Reconstruction of Absolute Absorption Spectrum of Reduced Heme a in Cytochrome c Oxidase from Bovine Heart

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A. V. Dyuba^*, T. V. Vygodina, and A. A. Konstantinov

Lomonosov Moscow State University, Belozersky Institute of Physico-Chemical Biology, 119991 Moscow, Russia; E-mail: dyubon@gmail.com; vygodina@genebee.msu.ru

^* To whom correspondence should be addressed.

Received September 13, 2013; Revision received September 20, 2013

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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 a²⁺ absolute absorption spectrum. Such an absolute spectrum was obtained for the mixed-valence cyanide complex of cytochrome oxidase (a²⁺a₃³⁺-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 a²⁺ 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 B_0x and B_0y in the porphyrin ring. The reconstructed spectrum in the Soret band differs significantly from the “classical” absorption spectrum of heme a²⁺ originally described by Vanneste (Vanneste, W. H. (1966) Biochemistry, 65, 838-848). The differences indicate that the overall γ-band of heme a²⁺ in cytochrome oxidase contains in addition to the B_0x and B_0y transitions extra components that are not sensitive to calcium ions, or, alternatively, that the Vanneste’s spectrum of heme a²⁺ contains significant contribution from heme a₃²⁺. The reconstructed absorption band of heme a²⁺ in the α-band with maximum at 605 nm and half-width of 18 nm (850 cm^–1) corresponds most likely to the individual Q_0y transition of heme a, whereas the Q_0x transition contributes only weakly to the spectrum.

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KEY WORDS: cytochrome c oxidase, Ca²⁺, heme a, absorption spectrum, spectral shift

DOI: 10.1134/S0006297913120067

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