PHOTOREVERSIBLE PROTON DISSOCIATION AND ASSOCIATION IN PEA PHYTOCHROME AND ITS CHROMOPEPTIDES

Abstract— Effect of red-light irradiation on the medium pH at 10d?C was measured and compared among unbuffered solutions of the 121-kDa native pea (Pisum sativum cv. Alaska) phytochrome and its 114- and 62-kDa fragments in a red-light-absorbing form (P_r), all of which converted to far-red-light-absorbing form (Pf_r) on red-light irradiation. Red-light irradiation induced alkalinization in the solutions of the phytochrome and the fragments in the pH range 6.6-7.2 and 6.2-7.8, respectively. The amount of protons taken up by the 121-kDa phytochrome was less than one half of that of the 114-kDa fragment. Red-light irradiation induced acidification in the solutions of the 114- and the 62-kDa fragments above pH 7.8. In the solutions of the 121-kDa phytochrome, however, the irradiation induced no pH change at pH 7.2-8.2, and only a slight acidification at pH 8.2-8.7, which may be ascribed to a small amount of contamination from the 114-kDa fragment. All these red-light-induced pH changes were reversible following exposure to far-red light. The 7-kDa polypeptide(s) of the native 121-kDa phytochrome, which is lacking in the 114-kDa fragment, thus, prohibited proton transfer between phytochrome and the medium. A red-light-induced pH change was also measured in unbuffered solutions of the 39-kDa fragment of the phytochrome and of the 114-kDa fragment in the presence of 0.8 mM soyasaponin I. The 39-kDa fragment showed partially photoreversible conversion between a spectral form having an absorption maximum at 659 nm (P₆₅₉) and a bleached form, P_***. The 114-kDa fragment in the presence of the saponin showed a photoreversible conversion between P65V and Pb,. Exposure of P₆₅₉ from the 39-kDa fragment and from the 114-kDa fragment in the presence of the saponin to red light, caused acidification of the medium in the pH range 6.8-8.8 and 7.2-9.0, respectively, but no change at pH 6.2-6.8 and 6.4-7.2, respectively. The acidification of the latter was reversible following a far-red-light irradiation, but that of the former was only partially photoreversible. Proton uptake of phytochrome was inhibited by tryptic degradation to the 39-kDa fragment and also by the presence of the saponin. Only proton release was observed during the photoconversion from P₆₅₉ and P_***hl. It is suggested that a phytochrome molecule has possible site(s) for both proton release and for uptake and that the proton release reaction may be correlated to the photoconversion process(es) prior to the bleached intermediate (I_***) of phytochrome.