title: Femtosecond resolution of ligand-heme interactions in the high-affinity quinol oxidase bd: A di-heme active site?
abstract: 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 femtosecond 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 cytochrome 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 recombination is observed, in the mixed-valence CO-liganded complex with heme b(595) initially oxidized, a significant part of photodissociated 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 b(595) and corroborate the possibility of a functional cooperation between the two hemes in the dioxygen-reducing center of cytochrome bd.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale U451, Laboratoire d'Optique Appliquée, Ecole Polytechnique-Ecole Nationale Supérieure des Techniques Avancées, 91761 Palaiseau Cedex, France. vos@enstay.ensta.fr
authors.forename: M H
authors.initials: MH
authors.lastname: Vos
authors.forename: V B
authors.initials: VB
authors.lastname: Borisov
authors.forename: U
authors.initials: U
authors.lastname: Liebl
authors.forename: J L
authors.initials: JL
authors.lastname: Martin
authors.forename: A A
authors.initials: AA
authors.lastname: Konstantinov
mesh_terms: D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry; D017936:Cytochrome a Group / Q000737:chemistry; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry; D045263:Cytochromes a1; D045222:Electron Transport Chain Complex Proteins; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006418:Heme / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D013053:Spectrophotometry
chemical_list: D017936:Cytochrome a Group; D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D006418:Heme; C080647:heme d; D002248:Carbon Monoxide; D045263:Cytochromes a1; D010088:Oxidoreductases; C020094:duroquinol oxidase; C040077:cytochrome bd terminal oxidase complex, E coli