Photoassembly of the photosystem II (Mn)4 cluster in site-directed mutants impaired in the binding of the manganese-stabilizing protein.

Photoactivation is the light-dependent ligation of Mn2+ into the H2O oxidation complex of photosystem II (PSII) and culminates in the formation of an enzymatically active complex containing Ca2+ and four Mn>/=3+. Previous kinetic analysis demonstrated that the genetic removal of the extrinsic manganese-stabilizing protein (MSP) increases the quantum yield of photoactivation 4-fold relative to that of the wild type, consistent with the hypothesis that MSP hinders access of Mn2+ to a site of photoligation [Burnap, R. L., et al. (1996) Biochemistry35, 874-882]. In this report, several Synechocystis sp. PCC6803 mutants with defined amino acid substitutions in the N-terminal region of MSP or the e-loop of intrinsic PSII protein CP47 [Putnam-Evans, C., et al. (1996) Biochemistry 35, 4046-4053] were characterized in terms of the binding of MSP to the intrinsic portion of the PSII complex and in terms of photoactivation kinetics. The charge-pair switch mutation, Arg384Arg385 --> Glu384Glu385 in the lumenal e-loop of CP47 (CP47 RR384385EE), exhibited the most severe impairment of MSP binding, whereas the Arg384Arg385 --> Gly384Gly385 (CP47 RR384385GG) mutation caused a more moderate impairment in binding. Single-substitution mutations at the highly conserved Asp9 or Asp10 positions in the amino-terminal region of MSP also resulted in a reduced binding affinity, but not as severe as that in CP47 RR384385EE. The relative quantum yield of photoactivation of hydroxylamine-extracted mutant PSII was generally found to correlate with the degree of MSP binding impairment, with the CP47 RR384385 mutants exhibiting the highest quantum yields. A two-locus, double-mutant construct involving deletion of MSP in the CP47 RR384385EE background was found to be only slightly more impaired in H2O oxidation activity than either of the corresponding single-locus mutant derivatives, indicating that mutations at these genetically separate loci encode physically interacting products affecting the same reaction parameter during H2O oxidation. Taken together, the results reinforce the concept that MSP interacts with the e-loop of CP47 at Arg384Arg385 and that disruption of this interaction causes significant alterations of the site of H2O oxidation in terms of assembly and enzymatic activity of the Mn cluster.