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 o xidation complex of photosystem II (PSII) and culminates in the format ion of an enzymatically active complex containing Ca2+ and four Mngrea ter than or equal to 3+. Previous kinetic analysis demonstrated that t he genetic removal of the extrinsic manganese-stabilizing protein (MSP ) increases the quantum yield of photoactivation if-fold relative to t hat 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 ) Biochemistry 35, 874-882]. in this report, several Synechocystis sp, PCC6803 mutants with defined amino acid substitutions in the N-termin al region of MSP or the e-loop of intrinsic PSII protein CP47 [Putnam- Evans, C,, et al. (1996) Biochemistry 35, 4046-4053] were characterize d in terms of the binding of MSP to the intrinsic portion of the PSII complex and in terms of photoactivation kinetics, The charge-pair swit ch mutation, Arg384Arg385 --> Glu384Glu385 in the lumenal e-loop of CP 47 (CP47 RR384385EE), exhibited the most severe impairment of MSP bind ing, whereas the Arg384Arg385 --> Gly384Gly385 (CF47 RR384385GG) mutat ion 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, bu t not as severe as that in CP47 RR384385EE. The relative quantum yield elf photoactivation of hydroxylamine-extracted mutant PSII was genera lly found to correlate with the degree of MSP binding impairment, with the CP47 RR384385 mutants exhibiting the highest quantum yields. A tw o-locus, double-mutant construct involving deletion of MSP in the CS47 RR384385EE background was found to be only slightly more impaired in H2O oxidation activity than either of the corresponding single-locus m utant derivatives, indicating that mutations at these genetically sepa rate loci encode physically interacting products affecting the same re action parameter during H2O oxidation. Taken together, the results rei nforce the concept that MSP interacts with the e-loop of CP47 at Arg38 4Arg385 and that disruption of this interaction causes significant alt erations of the site of H2O oxidation in terms of assembly and enzymat ic activity of the Mn cluster.