The pathway of O-2 to the active site in heme-copper oxidases

The route of O-2 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 Cu-B(+) on the pathway to and from the high-spin heme. The presence of CO on Cu-B(+) suggests that O-2 binding may be compromised in CO flow-flash experiments. Timeresolved optical absorption studies show that the rate of O-2 and NO binding in the bovine enzyme (1 x 10(8)M(-1) s(-1)) is unaffected by the presence of CO, which is consistent with the rapid dissociation (t(1/2) = 1.5 mu s) of CO from Cu-B(+) In contrast, in Therm us thermophilus (Tt) cytochrome ba(3) the O-2 and NO binding to heme a(3) slows by an order of magnitude in the presence of CO (from 1 x 10(9) to 1 x 10(8) M-1 s(-1)), but is still considerably faster (similar to 10 mu s at 1 atm O-2) than the CO off-rate from CUB in the absence of O-2 (milliseconds). These results show that traditional CO flow-flash experiments do not give accurate results for the physiological binding of O-2 and NO in Tt ba(3), 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 Cu-B(+) impedes the binding of O-2 to Cu-B(+) or, if O-2 does not bind to Cu-B(+) prior to heme a(3), whether the Cu-B(+)-CO complex sterically restricts access of O-2 to the heme. Both possibilities are discussed, and we argue that O-2 binds directly to heme a(3) in Tt ba(3), causing CO to dissociate from Cult in a concerted manner through steric and/or electronic effects. This would allow Cult to function as an electron donor during the fast (5 mu s) breaking of the O-O bond. These results suggest that the binding of CO to Cu-B(+) on the path to and from heme a(3) may not be applicable to O-2 and NO in all heme-copper oxidases. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems.