The kinetic and redox potentiometric resolution of the carotenoid shifts in Rhodopseudomonas spheroides chromatophores: Their relationship to electric field alterations in electron transport and energy coupling

Abstract

1. 1. Three principal phases of the carotenoid band shift in Rhodopseudomonas spheroides chromatophores elicited by a single-turnover flash can be resolved both kinetically and potentiometrically. Phase I (complete in <1 μs) is apparent over the redox potential limits of the light reaction, i.e. the potential range in which reaction centre bacteriochlorophyll is reduced and the primary electron acceptor oxidized before the flash; thus the band shift is consistent with its response to the formation of P +X −. Phase II (about 25% of the amplitude of Phase I) with an approximate 0.15-ms half time is observed if cytochrome c ( E m7.2 + 295 mV) is chemically reduced before the flash and hence may be in response to the photooxidation of cytochrome c and re-reduction of P +. A much slower phase (PHase III) can also be detected at positive potentials. It is enhanced both in extent and formation rate ( t 1 2 > 5 ms to about 1 ms) over the 150 mV potential range in which cytochrome b 155 ( E m7.2 + 155 mV) becomes chemically reduced. Between 50 and 100 mV this phase is approx. 80–100% of the extent of Phase I. Phase III is abolished by antimycin A as are the oxidation of cytochrome b 155 and the re-reduction of photooxidized cytochrome c. All phases are additive. Thus the formation of the carotenoid band shift is in response to pulsed electron transfer events. Further, using multiple, one-turnover flashes, the extent following each flash, and behaviour of the formation the carotenoid band shift can be clearly explained in terms of the electron flow patterns in the chromatophore. 2. 2. The decay of the carotenoid band shift is stimulated by agents such as the uncoupler carbonyl cyanide p-trifluoromethylphenylhydrazone, and the potassium ionophore valinomycin, irrespective of the suspension redox potential. The decay half time, which after five flashes is in the region of 500 ms without addition, can be reduced to < 1 ms by addition of the membrane ion carriers. The kinetic behaviour of cytochrome c is used to demonstrate that the course of carotenoid band shift decay has no obligate relationship to the redox state or electron transfer events of the electron carriers. 3. 3. The carotenoid band shifts appear to be generated by electrostatic field alterations resulting from oxidation-reduction reactions between adjacent electron transport carriers. Once formed, the retention of the fields is a function of membrane ion permeability. The location of the field-forming reactions, with respect to the kinetically and thermodynamically defined spans of electron transport, and to their topology within the membrane, is discussed.