Identification and Partial Characterization of the Denaturation Transition of the Photosystem II Reaction Center of Spinach Chloroplast Membranes.

Sensitive differential scanning calorimetry was employed to investigate thylakoid membrane structure. Calorimetric scans of chloroplast membranes suspended in a low ionic strength Hepesbuffered medium revealed endothermic transitions centered at the following temperatures ( degrees C): A (42.5), B (60.6), C(1) (64.9), C(2) (69.6), D (75.8), E (84.3), and F (88.9). The B transition was demonstrated by several different methods to originate from denaturation of the photosystem II reaction center complex. Evidence for this conclusion is as follows: (a) the isolated reaction center complex denatures near the temperature of the B transition; (b) inorganic phosphate destablizes the isolated reaction center complex and the B endotherm to a similar extent; (c) heat inactivation of the photosystem II-mediated 1,5-diphenylcarbazide --> dichloroindophenol photoreaction occurs at the temperature of the B transition and is influenced in a manner similar to B by the presence of phosphate; (d) thermal gel analysis indicates that the 43 and 47 kilodalton polypeptides of the photosystem reaction center complex denature at the temperature of the B transition, both in the presence and absence of phosphate; (e) low temperature (77 Kelvin) fluorescence reveals that a change in photosystem II emission at 695 nanometers occurs during the B transition; and (f) ioxynil, a specific inhibitor of photosystem II, selectively stabilizes the B endotherm. With the identification of the B transition established, the origins of six of the eight major transitions of the chloroplast membrane have now been determined.