MUTAGENESIS OF A POTENTIAL PHEOPHYTIN BINDING SITE RESIDUE IN THE PHOTOSYSTEM II COMPLEX OF CHLAMYDOMONAS.

Xiong L and Sayre RT.

Department of Plant Biology, Ohio State University, Columbus, OH. 43210

In the bacterial, quinone type reaction center and in the photosystem II (PSII) reaction center complex pheophytin (Phe) is the first stable electron acceptor reduced following charge separation. Both reaction center types have two Phes per reaction center complex yet only one is reduced. Our interest is to determine which Phe is reduced in PSII and to determine the effects of the protein environment on the efficiency of Phe reduction. It is apparent from the Rhodopsuedomonas viridis crystal structure that residue L-E104 is hydrogen bonded to the active branch Phe. The analogous residue in the chloroplastic PSII reaction center is D1-E130 while in the cyanobacterial PSII complex it is D1-Q130. The differences in amino acid side chains of the chloroplastic and cyanobacterial PSII D1-130 residue has been proposed to account for the apparent differences in the quantum yield for charge separation between the two PSII types. Previously, the cyanobacterial D1-Q130 residue had been mutated to a glutamate resulting in an increased efficiency of charge separation. To determine whether introduction of the reciprocal mutation into the chloroplastic PSII complex would reduce the efficiency of charge separation we mutated the D1-E130 residue of Chlamydomonas reharditii to glutamine, histidine, and leucine. The D1-E130H, D1-E130Q, and D1-E130L mutants evolve oxygen at 65%, 53%, and 12% of the wild type rate, respectively. Light-dependent and DPC supported rates of DCPIP reduction in Mn extracted thylakoids of the D1-E130H, -Q and -L mutants were 80%, 74%, and 62% of the wild type rate, respectively. The higher relative rates of DPC photooxidation relative to water oxidation in the mutants, particularly the D1-E130L mutant, suggest that the D1-E130 mutations have an indirect affect on the assembly or operation of the water-splitting, Mn cluster as well as on P680®Phe electron transfer. We also have measured Chl fluorescence rise and decay kinetics following a flash. The D1-E130H and the D1-E130Q mutants had 26 and 80% higher Fo levels than wild type, respectively, possibly associated with inactive PSII centers. Significantly, the D1-E130H mutant exhibited a more rapid Q_A^- to P680 back-reaction following charge separation. In contrast, the D1-E130Q mutant exhibited Chl fluorescence decay kinetics that were essentially identical to wild type. This result suggests that the D1-E130Q mutation has little effect on PSII donor side kinetics. In contrast to the D1-E130H/Q mutants, the D1-E130L mutant had essentially no variable Chl fluorescence and a high Fo value, indicative of both inactive PSII centers and reduced rates of electron transfer to Q_B, possibly at the level of Phe. We also have characterized the Phe Q_X absorption band in D1-E130 mutant, PSII reaction center complexes. Our results suggest that the D1-E130Q mutation causes a red-shift in the Q_X band of the functional Phe.