A study with O18 of adenosine triphosphate formation in oxidative phosphorylation.

In a previous communication from this laboratory (1) a reaction of inorganic phosphate with water was described which occurred during phosphorylation coupled to oxidation in the electron transport system in liver mitochondria. This reaction was followed by observing the replacement of 018 in labeled inorganic phosphate by normal oxygen from water concomitant with the occurrence of phosphorylation. The results indicated that this inorganic phosphate-oxygen replacement reaction was intimately related to the phosphorylation mechanism since it occurred only when phosphorylation associated with the electron transport system was proceeding and was suppressed by the same means as phosphorylation, for example addition of 2 ,hdinitrophenol. In order to gain further insight into the mechanism of oxidative phosphorylation, the present study was undertaken to follow simultaneously the kinetics of this inorganic phosphate-oxygen replacement reaction and of the transfer of the oxygen-labeled inorganic phosphate to adenosine triphosphate (ATP).l The results indicate that, in addition to the inorganic phosphate-oxygen replacement reaction already described, there is a further replacement of oxygen in the sequence of reactions leading to the formation of ATP in oxidative phosphorylation. The same pattern has been observed in our experiments on the inorganic phosphate-ATP exchange reaction described by Boyer and coworkers (2); namely, more oxygen has been replaced in the phosphate transferred to ATP than in the inorganic phosphate itself. The present study includes a comparison of the kinetics of the inorganic phosphate-oxygen replacement reaction and the transfer reaction to ATP with (a) normal inorganic phosphate in HzO1* and (b) 01*-labeled inorganic