METHIONINE-393 IS AN AXIAL LIGAND OF THE HEME B(558) 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 b(558), heme b(595), and a chlorin, called heme d). Both subunits are located within the bacterial cytoplasmic membrane, and each has multiple putative transmembrane helices. Heme b(558) 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 b(558). Since mutagenesis of none of the other histidines in subunit I perturbs heme b(558), 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 b(558). Replacement of M393 by leucine results in the conversion of heme b(558) 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 b(558) back to a low-spin configuration. The data strongly suggest that the sixth axial ligand of heme b(558) 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 b(558).