NEW TRANSIENTS IN THE ELECTRON-TRANSFER DYNAMICS OF PHOTOLYZED MIXED-VALENCE CO-CYTOCHROME-C OXIDASE

Electron transfer following photolysis of CO from mixed-valence (cytochrome a3+ Cu(A)2+ cytochrome a3(2+)-CO Cu(B)+) cytochrome oxidase (ferrocytochrome-c; oxygen oxidoreductase, EC 1.9.3.1) was studied on time scales of nanoseconds to milliseconds by multichannel time-resolved optical absorption spectroscopy. In this method, the optical absorption was measured at many wavelengths simultaneously by using an optical spectrometric multichannel analyzer system. The high-quality time-resolved difference spectra showed a large increase on a microsecond time scale in the visible region centered at almost-equal-to 520 nm and in the UV region centered at almost-equal-to 390 nm. These absorbance changes were not observed after photodissociation of CO from the fully reduced enzyme and therefore are attributed to intramolecular electron transfer. Simultaneously, there was a blue shift and a small increase in the alpha-band, which is attributed to the reduction of cytochrome a. Approximately one-third of the absorbance change at 520 nm can be attributed to reduction of cytochrome a. The absorbance changes associated with the 520- and the 390-nm bands are on the same time scale (t1/2 almost-equal-to 2-mu-s) as the dissociation of CO from Cu(B)+ reported previously by time-resolved infrared spectroscopy. The position and shape of these bands are reasonable for charge-transfer transitions involving copper(II). We suggest that the absorbance increase at 520 nm, which cannot be attributed to a reduction of cytochrome a, may represent a charge transfer involving Cu2(B)2+ accompanying the oxidation of Cu(B)+ to Cu(B)2+ The absorbance increase at 390 nm is also partially attributed to this transition. These results suggest that Cu(B)2+ may be observed spectrophotometrically in the electron-transfer dynamics of cytochrome oxidase.