The Reactions of O and NO with Mixed-Valence Ba Cytochrome C Oxidase from Thermus Thermophilus

Earlier CO flow-flash experiments on the fully reduced Thermus thermophilus ba (Tt ba) cytochrome oxidase revealed that O binding was slowed down by a factor of 10 in the presence of CO (Szundi et al., 2010, PNAS 107, 21010-21015). The goal of the current study is to explore whether the long apparent lifetime (∼50 ms) of the Cu-CO complex generated upon photolysis of the CO-bound mixed-valence Tt ba (Koutsoupakis et al., 2019, Acc. Chem. Res. 52, 1380-1390) affects O and NO binding and the ability of Cu to act as an electron donor during O-O bond splitting. The CO recombination, NO binding, and the reaction of mixed-valence Tt ba with O were investigated by time-resolved optical absorption spectroscopy using the CO flow-flash approach and photolabile O and NO carriers. No electron backflow was detected after photolysis of the mixed-valence CO-bound Tt ba. The rate of O and NO binding was two times slower than in the fully reduced enzyme in the presence of CO and 20 times slower than in the absence of CO. The purported long-lived Cu-CO complex did not prevent O-O bond splitting and the resulting P formation, which was significantly faster (5-10 times) than in the bovine heart enzyme. We propose that O binding to heme a in Tt ba causes CO to dissociate from Cu in a concerted manner through steric and/or electronic effects, thus allowing Cu to act as an electron donor in the mixed-valence enzyme. The significantly faster O binding and O-O bond cleavage in Tt ba compared to analogous steps in the aa oxidases could reflect evolutionary adaptation of the enzyme to the microaerobic conditions of the T. thermophilus HB8 species.