Cytochrome c2 and reaction center of Rhodospeudomonas spheroides Ga. membranes. Extinction coefficients, content, half-reduction potentials, kinetics and electric field alterations

Abstract

1. The reduced minus oxidized extinction coefficients ( Δϵ red-ox) of reaction center P605 when in the chromatophore is about 20% smaller than in the detergent-isolated state. Presumably the coupling of the reaction center protein to the antenna bacteriochlorophylls and carotenoids causes this hypochromism. The chromatophore values for P605 are 19.5 mM −1 · cm −1 with the spectrophotometer on single beam mode at 605 nm, and 29.8 mM −1 · cm −1 on dual wavelength mode set at 605 – 540 nm. Cytochrome c 2, which is not affected by detergent, has a Δϵ red-ox value at 550-540 nm of 19.0 mM −1 · cm −1. 2. The total bacteriochlorophyll to reaction center bacteriochlorophyll protein (P) ratio is about 100 : 1. The cytochrome c 2: reaction center protein ratio approaches 2. In current French press chromatophore preparations, about 70% of the reaction centers are each associated on a rapid kinetic basis with two cytochrome c 2 molecules (intact P- c 2 units). The remaining reaction center proteins are not associated with cytochrome c 2 on a kinetically viable basis and may be the result of damage incurred during mechanical rupture of the cells. 3. The half-reduction potential of cytochrome c 2 in the isolated state is 345 mV. In the chromatophore, two electrochemical species of cytochrome c 2 are recognized. The majority has a value of approx. 295 mV and is identifiable with cytochrome c 2 in a reaction center protein-associated state (kinetically active, intact P- c 2 units); the remainder has an approx. 350 mV half-reduction potential and is probably cytochrome c 2 in the “free” or reaction center-dissociated state (possibly from damaged P- c 2 units). It appears that there is no exchange of cytochrome c 2 between the reaction center-associated and the reaction center-dissociated state. 4. The half-reduction potential of cytochrome c 2 is pH independent (from pH 5 to 9) whether measured in the free state or when associated with the chromatophore membrane. This shows that a proton is not involved in the oxidation and reduction of cytochrome c 2 in the physiological pH range. 5. The kinetics of the intact reaction center, P, and cytochrome c 2 units in chromatophores and whole cells of Rhodopseudomonas spheroides are described. The two cytochrome c 2 molecules which are associated with one P exhibit similar oxidation kinetics; both are biphasic. The fast phase is estimated to be 20–40 μs in half time. The second slower phase is variable depending on the ionic strength of the medium used for the preparation of the chromatophores; it varies from 0.3 to 8 ms. 6. An equilibrium for cytochrome c 2 and the reaction center and/or the membrane is suggested. The two states of the equilibrium are described by a population of cytochrome c 2 functionally “close” to the P +, and a population functionally distant from the P +, which might be physically off the binding site, or orientated unfavorably to the P +. The former population is identified by the 20–40 μs oxidation rate; the latter variable and somewhat slower oxidation (0.3–8 ms) is that whose rate is governed by the diffusional processes of the equilibrium which brings the cytochrome to the close position. 7. Carotenoid bandshifts are kinetically compatible (a) with the P oxidation which is too fast to measure, and (b) with the two phases of cytochrome c 2 oxidation. These are interpreted as arising from local electric field alterations occurring during the electron transfer events in the reaction center and cytochrome c 2.