NqrM (DUF539) Protein Is Required for Maturation of Bacterial Na + -Translocating NADH:Quinone Oxidoreductase

Abstract

ABSTRACT Na + -translocating NADH:quinone oxidoreductase (Na + -NQR) catalyzes electron transfer from NADH to ubiquinone in the bacterial respiratory chain, coupled with Na + translocation across the membrane. Na + -NQR maturation involves covalent attachment of flavin mononucleotide (FMN) residues, catalyzed by flavin transferase encoded by the nqr -associated apbE gene. Analysis of complete bacterial genomes has revealed another putative gene ( duf539 , here renamed nqrM ) that usually follows the apbE gene and is present only in Na + -NQR-containing bacteria. Expression of the Vibrio harveyi nqr operon alone or with the associated apbE gene in Escherichia coli , which lacks its own Na + -NQR, resulted in an enzyme incapable of Na + -dependent NADH or reduced nicotinamide hypoxanthine dinucleotide (dNADH) oxidation. However, fully functional Na + -NQR was restored when these genes were coexpressed with the V. harveyi nqrM gene. Furthermore, nqrM lesions in Klebsiella pneumoniae and V. harveyi prevented production of functional Na + -NQR, which could be recovered by an nqrM -containing plasmid. The Na + -NQR complex isolated from the nqrM -deficient strain of V. harveyi lacks several subunits, indicating that nqrM is necessary for Na + -NQR assembly. The protein product of the nqrM gene, NqrM, contains a single putative transmembrane α-helix and four conserved Cys residues. Mutating one of these residues (Cys33 in V. harveyi NqrM) to Ser completely prevented Na + -NQR maturation, whereas mutating any other Cys residue only decreased the yield of the mature protein. These findings identify NqrM as the second specific maturation factor of Na + -NQR in proteobacteria, which is presumably involved in the delivery of Fe to form the (Cys) 4 [Fe] center between subunits NqrD and NqrE. IMPORTANCE Na + -translocating NADH:quinone oxidoreductase complex (Na + -NQR) is a unique primary Na + pump believed to enhance the vitality of many bacteria, including important pathogens such as Vibrio cholerae , Vibrio parahaemolyticus , Haemophilus influenzae , Neisseria gonorrhoeae , Pasteurella multocida , Porphyromonas gingivalis , Enterobacter aerogenes , and Yersinia pestis . Production of Na + -NQR in bacteria requires Na + -NQR-specific maturation factors. We earlier identified one such factor (ApbE) that covalently attaches flavin residues to Na + -NQR. Here we identify the other protein factor, designated NqrM, and show that NqrM and ApbE suffice to produce functional Na + -NQR from the Vibrio harveyi nqr operon. NqrM may be involved in Fe delivery to a unique Cys 4 [Fe] center during Na + -NQR assembly. Besides highlighting Na + -NQR biogenesis, these findings suggest a novel drug target to combat Na + -NQR-containing bacteria.