Interactions between Heme d and Heme b sub(595) in Quinol Oxidase bd from Escherichia coli: A Photoselection Study Using Femtosecond Spectroscopy

Femtosecond spectroscopy was performed on CO-liganded (fully reduced and mixed-valence states) and O sub(2)-liganded quinol oxidase bd from Escherichia coli. Substantial polarization effects, unprecedented for optical studies of heme proteins, were observed in the CO photodissociation spectra, implying interactions between heme d (the chlorin ligand binding site) and the close-lying heme b sub(595) on the picosecond time scale; this general result is fully consistent with previous work [Vos, M. H., Borisov, V. B., Liebl, U., Martin, J.-L., and Konstantinov, A. A. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1554-1559]. Analysis of the data obtained under isotropic and anisotropic polarization conditions and additional flash photolysis nanosecond experiments on a mutant of cytochrome bd mostly lacking heme b sub(595) allow to attribute the features in the well-known but unusual CO dissociation spectrum of cytochrome bd to individual heme d and heme b sub(595) transitions. This renders it possible to compare the spectra of CO dissociation from reduced and mixed-valence cytochrome bd under static conditions and on a picosecond time scale in much more detail than previously possible. CO binding/dissociation from heme d is shown to perturb ferrous heme b sub(595), causing induction/loss of an absorption band centered at similar to 435 nm. In addition, the CO photodissociation-induced absorption changes at 50 ps reveal a bathochromic shift of ferrous heme b sub(595) relative to the static spectrum. No evidence for transient binding of CO to heme b sub(595) after dissociation from heme d is found in the picosecond time range. The yield of CO photodissociation from heme d on a time scale of