The reduction kinetics of chlorophyll aI as an indicator for proton uptake between the light reactions in chloroplasts.
The flash-induced oxidation kinetics of the primary acceptor of light Reaction II (X-320) and the reduction kinetics of chlorophyll aI (P-700) after far-red preillumination have been studied with high time resolution in spinach chloroplasts. 1. The kinetics of chlorophyll aI exhibits a pronounced lag phase of 2--3 ms at the onset of reduction as would be expected for the final product of consecutive reactions. Because the oxidation of the plastoquinone pool is the rate-limiting step for the electron transport between the two light reactions, the lag indicates the maximal electron transfer time over all preceding reactions after light Reaction II. 2. The observation that the lag phase decreases with decreasing pH is evidence of an electron transfer step coupled to a proton uptake reaction. 3. Protonation of X-320 after reduction in the flash is excluded because a slight increase of the decay time is found at decreasing pH values. 4. The time course of plastohydroquinone formation is deduced from the first derivative of the reduction kinetics of chlorophyll aI. This approach covers those plastohydroquinone molecules being available to the electron carriers of System I via the rate-limiting step. Direct measurements of absorbance changes would not allow to discriminate between these and functionally different plastohydroquinone molecules. 5. The derived time course of plastohydroquinone at different pH gives evidence for an additional electron transfer step with a half time of about 1 ms following the proton uptake and preceding the rate-limiting step. It is tentatively attributed to the diffusion of neutral plastohydroquinone across the hydrophobic core of the thylkaloid membrane. 6. The lower limit of the rate constant for proton uptake by an electron carrier, consistent with the lag of chlorophyll aI reduction, is estimated as greater than 10(11) M-1s-1. The value is higher than that of the fastest diffusion controlled protonations of organic molecules in solution. Possible mechanisms of linear electron transport between light Reaction II and the rate-limiting oxidation of neutral plastohydroquinone are thoroughly discussed.