Two functionally distinct manganese clusters formed by introducing a mutation in the carboxyl terminus of a photosystem II reaction center polypeptide, D1, of the green alga Chlamydomonas reinhardtii

To study the function of the carboxyl-terminal domain of a photosystem II (PSII) reaction center polypeptide, D1, chloroplast mutants of the green alga Chlamydomonas reinhardtii have been generated in which Leu-343 and Ala-344 have been simultaneously or individually replaced by Phe and Ser, respectively. The mutants carrying these replacements individually, L343F and A344S, showed a wild-type phenotype. In contrast, the double mutant, L343FA344S, evolved O₂ at only 20-30% of the wild-type rate and was unable to grow photosynthetically. In this mutant, PSII accumulated to 60% of the wild-type level, indicating that the O₂-evolving activity per PSII was reduced to approximately half that of the wild-type. However, the amount of Mn atom detected in the thylakoids suggested that a normal amount of Mn cluster was assembled. An investigation of the kinetics of flash-induced fluorescence yield decay revealed that the electron transfer from Q^-_A to Q_B was not affected. When a back electron transfer from Q^-_A to a donor component was measured in the presence of 3-(3,4-dichlorophenol)-1,1-dimethylurea, a significantly slower component of the Q^-_A oxidation was detected in addition to the normal component that corresponds to the back electron transfer from the Q^-_A to the S₂-state of the Mn cluster. Thermoluminescence measurements revealed that L343FA344S cells contained two functionally distinct Mn clusters. One was equivalent to that of the wild-type, while the other was incapable of water oxidation and was able to advance the transition from the S₁-state to the S₂-state. These results suggested that a fraction of the Mn cluster had been impaired by the L343FA344S mutation, leading to decreased O₂ evolution. We concluded that the structure of the C-terminus of D1 is critical for the formation of the Mn cluster that is capable of water oxidation, in particular, transition to higher S-states.