Kinetic distinction between cytochromes a and a3 in cytochrome c oxidase. Rapid scanning stopped flow study of anaerobic reduction by a neutral and a negatively charged donor.
Anaerobic reduction of cytochrome c oxidase by 5,10-dihydro-5-methylphenazine (reduced PMS) and by sodium dithionite were studied by rapid scanning stopped flow spectrophotometry. In both cases the decay of the Soret band of the oxidized oxidase is not uniform. With reduced PMS, the reduction involves two molecules of reductant (4 electrons)/oxidase molecule. The first stage of the reduction exhibits an isosbestic point in the Soret region at 437 nm. This shifts to 428 nm in later stages of the reaction. The reduction of the oxidase by sodium dithionite is also complete and apparently involves SO2 radical. In this case the spectra show an isosbestic point at approximately 420 nm which shifts to 432 nm as the reaction proceeds. For each of the reductants the reaction is best described by three phases: the first is a second order reaction between the oxidase and the reductant, followed by two first order processes which appear to describe the intramolecular electron redistribution within the oxidase molecule. The results agree with the assignment of the Soret band of the oxidase molecule to cytochrome a3 with an absorption maximum near 410 nm and to cytochrome a which has its maximum absorption hear 430 nm. If these assignments are correct, the present data show that reduced PMS, an uncharged molecule, reacts more rapidly with cytochrome a than it does with cytochrome a3, while the negatively charged radical anion, SO2, appears to have more direct access to cytochrome a3.
D000693:Anaerobiosis; D017936:Cytochrome a Group; D003574:Cytochrome c Group / Q000378:metabolism*; D003580:Cytochromes / Q000378:metabolism*; D004227:Dithionite; D003576:Electron Transport Complex IV / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D013053:Spectrophotometry
D017936:Cytochrome a Group; D003574:Cytochrome c Group; D003580:Cytochromes; D004227:Dithionite; D003576:Electron Transport Complex IV