Photophosphorylation accompanying the hill reaction with ferricyanide

Abstract

A study of the dependence upon pH of ferricyanide photoreduction revealed that the reaction was most rapid but least stable at high pH (above pH 8) and that inadequate buffering could readily lead to differences in rate due to pH changes. Since the occurrence of phosphorylation may be associated with a pH change, precautions are necessary to prevent artifacts due to pH when determining the effect of phosphorylation on Hill reaction rates. Depending upon circumstances one may overestimate or underestimate the effect. For this reason a reappraisal has been made of the relationship between photophosphorylation and the photoreduction of ferricyanide as catalyzed by spinach chloroplasts. A study of the stoichiometry of photophosphorylation in relation to the Hill reaction revealed that the quantity of ATP formed approached a maximum of 1 mole for 2 moles of ferricyanide reduced and for 0.5 mole of O 2 evolved, in agreement with previous reports from other laboratories. The effects of concomitant phosphorylation upon rates of ferricyanide photoreduction were smaller than those reported previously by other workers. They may best be described as an inhibition of ferricyanide photoreduction by added ADP and the relief of this inhibition by added phosphate. When phosphate was added to the ADP-inhibited system the extra ferricyanide reduced was equal to the amount of phosphate consumed. A scheme for the mechanism of the Hill reaction which can accommodate all of the observed stoichiometry is presented and discussed. It is pointed out that the only mechanism for the Hill reaction which can easily be brought into congruence with this scheme appears to be that proposed by Warburg.