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

摘要

Interaction of the two high-spin hemes in the oxygen reduction site of the bd-type quinol oxidase from Escherwhia coli has been studied by femtosecond multicolor transient absorption spectros- copy. 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 cyto- chrome 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 absorption changes induced by photodissociation of CO from heme d.In the reduced enzyme, CO photodissociation from heme d perturbs the spectrum of ferrous cytochrome b_595 within a few ps, pointing to a direct interaction between hemes b_595 and d. Whereas in the reduced enzyme no heme d-CO geminate recom- bination is observed, in the mixed-valence CO-liganded complex with heme bs95 initially oxidized, a significant part of photodisso- ciated CO does not leave the protein and recombines with heme d within a few hundred ps. This 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 dissociated ligand leaves the protein. Taken together, the data indicate physical proximity of the hemes d and bs9s and corroborate the possibility of a functional cooperation between the two hemes in the dioxy- gen-reducing center of cytochrome bd.