Methionine-393 Is an Axial Ligand of the Heme b₅₅₈ Component of the Cytochrome bd Ubiquinol Oxidase from Escherichia coli

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 His 186, 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.