The pathway of O?to the active site in heme-copper oxidases.

ABSTRACT: The route of O?to 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 O?binding may be compromised in CO flow-flash experiments. Time-resolved optical absorption studies show that the rate of O?and NO binding in the bovine enzyme (1 × 10?M?¹s?¹) is unaffected by the presence of CO, which is consistent with the rapid dissociation (t½ = 1.5?s) of CO from CuB?. In contrast, in Thermus thermophilus (Tt) cytochrome ba3 the O?and NO binding to heme a3 slows by an order of magnitude in the presence of CO (from 1 × 10? to 1 × 10?M?¹s?¹), but is still considerably faster (~10?s at 1atm O?) than the CO off-rate from CuB in the absence of O?(milliseconds). These results show that traditional CO flow-flash experiments do not give accurate results for the physiological binding of O?and 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 O?to CuB? or, if O?does not bind to CuB? prior to heme a3, whether the CuB?-CO complex sterically restricts access of O?to the heme. Both possibilities are discussed, and we argue that O?binds 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 (5?s) 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 O?and NO in all heme-copper oxidases. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems.

SUBMITTER: Einarsdottir O 

PROVIDER: S-EPMC4252355 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature