title: The pathway of O₂to the active site in heme-copper oxidases.
abstract: The route of O₂to and from the high-spin heme in heme-copper oxidases has generally been believed to emulate that of carbon monoxide (CO). Time-resolved and stationary infrared experiments in our laboratories of the fully reduced CO-bound enzymes, as well as transient optical absorption saturation kinetics studies as a function of CO pressure, have provided strong support for CO binding to CuB⁺ on the pathway to and from the high-spin heme. The presence of CO on CuB⁺ suggests that O₂binding may be compromised in CO flow-flash experiments. Time-resolved optical absorption studies show that the rate of O₂and NO binding in the bovine enzyme (1 × 10⁸M⁻¹s⁻¹) is unaffected by the presence of CO, which is consistent with the rapid dissociation (t½ = 1.5μs) of CO from CuB⁺. In contrast, in Thermus thermophilus (Tt) cytochrome ba3 the O₂and NO binding to heme a3 slows by an order of magnitude in the presence of CO (from 1 × 10⁹ to 1 × 10⁸M⁻¹s⁻¹), but is still considerably faster (~10μs at 1atm O₂) than the CO off-rate from CuB in the absence of O₂(milliseconds). These results show that traditional CO flow-flash experiments do not give accurate results for the physiological binding of O₂and NO in Tt ba3, namely, in the absence of CO. They also raise the question whether in CO flow-flash experiments on Tt ba3 the presence of CO on CuB⁺ impedes the binding of O₂to CuB⁺ or, if O₂does not bind to CuB⁺ prior to heme a3, whether the CuB⁺-CO complex sterically restricts access of O₂to the heme. Both possibilities are discussed, and we argue that O₂binds directly to heme a3 in Tt ba3, causing CO to dissociate from CuB⁺ in a concerted manner through steric and/or electronic effects. This would allow CuB⁺ to function as an electron donor during the fast (5μs) breaking of the OO bond. These results suggest that the binding of CO to CuB⁺ on the path to and from heme a3 may not be applicable to O₂and NO in all heme-copper oxidases. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA. Electronic address: olof@ucsc.edu.
authors.forename: Olöf
authors.initials: O
authors.lastname: Einarsdóttir
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA.
authors.forename: William
authors.initials: W
authors.lastname: McDonald
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA.
authors.forename: Chie
authors.initials: C
authors.lastname: Funatogawa
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA.
authors.forename: Istvan
authors.initials: I
authors.lastname: Szundi
authors.affiliation: Santa Fe Institute, 1399 Hyde Park Road, Santa Fe NM 87501.
authors.forename: William H
authors.initials: WH
authors.lastname: Woodruff
authors.affiliation: Department of Chemistry, Emory University, Atlanta, GA 30322, USA.
authors.forename: R Brian
authors.initials: RB
authors.lastname: Dyer
mesh_terms: D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism; D020134:Catalytic Domain; D002417:Cattle; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D010088:Oxidoreductases / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared; D016963:Thermus thermophilus / Q000378:metabolism
chemical_list: D006418:Heme; D002248:Carbon Monoxide; D010088:Oxidoreductases; C024748:copper oxidase; D010100:Oxygen
keywords: CO photodissociation and recombination dynamics; Photolabile O(2) and NO complex; Thermus thermophilus ba(3); Time-resolved infrared linear dichroism; Time-resolved infrared spectroscopy