Glutamate-89 in subunit II of cytochrome bo3 from Escherichia coli is required for the function of the heme-copper oxidase.

Recent electrostatics calculations on the cytochrome c oxidase from Paracoccus denitrificans revealed an unexpected coupling between the redox state of the heme-copper center and the state of protonation of a glutamic acid (E78II) that is 25 A away in subunit II of the oxidase. Examination of more than 300 sequences of the homologous subunit in other heme-copper oxidases shows that this residue is virtually totally conserved and is in a cluster of very highly conserved residues at the "negative" end (bacterial cytoplasm or mitochondrial matrix) of the second transmembrane helix. The functional importance of several residues in this cluster (E89II, W93II, T94II, and P96II) was examined by site-directed mutagenesis of the corresponding region of the cytochrome bo(3) quinol oxidase from Escherichia coli (where E89II is the equivalent of residue E78II of the P. denitrificans oxidase). Substitution of E89II with either alanine or glutamine resulted in reducing the rate of turnover to about 43 or 10% of the wild-type value, respectively, whereas E89D has only about 60% of the activity of the control oxidase. The quinol oxidase activity of the W93V mutant is also reduced to about 30% of that of the wild-type oxidase. Spectroscopic studies with the purified E89A and E89Q mutants indicate no perturbation of the heme-copper center. The data suggest that E89II (E. coli numbering) is critical for the function of the heme copper oxidases. The proximity to K362 suggests that this glutamic acid residue may regulate proton entry or transit through the K-channel. This hypothesis is supported by the finding that the degree of oxidation of the low-spin heme b is greater in the steady state using hydrogen peroxide as an oxidant in place of dioxygen for the E89Q mutant. Thus, it appears that the inhibition resulting from the E89II mutation is due to a block in the reduction of the heme-copper binuclear center, expected for K-channel mutants.

Using matrix-assisted laser desorption ionization mass spectrometry to map the quinol binding site of cytochrome bo3 from Escherichia coli.

The cytochrome bo3 ubiquinol oxidase contains at least one and possibly two binding sites for ubiquinol/ubiquinone. Previous studies used the photoreactive affinity label 3-[3H]azido-2-methyl-5-methoxy-6-geranyl-1,4-benzoquinone (azido-Q), a substrate analogue, to demonstrate that subunit II contributes to at least one of the quinol binding sites. In the current work, mass spectroscopy is used to identify a peptide within subunit II that is photolabeled by the azido-Q. Purified cytochrome bo3 was photolabeled as previously described using azido-Q that was not tritiated (i.e., not radiolabeled). Subunit II was then isolated from an SDS-PAGE gel and proteolyzed in situ with trypsin. The resulting peptides were eluted from the gel and then identified using matrix-assisted laser desorption ionization mass spectrometry. The resulting mass spectrum was compared to that obtained by analysis of subunit II that had not been exposed to the photolabel. Using the amino acid sequence, each peak in the mass spectrum of the unlabeled subunit II could be assigned to an expected trypsin fragment. Two additional peaks were observed in the mass spectrum of the photolabeled subunit with m/z 1931.9 and 2287.7. Subtraction of the mass of azido-Q from the peak at m/z 1931.9 results in a mass equivalent to that of a peptide consisting of amino acids 165-178. The assignment of the peak at m/z 2287.7 cannot be made unequivocally and may correspond either to the covalent attachment of azido-Q to peptide 254-270 or to a peptide resulting from incomplete proteolysis. The labeled peptide, 165-178, is within the water-soluble domain of subunit II, whose X-ray structure is known. This peptide is located near the site where CuA is located in the homologous cytochrome c oxidases and can be placed near the interface between subunits I and II.

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.