Femtosecond resolution of ligand-heme interactions in the high-affinity quinol oxidase bd: A di-heme active site?

Interaction of the two high-spin hemes in the oxygen reduction site of the bd-type quinol oxidase from Escherichia coli has been studied by femtosecon d multicolor transient absorption spectroscopy. The previously unidentified Soret band of ferrous heme b(595) was determined to be centered around 440 nm by selective excitation of the fully reduced unliganded or CO-bound cyt ochrome bd in the alpha-band of heme b(595). The redox state of the b-type hemes strongly affects both the line shape and the kinetics of the absorpti on changes induced by photodissociation of CO from heme d. In the reduced e nzyme, CO photodissociation from heme d perturbs the spectrum of ferrous cy tochrome b(595) within a few ps, pointing to a direct interaction between h emes b(595) and d. Whereas in the reduced enzyme no heme d-CO geminate reco mbination is observed, in the mixed-valence CO-liganded complex with heme b (595) initially oxidized, a significant part: of photodissociated CO does n ot leave the protein and recombines with heme d within a few hundred ps. Th is caging effect may indicate that ferrous heme b(595) provides a transient binding site for carbon monoxide within one of the routes by which the dis sociated ligand leaves the protein. Taken together, the data indicate physi cal proximity of the hemes d and b(595) and corroborate the possibility of a functional cooperation between the two hemes in the dioxygen-reducing cen ter of cytochrome bd.