Reaction of CO with cytochrome c oxidase. Titration of the reaction site with chemical oxidant and reductant

The number of reducing equivalents required to form the reduced cytochrome a₃-CO compound has been determined for suspensions of submitochondrial particles and for isolated cytochrome c oxidase. Anaerobic preparations were titrated reductively with NADH and oxidatively with O₂ in the presence of high concentrations of CO (0.4 to 0.8 mM) while monitoring reduction of cytochrome a and the formation of the reduced cytochrome a₃-CO compound by their characteristic absorbance changes. Analysis of the titration data show that 2.0±0.3 and 2.1±0.2 reducing equivalents per mol of cytochrome oxidase (per cytochrome a) are required for formation of the reduced cytochrome a₃-CO compound in submitochondrial particles and isolated cytochrome c oxidase, respectively. In each case, the formation of the CO compound is proportional to the number of equivalents accepted by the preparation, indicating that the two equivalents are equal and the effective n value for the reaction is 2.0. Potentiometric titrations of cytochrome c oxidase using the cobalt orthophenanthrolene complex (E_{m, 7.0} = 0.37 V) as mediator give the same half-reduction potential values for cytochrome a and a₃ as those obtained using the ferro-ferricyanide couple. The formation of the reduced cytochrome a₃-CO compound at pH 7.0, in the presence of 0.6 mM CO and with CO-orthophenanthrolene as mediator occurs with a half-reduction potential of 0.45 V and requires two electrons. These data confirm and extend the observation of Lindsay et al. (Arch. Biochim. Biophys. (1975) 169, 492–505) that both the “invisible” copper and cytochrome a₃ must be reduced in order for CO to bind with high affinity.