2007

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Each of two hydrophobic subunits of Na⁺-translocating NADH:quinone oxidoreductase (NQR), NqrD and NqrE, contain a pair of strictly conserved cysteine residues within their transmembrane alpha-helices. Site-directed mutagenesis showed that substitutions of these residues in NQR of Vibrio harveyi blocked the Na⁺-dependent and 2-n-heptyl-4-hydroxyquinoline N-oxide-sensitive quinone reductase activity of the enzyme. However, these mutations did not affect the interaction of NQR with NADH and menadione. It was demonstrated that these conserved cysteine residues are necessary for the correct folding and/or the stability of the NQR complex. Mass and EPR spectroscopy showed that NQR from V. harveyi bears only a 2Fe-2S cluster as a metal-containing prosthetic group.

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KEY WORDS: Na⁺-translocating NADH:quinone oxidoreductase, sodium potential, Vibrio, respiratory chain

DOI: 10.1134/S0006297908020028

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