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.

A novel experiment has been used to show proximity relationships between sites on the surface of the cytochrome bd quinol oxidase of Escherichia coli. The artificial protease iron (S)-1-[p-(bromoacetamido)benzyl]-EDTA (Fe--BABE) was conjugated to selected reactive cysteines placed in subunit I or subunit II, with the aim of identifying amino acid residues within approximately 12 A of each site of attachment. The protease was activated with H2O2 and ascorbate for a few seconds, and hydrolysis products were isolated and analyzed by N-terminal sequencing. Among other results, we found that residue 39 of subunit II is near residue 255 of subunit I in the putative quinone-binding domain (Q loop) of the oxidase. Since this technique is insensitive to the nature of the amino acid side chains, it should prove generally valuable in revealing spatial relationships both within and between subunits in complex proteins where high-resolution structural information is not available.

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry has been used to obtain accurate molecular weight information for each subunit of several hydrophobic integral membrane proteins: cytochrome bo3 (4 subunits) and cytochrome bd (2 subunits) from E. coli, and the bc1 complex (3 subunits) and the cytochrome c oxidase (3 subunits) from Rhodobacter sphaeroides. The results demonstrate that the MALDI method is a convenient, quick, sensitive and reliable means for obtaining the molecular masses of the subunits of purified multisubunit membrane proteins.

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