Abstract
The cytochrome bd quinol oxidase is one of two respiratory oxidases in Escherichia coli. It oxidizes dihydroubiquinol or dihydromenaquinol 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 O(2). 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 R391Q. 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 where O(2) 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 b(595), 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 Met(393), one of the axial ligands to heme b(558), it is to be expected that the R391A mutation might destabilize the reduced form of heme b(558). The fact that the midpoint potentials of heme d and heme b(595) are also significantly lowered in the R391A mutant is consistent with these hemes being physically close together on the periplasmic side of the membrane.
Highlights
The cytochrome bd respiratory oxidase has a very high affinity for O2 and is found in many prokaryotes [1,2,3,4]
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 Arg391 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
Summary
The cytochrome bd respiratory oxidase has a very high affinity for O2 (in nM range) and is found in many prokaryotes [1,2,3,4]. The role of Arg391 in the CydA subunit of the Escherichia coli oxidase is investigated This arginine is very highly conserved and is only two residues away from Met393, one of the axial ligands to heme b558 (Fig. 1). The positive charge on Arg391 appears to stabilize the reduced form of heme b558 in the E. coli oxidase by about 210 mV (ϩ80 mV versus Ϫ130 mV) Substitution by another amino acid residue, other than lysine, makes it much harder to reduce heme b558 and inactivates the enzyme. A local conformational change due to the R391A mutation cannot be ruled out as the cause of the lower midpoint potentials of hemes b595 and d This is unlikely, since the optical and EPR spectra appear unperturbed, and the TMPD oxidase activity is unimpaired
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