The Kinetics of Reactions in and Near the Cytochrome b/f Complex of Chloroplasts. II. Cytochrome b-563 Reduction

Abstract

The reduction of cytochrome b-563 was measured following flash-induced electron transfer from duroquinol to methyl viologen, in the presence of 3,(3,4-dichlorophenyl)-1,1-dimethyl urea and 1 µM nonactin or 1 µM valinomycin (plus 10 mM K⁺). The apparent rate constant of this reduction (nonactin present) increased from about 100 s^-1 to 460 s^-1 as the external concentration of duroquinol was varied from 0.01 to 0.5 mM. The corresponding maximum extent of reduction of cytochrome b-563 varied from 0.13 to 0.27 molecules per b/f complex. Rate constants in the presence of valinomycin were lower at all concentrations of duroquinol by a factor of about 1.5.

The mean enthalpy of activation calculated from Arrhenius plots of apparent rate constant for cytochrome b-563 reduction was 60 kJ mol^-1, for temperature variation between 23 and 4°C.

The above, and further data in oxidising conditions, and with added 2-n-nonyl-4-hydroxyquinoline N-oxide, together with data on proton deposition, were compared with the predictions of a kinetic model. In this model, flash-generated oxidised plastocyanin oxidised Rieske centres during random diffusion, and plastoquinol reduced the Rieske centres and cytochrome b-563 sequentially; subsequent electron and proton transfers followed those in a Q-cycle.

Many observations were predicted by the model, in which the rate-limiting step was the first electron transfer from plastoquinol to the Rieske centre, subsequent steps being much faster. The rate and extent of reduction of cytochrome b-563 were fully consistent with a reaction between it and a radical form of plastoquinone formed after oxidation of the latter by Rieske centres (referred to as 'oxidant-induced reduction'), under a wide range of conditions.