Methionine-393 is an axial ligand of the heme b558 component of the cytochrome bd ubiquinol oxidase from Escherichia coli

Abstract

The cytochrome bd oxidase is one of two terminal oxidases in the aerobic respiratory chain of Escherichia coli. The complex is composed of two subunits (I and II) and three heme prosthetic groups (heme b558, heme b595, and a chlorin, called heme d). Both subunits are located within the bacterial cytoplasmic membrane, and each has multiple putative transmembrane helices. Heme b558 is a six-coordinate, low-spin heme component of the oxidase which has been shown to be contained within subunit I and has been implicated in the oxidation of the substrate, ubiquinol-8, in the cytoplasmic membrane. Previous site-directed mutagenesis studies identified His186, predicted to be near the periplasmic side of transmembrane helix D of subunit I, as one of the axial ligands of heme b558. Since mutagenesis of none of the other histidines in subunit I perturbs heme b558, it was concluded that this heme cannot have bis(histidine) ligation. In this work, the properties of 14 mutants are reported, including substitutions for each of 10 methionine residues within subunit I. Among this set of mutants, only the replacement of M393 perturbs heme b558. Replacement of M393 by leucine results in the conversion of heme b558 to a high-spin state. Surprisingly, the M393L mutation does not eliminate enzymatic activity, and the mutant oxidase has sufficient turnover to support aerobic growth of the cells. The addition of imidazole to the purified M393L oxidase converts heme b558 back to a low-spin configuration. The data strongly suggest that the sixth axial ligand of heme b558 is methionine-393, and that this heme, therefore, has histidine-methionine ligation. The results are consistent with recent cryogenic near-infrared magnetic circular dichroism spectra that also indicate histidine-methionine ligation of heme b558.

Discovery of the True Peroxy Intermediate in the Catalytic Cycle of Terminal Oxidases by Real-time Measurement

“…coli cells were obtained from GO105/pTK1 strain according to Ref. 46, and cytochrome bd oxidase was isolated from cell membranes as described (37,47). Cytochrome bd from A. vinelandii strain MK8 was isolated as reported in Ref.…”

Femtosecond resolution of ligand-heme interactions in the high-affinity quinol oxidase bd : A di-heme active site?

“…coli cells (strain GO 105͞pTK1; ref. 21) overproducing cytochrome bd and deleted in cytochrome bo 3 , kindly provided by R. Gennis (University of Illinois, Urbana-Champaign) were grown in 10-liter flasks at 37°C in a medium containing 80 mM potassium phosphate, 2.5 mM sodium citrate, 19 mM ammoAbbreviations: R, fully reduced; MV, mixed valence. ‡ To whom reprint requests should be addressed.…”

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

“…Since it is only two residues away from Met 393 , which has been shown to be an axial ligand to heme b 558 (17), it is reasonable to speculate that Arg 391 might be important in determining the properties of this heme component of the enzyme. The current work confirms this speculation.…”

“…Strains and Plasmids-E. coli strain GO105 (cyd AB::kan, cyo, recA), which lacks both cytochrome bo 3 and cytochrome bd quinol oxidases (17), was used as the host strain for expressing both the wild type and mutant cytochrome bd from a plasmid. To obtain wild type cytochrome bd, plasmid pTK1 (24) was introduced into the strain.…”

“…Heme b 558 is ligated to His 186 and Met 393 in CydA (see Fig. 1) (16,17) and appears to be directly involved in the oxidation of quinol (18). Heme b 595 and heme d are both high spin hemes that appear to form a binuclear center where O 2 is reduced to water (19).…”