Site-Directed Mutation of the Highly Conserved Region near the Q-Loop of the Cytochrome bd Quinol Oxidase from Escherichia coli Specifically Perturbs Heme b₅₉₅

Abstract

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 b(595), 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 heme b(595). Detailed spectroscopic and chemical analyses, indeed, show that one of the three heme prosthetic groups in the enzyme, heme b(595), is specifically perturbed and mostly missing from this mutant. Surprisingly, heme d, while known to interact with heme b(595), appears relatively unperturbed, whereas the low-spin heme b(558) shows some modification. This is the first report of a mutation that specifically affects the binding site of heme b(595).

Time-resolved electrometric and optical studies on cytochrome bd suggest a mechanism of electron-proton coupling in the di-heme active site

“…This is the E445A mutant of subunit I (CydA) of cytochrome bd from Escherichia coli (35). Previous analysis concluded that heme b 595 is either partially or completely absent from the E445A mutant (35). In the current work, it is shown that the E445A oxidase preparations actually still retain the heme, but this heme remains in the ferric state, even in the presence of a strong reductant (dithionite).…”

“…E. coli cells were obtained from GO105͞ pTK1 and GO105͞pTK1͞E445A strains (35). Both the WT and the E445A mutant cytochrome bd oxidases were purified as described (36), but the second (hydroxyapatite) chromatographic step was omitted as it can cause destabilization and substantial degradation of heme b 595 in the E445A mutant enzyme (35).…”

“…E. coli cells were obtained from GO105͞ pTK1 and GO105͞pTK1͞E445A strains (35). Both the WT and the E445A mutant cytochrome bd oxidases were purified as described (36), but the second (hydroxyapatite) chromatographic step was omitted as it can cause destabilization and substantial degradation of heme b 595 in the E445A mutant enzyme (35). Besides, in this work for solubilization of the membranes sucrose monolaurate detergent was used instead of N-dodecyl-N,N-dimethylammonio-3-propane-sulfonate used in an earlier report (11).…”

“…The conventional procedure of using the dithionitereduced minus air-oxidized difference absorption spectrum appears to be error-prone because the mutant enzyme in the air-oxidized form has a much lower amount of the ferrous oxy species. Hence, the use of the ⌬ 628 -607 value of 10.8 mM Ϫ1 ⅐cm Ϫ1 , which correctly quantifies the content of heme d in the WT enzyme (20), results in an incorrect value of heme d in the E445A mutant enzyme (35). The use of this traditional method results in overestimation of heme d in the mutant enzyme.…”

“…This is the E445A mutant of subunit I (CydA) of cytochrome bd from Escherichia coli (35). Previous analysis concluded that heme b 595 is either partially or completely absent from the E445A mutant (35).…”