New transients in the electron-transfer dynamics of photolyzed mixed-valence CO-cytochrome c oxidase.

Abstract

Electron transfer following photolysis of CO from mixed-valence (cytochrome a3+ Cu2+A cytochrome a2+3-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 approximately 520 nm and in the UV region centered at approximately 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 alpha. 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 approximately 2 microseconds) 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 accompanying the oxidation of Cu+B to Cu2+B. The absorbance increase at 390 nm is also partially attributed to this transition. These results suggest that Cu2+B may be observed spectrophotometrically in the electron-transfer dynamics of cytochrome oxidase.