Amino acid residues associated with cluster N3 in the NuoF subunit of the proton-translocating NADH-quinone oxidoreductase from Escherichia coli

Abstract

The NuoF subunit, which harbors NADH-binding site, of Escherichia coli NADH-quinone oxidoreductase (NDH-1) contains five conserved cysteine residues, four of which are predicted to ligate cluster N3. To determine this coordination, we overexpressed and purified the NuoF subunit and NuoF + E subcomplex in E. coli. We detected two distinct EPR spectra, arising from a [4Fe–4S] cluster ( g x,y,z = 1.90, 1.95, and 2.05) in NuoF, and a [2Fe–2S] cluster ( g x,y,z = 1.92, 1.95, and 2.01) in NuoE subunit. These clusters were assigned to clusters N3 and N1a, respectively. Based on the site-directed mutagenesis experiments, we identified that cluster N3 is ligated to the 351Cx 2Cx 2Cx 40C 398 motif.