Site-directed mutation of the highly conserved region near the Q-loop of the cytochrome bd quinol oxidase from Escherichia coli specifically perturbsheme b(595)

Cytochrome bd is one of the two quinol oxidases in the respiratory chain of Escherichia coli. The enzyme contains three heme prosthetic groups. The di oxygen binding site is heme d, which is thought to be part of the heme-heme binuclear center along with heme b(595), which is a high-spin heme whose f unction is not known. Protein sequence alignments [Osborne, J. P., and Genn is, R. B. (1999) Biochim. Biophys Acta 1410, 32-50] of cytochrome bd quinol oxidase sequences from different microorganisms have revealed a highly con served sequence (GWXXXEXGRQPW; bold letters indicate strictly conserved res idues) predicted to be on the periplasmic side of the membrane between tran smembrane helices 8 and 9 in subunit I. The functional importance of this r egion is investigated in the current work by site-directed mutagenesis. Sev eral mutations in this region (W441A, E445A/Q, R448A, Q449A, and W451A) res ulted in a catalytically inactive enzyme with abnormal W-vis spectra. E445A was selected for detailed analysis because of the absence of the absorptio n bands from heme b(595). Detailed spectroscopic and chemical analyses, ind eed, show that one of the three heme prosthetic groups in the enzyme, heme b(595), is specifically perturbed and mostly missing from this mutant. Surp risingly, heme d, while known to interact with heme b(595), appears relativ ely unperturbed, whereas the low-spin heme b(558) shows some modification. This is the first report of a mutation that specifically affects the bindin g site of heme b(595).