THE EFFECT OF DEUTERIUM OXIDE ON THE FLUORESCENCE AND PHOTOTRANSFORMATION OF 124 kDALTON PHYTOCHROME*

To probe the nature of primary photoprocess and the mechanism of the phototransformation of undegraded 124 kDa oat phytochrome, solvent deuterium isotope effects on the fluorescence and phototransformation of phytochrome have been investigated. The fluorescence intensity and lifetime of phytochrome (Pr form) are greater in D20‐buffer than in H2O‐buffer, suggesting a possible involvement of proton transfer in the primary photoprocess of phytochrome. Although the photostationary equilibrium (Pr to Pfr ratio) was not altered by deuterium oxide, in contrast to degraded phytochrome, the rate constants of both transformations, Pr→ Pfr and Pfr→ Pr were enhanced by up to 24%. The Pr to Pfr phototransformation of degraded phytochrome, however, was retarded by about the same percentage in D2O. These opposite effects of D2O with degraded and undegraded phytochromes underscore the fact that the Pr form from the former reverts to the Pr form in the dark, apparently catalyzed by deuterated general and/or conjugate acidic group(s). With the degraded phytochrome the deuterium oxide enhancement of the rate of dark reversion was approximately 2‐fold (Sarkar and Song, 1981). Both the fluorescence intensity and the rates of phototransformation of phytochrome were enhanced in D2O with successive photocyclings (Pr→ Pfr→ Pr→ Pfr→ Pr etc.) with alternating red and far‐red irradiation. It has been proposed that successive photocycling of phytochrome in D2O results in proton‐deuteron exchange in the partially exposed Ptr chromophore and/or its surrounding amino acid residues.