Reconstruction of absolute absorption spectrum of reduced heme a in cytochrome c oxidase from bovine heart

Abstract

This paper presents a new experimental approach for determining the individual optical characteristics of reduced heme a in bovine heart cytochrome c oxidase starting from a small selective shift of the heme a absorption spectrum induced by calcium ions. The difference spectrum induced by Ca2+ corresponds actually to a first derivative (differential) of the heme a(2+) absolute absorption spectrum. Such an absolute spectrum was obtained for the mixed-valence cyanide complex of cytochrome oxidase (a(2+)a3(3+)-CN) and was subsequently used as a basis spectrum for further procession and modeling. The individual absorption spectrum of the reduced heme a in the Soret region was reconstructed as the integral of the difference spectrum induced by addition of Ca2+. The spectrum of heme a(2+) in the Soret region obtained in this way is characterized by a peak with a maximum at 447 nm and half-width of 17 nm and can be decomposed into two Gaussians with maxima at 442 and 451 nm and half-widths of ~10 nm (589 cm(-1)) corresponding to the perpendicularly oriented electronic π→π* transitions B0x and B0y in the porphyrin ring. The reconstructed spectrum in the Soret band differs significantly from the "classical" absorption spectrum of heme a(2+) originally described by Vanneste (Vanneste, W. H. (1966) Biochemistry, 65, 838-848). The differences indicate that the overall γ-band of heme a(2+) in cytochrome oxidase contains in addition to the B0x and B0y transitions extra components that are not sensitive to calcium ions, or, alternatively, that the Vanneste's spectrum of heme a(2+) contains significant contribution from heme a3(2+). The reconstructed absorption band of heme a(2+) in the α-band with maximum at 605 nm and half-width of 18 nm (850 cm(-1)) corresponds most likely to the individual Q0y transition of heme a, whereas the Q0x transition contributes only weakly to the spectrum.