Magnetic Circular Dichroism Used To Examine the Interaction ofEscherichia coliCytochromebdwith Ligands

Abstract

The interactions of the fully reduced and fully oxidized cytochrome bd from E. coli with ligands CO, NO, and CN- have been studied by a combination of absorption and magnetic circular dichroism (MCD) spectroscopy. In the reduced cytochrome bd, MCD resolves individual bands due to the high-spin heme b595 and the low-spin heme b558 components of the enzyme, allowing one to separately monitor their interactions along with ligand binding to the heme d component. The data show that at low concentrations, the ligands bind almost exclusively to heme d. At high concentrations, the ligands begin to interact with the low-spin heme b558. At the same time, no evidence for significant binding of the ligands to the high-spin heme b595 is revealed in either the reduced or the fully oxidized cytochrome bd complex. The data support the model [Borisov, V. B., Gennis, R. B., and Konstantinov, A. A. (1995) Biochemistry (Moscow) 60, 231-239] according to which the two high-spin hemes d and b595 share a high-affinity ligand binding site with a capacity for only a single molecule of the ligand; i.e., there is a strong negative cooperativity with respect to ligand binding to these two hemes with cytochrome d having an intrinsic ligand affinity much higher than that of heme b595.

Femtosecond resolution of ligand-heme interactions in the high-affinity quinol oxidase bd : A di-heme active site?

“…1 A, curve a) is characterized by an asymmetric band with a maximum at Ϸ430 nm attributed provisionally to low-spin heme b 558 (11,14) and a shoulder around 440 nm, the origin of which is controversial. The absorption changes around 440 nm induced by binding of CO or NO to reduced cytochrome bd have been assigned earlier either to heme d (29,30,32) or heme b 595 (11,23). This work, exploiting the combination of selective heme excitation and ultrafast spectroscopy, provides direct evidence that the Soret shoulder at Ϸ440 nm corresponds to heme b 595 .…”

“…Second, the picosecond curve reveals increased absorbance around 420 nm relative to the peak around 440 nm. Whereas the various reported static CO-induced difference spectra of cytochrome bd show some variation (23,29,45,46), the long wavelength extreme at 443-445 nm is invariably more intense than the short wavelength one at 425-430 nm (Fig. 4, curve a).…”

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

“…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.…”

“…In the mutant, the 630-nm peak is broader on its blue side than that in the WT, whereas the lineshapes of the red side of the peak are virtually identical in the WT and mutant enzymes (A. M. Arutyunyan, V.B.B., R.B.G., and A.A.K., unpublished work). For this reason the content of heme d for the WT and mutant enzyme was determined from the ferrous heme d absorption spectra by using the wavelength pair 630-nm minus 670-nm (⌬ 628 -670 of 25 mM Ϫ1 ⅐cm Ϫ1 ) instead of the frequently used pair 630-nm minus 607-nm (20). This extinction coefficient for measuring heme d gives the same result obtained by an independent method based on the intensity of the dithionite-reduced absolute CD spectrum in the 600-to 700-nm range (A. M. Arutyunyan, V.B.B., R.B.G., and A.A.K., unpublished work).…”

“…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).…”