Arginine 391 in subunit I of the cytochrome bd quinol oxidase from Escherichia coli stabilizes the reduced form of the hemes and is essential for quinol oxidase activity

The cytochrome bd quinol oxidase is one of two respiratory oxidases in Escherichia coli. It oxidizes dihydroubiquinol or dihyclromenaquinol while reducing dioxygen to water. The bd-type oxidases have only been found in prokaryotes and have been implicated in the survival of some bacteria, including pathogens, under conditions of low aeration. With a high affinity for dioxygen, cytochrome bd not only couples respiration to the generation of a proton motive force but also scavenges O₂. In the current work, the role of a highly conserved arginine residue is explored by site-directed mutagenesis. Four mutations were made: R391A, R391K, R391M, and H391Q. All of the mutations except R391K result in enzyme lacking ubiquinol oxidase activity. Oxidase activity using the artificial reductant N,N,N′,N′-tetramethyl-p- phenylenediamine in place of ubiquinol was, however, unimpaired by the mutations, indicating that the catalytic center wliere O₂ is reduced is intact. UV-visible spectra of each of the mutant oxidases show no perturbations to any of the three heme components (heme b_558, heme b₅₉₅, and heme d). However, spectroelectrochemical titrations of the R391A mutant reveal that the midpoint potentials of all of the heme components are substantially lower compared with the wild type enzyme. Since Arg ³⁹¹ is close to Met³⁹³, one of the axial ligands to heme b₅₅₈, it is to be expected that the R391A mutation might destabilize the reduced form of heme b₅₅₈. The fact that the midpoint potentials of heme d and heme b₅₉₅ are also significantly lowered in the M391A mutant is consistent with these hemes being physically close together on the periplasmic side of the membrane.