Evaluation of the buffer capacity and permeability constant for protons in chromatophores from Rhodobacter capsulatus

The kinetics of decay in the dark of the transmembrane pH difference (ΔpH) induced by light in non‐phosphorylating chromatophores of Rhodobacter capsulatus were studied using the fluorescent probe 9‐aminoacridine, in the presence of 50 mM KCl and 2μM valinomycin. The transient fluorescence changes induced by acid to base transitions of chromatophore suspensions were used as an empirical calibration [Casadio, R. & Melandri, B. A. (1985) Arch. Biophys. Biochem. 238, 219–228]. The kinetic competence of the probe response was tested by accelerating the Δφ decay with the ionophore nigericin. The time course in the dark of the increase in the internal pH in pre‐illuminated chromatophores was analyzed on the basis of a model which assumes a certain number of internal buffers in equilibrium with the free protons and a diffusion‐controlled H+ efflux [Whitmarsh, J. (1987) Photosynt. Res. 12, 43–62]. This model was extended to include the effects of the transmembrane electric potential difference on the H+ efflux. The diffusion constant for proton efflux was measured at different values of the internal pH by evaluating the frequency of trains of single‐turnover flashes capable of maintaining different Δφ in a steady state. The steady‐state equation derived from the model does not include any parameter relative to the internal buffers and allows unequivocal determination of the diffusion constant on the basis of the known H+/e− ratio (equal to two) for the active proton translocation by the bacterial photosynthetic chain. A value for the first‐order diffusion constant corresponding to a permeability coefficient, PH= 0.2 μm · s−1, was obtained at an external pH of 8.0; this value was constant for an internal pH ranging over 7.0–4.7. Using the value of the diffusion constant determined experimentally, a satisfactory fitting of the kinetics of Δφ decay in the dark could be obtained when the presence of two internal buffers (with pK values of 3.6 and 6.7, respectively) was assumed. For these calculations, the time course of the transmembrane electric potential difference was evaluated from the electrochromic signal of carotenoids, calibrated with K+‐induced diffusion potentials. The two internal buffers, suitable for modelling the behaviour of the system, were at concentrations of 250 mM (pK= 3.6) and 24 mM (pK= 6.7) respectively. The ability of the chromatophore suspension to store free and bound protons in the internal compartment, evaluated theoretically from the model on the basis of the above concentrations and pK values of the internal buffers, was compared with the quantity of protons disappearing from the outer suspending buffer measured with a glass electrode. Different steady states for the internal pH and for proton uptake were obtained in continuous light, adding subsaturating concentrations of nigericin. A close agreement between these two sets of data could be demonstrated. From the results of this study, a complete phenomenological description of the properties of the H+‐binding groups present in the internal compartment of chromatophores can be obtained. This allows a quantitative estimate of the quantify of inner bound protons as a function of the internal pH, information useful for evaluating the storage capacity for chemiosmotic energy of this system. Copyright © 1990, Wiley Blackwell. All rights reserved