Studies on the Energy-coupling Sites of Photophosphorylation

Abstract

The rate of Hill reaction can be measured accurately as O₂ uptake (the Mehler reaction) if a rapidly autoxidizable electron acceptor (e.g., methylviologen) is used. However, when an artificial electron donor-ascorbate couple (or ascorbate alone) replaces the natural donor, water, the rate of O₂ consumption is no longer a reliable measure of the electron flux, because superoxide radical reactions contribute to O₂ uptake. Such radical reactions, however, can be suppressed by adding enough superoxide dismutase to the reaction mixture. Indeed in all of the photosystem I- and photosystem II-donor reactions tested (except with benzidine which was tested without ascorbate added), the O₂ uptake was inhibited by 30 to 50% by the addition of superoxide dismutase. The rate of phosphorylation was totally unaffected by the enzyme. The reasessment of the phosphorylation efficiencies thus made by the use of superoxide dismutase led us to the following conclusions. The phosphorylation efficiency associated with the transfer of electrons from a donor to methlylviologen (than to O₂) through both photosystems II and I is practically independent of the donor used—catechol, benzidine, p-aminophenol, dicyanohydroquinone, or water. The P/e₂ ratio is 1.0 ± 0.1. Only ascorbate gives a slightly lower value (P/e₂ = 0.9). (NH₂OH-treated, non-water-splitting chloroplasts were used for reactions with these artificial donors.) The phosphorylation efficiency associated with DCMU-insensitive, photosystem I-mediated transfer of electrons from a donor to methylviologen (then to O₂) is again largely independent of the donor used, such as diaminodurene, diaminotoluene, and reduced 2,6-dichlorphenol-indophenol. The P/e₂ ratio is 0.6 ± 0.08.