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 alg a Chlamydomonas reinhardtii have been generated in which Leu-343 and Ala-34 4 have been simultaneously or individually replaced by Phe and Ser, respect ively. The mutants carrying these replacements individually, L343F and A344 S, showed a wild-type phenotype. In contrast, the double mutant, L343FA344S , evolved O-2 at only 20-30% of the wild-type rate and was unable to grow p hotosynthetically. In this mutant, PSII accumulated to 60% of the wild-type level, indicating that the O-2-evolving activity per PSII was reduced to a pproximately half that of the wild-type. However, the amount of Mn atom det ected in the thylakoids suggested that a normal amount of Mn cluster was as sembled. An investigation of the kinetics of flash-induced fluorescence yie ld decay revealed that the electron transfer from Q(A)(-) to Q(B) was not a ffected. When a back electron transfer from Q(A)(-) to a donor component wa s measured in the presence of 3-(3,4-dichlorophenol)-1,1 -dimethylurea, a s ignificantly slower component of the Q(A)(-) oxidation was detected in addi tion to the normal component that corresponds to the back electron transfer from the Q(A)(-) to the S-2-state of the Mn cluster. Thermoluminescence me asurements revealed that L343FA344S cells contained two functionally distin ct Mn clusters. One was equivalent to that of the wild-type, while the othe r was incapable of water oxidation and was able to advance the transition f rom the S-1-state to the S-2-state. These results suggested that a fraction of the Mn cluster had been impaired by the L343FA344S mutation, leading to decreased O-2 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 w ater oxidation, in particular, transition to higher S-states. (C) 2001 Else vier Science B.V. All rights reserved.