Kinetic studies on cytochrome c oxidase by combined EPR and reflectance spectroscopy after rapid freezing

Participation of the iron-sulphur cluster and of the covalently bound coenzyme of trimethylamine dehydrogenase in catalysis

“…These observations suggest that the transfer of electrons to the iron-sulphur centre is accompanied by slow conformational changes in the enzyme, which bring the flavin and iron-sulphur loci in close contact with each other, or by the slow making and breaking of covalent bonds between enzyme-bound intermediates originating from the substrate and the flavin component. It is noteworthy that intramolecular electron transfer is generally not rate-limiting in complex flavoproteins (Edmondson et al, 1973;Olson et al, 1974;Beinert et al, 1976) and that, hence, the mechanism of action of this enzyme may be somewhat unique.…”

“…Low-temperature optical reflectance and e.p.r. spectra were measured and anaerobic rapid kinetic experiments by freeze-quenching were carried out and evaluated as described previously (Orme-Johnson et al, 1974a,b;Beinert et al, 1976). Specific details are given in the Figure legends.…”

The involvement of the fully oxidized state in cytochrome oxidase reaction with oxygen studied with the 655 nm band as a probe

“…We must point out that the decrease at 606 nm is accompanied also by a significant absorbance decrease at 549 nm due to oxidation of cytochrome c. We shall demonstrate elsewhere (manuscript 4. Discussion tic of the a3 form [10] we have demonstrated on the hand that the reaction with oxygen of mitochondrial cytochrome oxidase in the mixed valence state does not lead to the formation of the fully oxidized state. This result is consistent with the prominent absorbance increase observed at 606 nm [4,5,12] when Compound C forms which strongly supports a reduced state of the iron.…”

“…A dotted line has been drawn as an empirical correction [ 13] in order to give a better representation of this band. Although the Area x appears to be closer to 660 nm than 655 nm we keep the previous 655 nm band label [10] to avoid confusion. The investigation has been carried out mainly in the range -40°C to -80°C.…”

“…On the basis of their heme-heme interaction model, Wikstrom et al [8] suggested that the mechanism of oxygen reduction by cytochrome oxidase does not involve the fully oxidized form. Recently Beinert et al [10] characterized specifically the aT ÷ form with the 655 nm band that they pointed out previously by reflectance spectroscopy [ 11 ]. In this study this 655 nm band is used as a probe of the The lack of the 655 nm band has been pointed out already [12] in Compound C formation.…”