The Kinetics of Reactions in and Near the Cytochrome b/f Complex of Chloroplast Thylakoids. I. Proton Deposition

Abstract

The kinetics of proton deposition in the intrathylakoid spaces of pea chloroplasts were measured under a wide range of conditions. With duroquinol added to reduce the plastoquinone pool, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea added to inhibit photosystem II, but no ionophore present, the proton deposition, attributed to plastoquinol oxidation, was biphasic. About half the deposition had an apparent rate constant (k) of 150-200 s^-1, the other half about 10 s^-1. Valinomycin or nonactin (<0.1 µM) plus potassium ions made the deposition almost monophasic, with k = 140 s^-1.

When the state of reduction of the plastoquinone pool was varied by the addition of varied concentrations of duroquinol, in the presence of 1 µM nonactin, k for proton deposition varied from about 20 (0.01 mM duroquinol) up to a maximum of 140 s^-1 (0.5 mM duroquinol).

When temperature was varied between 4 and 23°C, with 1 µM nonactin, an Arrhenius plot of ln(k) for proton deposition was linear; the activation enthalpy was 67 kJ mol^-1, the entropy of activation, 23 J K^-1 mol^-1.

The data are analysed in terms of a bimolecular reaction between a varying concentration of plastoquinol and a fixed concentration of oxidised Rieske centre. The results are consistent with a rate constant, for the first electron donation by plastoquinol, of 28 s^-1 (the rate-limiting step), followed by a relatively fast second electron donation to cytochrome b563 (low potential), followed by deposition of two protons. The speed of the second proton deposition is dependent on the membrane potential difference.