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 (MSP) 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) were created by single and double amino acid substitution mutagenesis. The modified psbO genes encoding the mutants forms of MSP were used to transform a single-deletion mutant psO strain lacking MSP as well as a double-deletion strain psbO:psbV lacking both MSP and cyt. c-550. The mutant forms of MSP were expressed in each case and all permitted autotrophic growth in strains expressing cyt. c-550. However, when the MSP mutations were introduced into a strain which lacks cyt. c-550 (psbV), the two single amino acid substitution mutants (psbV:MSP-D159N and psbV:MSP-R163L) failed to grow photoautotrophically. These strains exhibited coupled O₂-evolving activity of 68–77% compared to the wild-type control using CO₂ as an electron acceptor and maximal uncoupled O₂-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 together in the absence of cyt. c-550 (psbV:MSP-D159N/R163L), the mutant grew 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 of cyt. c-550. The possibilities of a direct (ionic) or indirect interaction between D159 and R163 of MSP are discussed.