The pathway of O2 to the active site in heme–copper oxidases

Abstract 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. Time-resolved optical absorption studies show that the rate of O 2 and NO binding in the bovine enzyme (1 × 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 μs) of CO from Cu B + . In contrast, in Thermus 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 × 10 9 to 1 × 10 8  M − 1  s − 1 ), but is still considerably faster (~ 10 μs at 1 atm O 2 ) than the CO off-rate from Cu B 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 ba 3 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 Cu B + in a concerted manner through steric and/or electronic effects. This would allow Cu B + to function as an electron donor during the fast (5 μs) 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.