The role of cytochrome b-566 in the electron-transfer chain of Rhodopseudomonas sphaeroides

Abstract

Spectrophotometric, kinetic, thermodynamic and stoichiometric properties of the low-potential b-type cytochrome of chromatophores from Rhodopseudomonas sphaeroides are reported. Cytochrome b-566 has a double α-band with maxima at 559 and 566 nm. Resolution of the spectrum by full-spectral redox potentiometry showed no indication that the two peaks represent more than one component. The component titrated with E m,7 ≈ −80 ± 10 mV. By appropriate choice of wavelength pairs and by subtraction of the contribution due to other components, the kinetics of cytochrome b-566 absorbance changes following flash excitation have been resolved from those of other components. Time-resolved flash spectra corrected for the contributions of other components are consistent with the behavior of both peaks of the α-band as a single kinetic species. The kinetics of cytochrome b-566 in the presence of antimycin show that the reduction of this cytochrome occurred only if cytochrome b-561 was reduced before the flash, either chemically, by poising the ambient redox potential ( E h) below the E m of cytochrome b-561 ( E m,7 ≈ 50 mV), or photochemically at higher redox potentials by a previous flash. The rate of reduction of cytochrome b-566 varied with E h. At low E h (approx. 0 mV) reduction on the first flash showed t 1 2 ≈ 1.25 ms ; at high E h (approx. 180 mV) reduction on the second flash showed t 1 2 ≈ 10 ms . In the absence of antimycin at E h ≈ 0 mV, cytochrome b-566 was observed to become rapidly reduced ( t 1 2 ≈ 500 μ s ) and then reoxidized ( t 1 2 ≈ 2 ms ) after a single flash. At higher redox potentials ( E h > 80 mV) no kinetic changes which could be unambiguously attributed to cytochrome b-566 were observed following a single flash. The results are interpreted in terms of a Q-cycle mechanism in which the reductant for cytochrome b-566 is the semiquinone formed on oxidation of ubiquinol from the quinone pool. The oxidation of the ubiquinol occurs by a concerted reaction in which one electron is accepted by the Rieske-type FeS center and the other by cytochrome b-566. We suggest that the kinetic characteristics may indicate a pathway for reduction of the b-type cytochromes in which cytochrome b-566 is the immediate electron acceptor and donates to cytochrome b-561 in a serial pathway. The experimental results in the presence of antimycin are compared with data from a computer simulation of the thermodynamic behavior of the chain, and the computer model is shown to provide an excellent fit.