Strong excitonic interactions in the oxygen-reducing site of bd-type oxidase: the Fe-to-Fe distance between hemes d and b595 is 10 A.

Absorption and circular dichroism (CD) spectra of cytochrome bd from Escherichia coli have been compared for the wild type enzyme and an inactive mutant in which a highly conserved E445 in subunit I has been replaced by alanine [Zhang, J., Hellwig, P., Osborne, J.

Matrix-assisted laser desorption ionization mass spectrometry of membrane proteins: demonstration of a simple method to determine subunit molecular weights of hydrophobic subunits.

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.

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.

Proximity mapping the surface of a membrane protein using an artificial protease: demonstration that the quinone-binding domain of subunit I is near the N-terminal region of subunit II of cytochrome bd.

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.