Role of the conserved arginine 274 and histidine 224 and 228 residues in the NuoCD subunit of complex I from Escherichia coli.
The conserved arginine 274 and histidine 224 and 228 residues in subunit NuoCD of complex I from Escherichia coli were substituted for alanine. The wild-type and mutated NuoCD subunit was expressed on a plasmid in an E. coli strain bearing a nuoCD deletion. Complex I was fully expressed in the H224A and H228A mutants, whereas the R274A mutation yielded approximately 50% expression. Ubiquinone reductase activity of complex I was studied in membranes and with purified enzyme and was 50% and 30% of the wild-type activity in the H224A and H228A mutants, respectively. The activity of R274A was less than 5% of the wild type in membranes but 20% in purified complex I. Rolliniastatin inhibited quinone reductase activity in the mutants with similar affinity as in the wild type, indicating that the quinone-binding site was not significantly altered by the mutations. Ubiquinone-dependent superoxide production by complex I was similar to the wild type in the R274A mutant but slightly higher in the H224A and H228A mutants. The EPR spectra of purified complex I from the H224A and H228A mutants did not differ from the wild type. In contrast, the signals of the N2 cluster and another fast-relaxing [4Fe-4S] cluster, tentatively assigned as N6b, were drastically decreased in the NADH-reduced R274A mutant enzyme but reappeared on further reduction with dithionite. These findings show that the redox potential of the N2 and N6b centers is shifted to more negative values by the R274A mutation. Purified complex I was reconstituted into liposomes, and electric potential was generated across the membrane upon NADH addition in all three mutant enzymes, suggesting that none of the mutations directly affect the proton-pumping machinery.
Belevich
Galina
G
Helsinki Bioenergetics Group, Institute of Biotechnology, University of Helsinki, P.O. Box 65 (Viikinkaari 1), Helsinki FIN-00014, Finland.
Euro
Liliya
L
Wikström
Mårten
M
Verkhovskaya
Marina
M
D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D021122:Protein Subunits
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C000596778:NuoCD protein, E coli; D021122:Protein Subunits; D006639:Histidine; D001120:Arginine; D042967:Electron Transport Complex I