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

A detailed study is presented of the room-temperature absorption, natural and magnetic circulation-dichroism (c.d. and m.c.d.) 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 c.d. band peaking at 1100nm arising from low-spin ferrous haem a and a positive m.c.d. peak at 780nm assigned to high-spin ferrous haem a3. Addition of cyanide ion doubles the intensity of the low-spin ferrous haem c.d. band and abolishes reduced carbonmonoxy derivative the haem a32+-CO group shows no c.d. or m.c.d. bands at wavelengths longer than 700nm. A comparison of the m.c.d. spectra of the oxidized and cyanide-bound oxidized forms enables bands characteristic of the high-spin ferric form of haem a33+ to be identified between 700 and 1300nm. At wavelengths longer than 1300nm a broad positive m.c.d. spectrum, peaking at 1600nm, is observed. By comparison with the m.c.d. spectrum of an extracted haem a-bis-imidazole complex this m.c.d. peak is assigned to one low-spin ferric haem, namely haem a3+. On binding of cyanide to the oxidized form of the enzyme a new, weak, m.c.d. signal appears, which is assigned to the low-spin ferric haem 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 haem a2+ is detected by means of an intense negative c.d. peak at 1100 nm and low-spin ferric haem a33+-CN gives a sharp positive m.c.d. peak at 1550nm. The c.d. and m.c.d. characteristics of the 830nm absorption band in oxidized cytochrome c oxidase are not typical of type 1 blue cupric centres.