Cytochrome bd is one of the two quinol oxidases in the respiratory chain of Escherichia coli. The enzyme contains three heme prosthetic groups. The dioxygen binding site is heme d, which is thought to be part of the heme-heme binuclear center along with heme b595, which is a high- spin heme whose function is not known. Protein sequence alignments [Osborne, J. P., and Gennis, R. B. (1999) Biochim. Biophys Acta 1410, 32-50] of cytochrome bd quinol oxidase sequences from different microorganisms have revealed a highly conserved sequence (GWXXXEXGRQPW; bold letters indicate strictly conserved residues) predicted to be on the periplasmic side of the membrane between transmembrane helices 8 and 9 in subunit I. The functional importance of this region is investigated in the current work by site-directed mutagenesis. Several mutations in this region (W441A, E445A/Q, R448A, Q449A, and W451A) resulted in a catalytically inactive enzyme with abnormal UV-vis spectra. E445A was selected for detailed analysis because of the absence of the absorption bands from heine b595. Detailed spectroscopic and chemical analyses, indeed, show that one of the three heine prosthetic groups in the enzyme, heme b595, is specifically perturbed and mostly missing from this mutant. Surprisingly, heme d, while known to interact with heme b595, appears relatively unperturbed, whereas the low-spin heme b558 shows some modification. This is the first report of a mutation that specifically affects the binding site of heme b595.
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