Characterization of site-directed mutants in manganese-stabilizing protein(MSP) of Synechocystis sp PCC6803 unable to grow photoautotrophically in the absence of cytochrome c-550

To investigate the interaction between the manganese-stabilizing protein (M SP) and cytochrome c-550 (cyt. c-550) of the photosystem II (PSII) complex in the cyanobacterium Synechocystis sp. PCC6803, three site-directed amino acid substitution mutants in MSP (MSP-D159N, MSP-R163L, MSP-D159N/R163L) we re created by single and double amino acid substitution mutagenesis. The mo dified psbO genes encoding the mutants forms of MSP were used to transform a single-deletion mutant Delta psO strain lacking MSP as well as a double-d eletion strain Delta psbO:Delta psbV lacking both MSP and cyt. c-550. The m utant forms of MSP were expressed in each case and all permitted autotrophi c growth in strains expressing cyt. c-550. However, when the MSP mutations were introduced into a strain which lacks cyt. c-550 (Delta psbV), the two single amino acid substitution mutants (Delta psbV:MSP-D159N and Delta psbV :MSP-R163L) failed to grow photoautotrophically. These strains exhibited co upled O-2-evolving activity of 68-77% compared to the wild-type control usi ng CO2 as an electron acceptor and maximal uncoupled O-2-evolution rates of 42-57% using 2,6-dichloro-p-benzoquinone (DCBQ) as an artificial electron acceptor. Interestingly, when the two amino acid substitutions were togethe r in the absence of cyt. c-550 (Delta psbV:MSP-D159N/R163L), the mutant gre w photoautotrophically and the oxygen-evolving activities were higher than in the single mutants. This indicates that the MSP-D159N mutant suppresses the non-autotrophic phenotype of MSP-R163L (or vice versa) in the absence o f cyt. c-550. The possibilities of a direct (ionic) or indirect interaction between D159 and R163 of MSP are discussed.