Near-infrared magnetic and natural circular dichroism of cytochrome c oxidase

Abstract

A detailed study is presented of the room-temperature absorption, natural and magnetic circular-dichroism (CD and MCD) spectra of cytochrome c oxidase and a number of its derivatives in the wavelength range 700-1900 nm. The spectra of the reduced enzyme show a strong negative CD band peaking at 1100 nm arising from low-spin ferrous heme a and a positive MCD peak at 780 nm assigned to high-spin ferrous heme a3. Addition of cyanide ion doubles the intensity of the low-spin ferrous heme CD band and abolishes the 780 nm MCD peak because heme a3 is switched to the low-spin ferrous state. In the reduced CO derivative the heme a32+-CO group shows no CD or MCD bands at wavelengths longer than 700 nm. A comparison of the MCD spectra of the oxidized and cyanide-bound oxidized forms enables bands characteristic of the high-spin ferric form of heme a33+ to be identified between 700 and 1300 nm. At wavelengths longer than 1300 nm a broad positive MCD spectrum, peaking at 1600 nm, is observed. By comparison with the MCD spectrum of an extracted heme a-bis-imidazole complex this MCD peak is assigned to 1 low-spin ferric heme, namely heme a3+. On binding of cyanide to the oxidized form of the enzyme a new, weak, MCD signal appears, which is assigned to the low-spin ferric heme a33+-CN species. A reductive titration, with sodium dithionite, of the cyanide-bound form of the enzyme leads to a partially reduced state in which low-spin heme a2 is detected by means of an intense negative CD peak at 1100 nm and low-spin ferric heme a33+-CN gives a sharp positive MCD peak at 1550 nm. The CD and MCD characteristics of the 830 nm absorption band in oxidized cytochrome c oxidase are not typical of type 1 blue cupric centers.