Cyanide-binding Site of bd-type Ubiquinol Oxidase from Escherichia coli

Abstract

We extended our investigation on the structure of the redox centers of bd-type ubiquinol oxidase from Escherichia coli using cyanide as a monitoring probe. We found that addition of cyanide to the air-oxidized O 2 -bound enzyme caused appearance of an infrared C-N stretching band at 2161 cm

Discovery of the True Peroxy Intermediate in the Catalytic Cycle of Terminal Oxidases by Real-time Measurement

“…This is in line with the proposal of physical proximity of hemes d and b 595 and their functional cooperation in the O 2 -reducing site (31)(32)(33)(34)(35)(36)(37).…”

A Cytochrome bb′-type Quinol Oxidase inBacillus subtilis Strain 168

“…6A). The difference spectra closely resemble those observed for the "aerobically oxidized" ("as isolated") E. coli cytochrome bd (57,58). The trough at 650 nm originates from cyanide-induced decay of the oxycomplex of heme d. The concomitant red shift in the ␥-band (trough at 410 nm, peak at 438 nm) is very similar to those observed for the purified E. coli cytochrome bd (a trough at 408 nm and a peak at 437 nm) in parallel with the cyanide-induced decomposition of the oxycomplex (cf.…”

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

“…35, evaluation of heme d content was based on the reduced minus air-oxidized difference spectrum of cytochrome bd, whereas in the current work, the absolute spectra of the reduced enzyme (using the red side of the 630-nm peak) have been used to quantify the amount of heme d. This method allows us to avoid the problems associated with (i) interference of heme b 595 at the wavelengths traditionally used to quantify heme d and (ii) the different yields of the oxyferrous species of heme d in the air-oxidized preparations of the WT and mutant cytochromes bd (see Materials and Methods for details). In addition, the heme d content of the WT and mutant oxidases, determined by using ⌬ 628 -670 for the dithionite-reduced absolute spectra, is equal to the content of heme b 558 of each enzyme (⌬ 561-580 of 21.0 mM Ϫ1 ⅐cm Ϫ1 ) for the dithionite-reduced minus air-oxidized difference spectrum (19). Hence, for the WT and mutant enzymes, the heme b 558 ͞heme d ratio is 1.0, and the total heme b͞heme d ratio is 2.0.…”

“…However the role of high-spin heme b 595 is still a matter of debate. Together with heme d, heme b 595 is proposed to form a binuclear site for the effective reduction of oxygen analogous to the high-spin heme͞ Cu B binuclear center in the heme-copper oxidases (17)(18)(19)(20)(21)(22)(23). The electron transfer sequence in cytochrome bd is thought to be QH 2 3 b 558 3 [b 595 3 d] 3 O 2 (24)(25)(26).…”