METHIONINE-393 IS AN AXIAL LIGAND OF THE HEME B(558) COMPONENT OF THECYTOCHROME BD UBIQUINOL OXIDASE FROM ESCHERICHIA-COLI

The cytochrome bd oxidase is one of two terminal oxidases in the aerob ic respiratory chain of Escherichia coli. The complex is composed of t wo 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 putat ive transmembrane helices. Heme b(558) is a six-coordinate, low-spin h eme component of the oxidase which has been shown to be contained with in subunit I and has been implicated in the oxidation of the substrate , ubiquinol-8, in the cytoplasmic membrane. Previous site-directed mut agenesis studies identified His186, predicted to be near the periplasm ic side of transmembrane helix D of subunit I, as one of the axial lig ands 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 ca nnot have bis(histidine) ligation. In this work, the properties of 14 mutants are reported, including substitutions for each of 10 methionin e residues within subunit I. Among this set of mutants, only the repla cement of M393 perturbs heme b(558). Replacement of M393 by leucine re sults in the conversion of heme b(558) to a high-spin state. Surprisin gly, the M393L mutation does not eliminate enzymatic activity, and the mutant oxidase has sufficient turnover to support aerobic growth of t he cells. The addition of imidazole to the purified M393L oxidase conv erts heme b(558) back to a low-spin configuration. The data strongly s uggest that the sixth axial ligand of heme b(558) is methionine-393, a nd that this heme, therefore, has histidine-methionine ligation. The r esults are consistent with recent cryogenic near-infrared magnetic cir cular dichroism spectra that also indicate histidine-methionine ligati on of heme b(558).