Heme/heme redox interaction and resolution of individual optical absorption spectra of the hemes in cytochrome bd from Escherichia coli

Cytochrome bd is a terminal component of the respiratory chain of Escherichia coli catalyzing reduction of molecular oxygen to water. it contains three hemes, b(558), b(595), and d. The detailed spectro-electrochemical redox titration and numerical modeling of the data reveal significant redox interaction between the low-spin heme b(558) and high-spin heme b(595), whereas the interaction between heme d and either hemes b appears to be rather weak. However, the presence of heme d itself decreases much larger interaction between the two hemes b. Fitting the titration data with a model where redox interaction between the hemes is explicitly included makes it possible to extract individual absorption spectra of all hemes. The alpha- and beta-band reduced-minus-oxidized difference spectra agree with the data published earlier ([22] J.G. Koland, MJ. Miller, R.B. Gennis, Potentiometric analysis of the purified cytochrome d terminal oxidase complex from Escherichia coli, Biochemistry 23 (1984) 1051-1056., and [23] R.M. Lorence, J.G. Koland, R.B. Gennis, Coulometric and spectroscopic analysis of the purified cytochrome d complex of Escherichia coli: evidence for the identification of cytochrome a(1) as cytochrome b(595), Biochemistry 25 (1986) 2314-2321.). The Soret band spectra show lambda(max) = 429.5 nm, lambda(min) approximate to 413 nm (heme b(558)), lambda(max) = 439 nm, lambda(min) approximate to 400 +/- 1 nm (heme b(595)), and lambda(max) = 430 nm, lambda(min) = 405 nm (heme d). The spectral contribution of heme d to the complex Soret band is much smaller than those of either hemes b; the Soret/alpha (Delta A(430):Delta A(629)) ratio for heme d is 1.6. (C) 2009 Elsevier B.V. All rights reserved.