Modification of the protein matrix around active- and inactive pheophytins by site -directed mutagenesis, affects on energy and electron transfer processes in photosystem II

Photosystem II reaction centers (PS II RCs) have two parallel pathways for electron transfer, but only one of these pathways is active.;We perturbed the protein matrix of the active branch pheophytin (Pheo active) by mutagenesis of D1-E130. The HF-EPR measurements indicate that the replacement glutamate with His, Gln, and Leu results in the shift of the gx value towards the high field direction, verifying the H-bond strength changes in the mutants. The D1-E130L mutants evolve O 2 at 10% of wild-type (WT) rates. This mutation induces a blue shift in both the ground state and transiently reduced state of the Pheo Qx absorption band. The D1-E130Q mutants display a distinct blue shift only in the spectrum of transiently reduced Pheo Qx band, and have intermediate rates of evolving O2 relative to WT. The D1-E130H mutants have similar phenotype as WT. There were no apparent differences in primary charge separation kinetics between WT and the D1-E130 mutants.;We provided a Mg ligand for Pheo to determine the function on the inactive branch. We mutated the D1-L210 to His to convert Pheoinactive into a Chl. Pigment composition analyses of PS II RCs indicate that the His substitution results in the incorporation of a Chl in place of Pheoinactive. We locate the Pheoactive Qx absorption band at 544 nm using the modified PS II RCs. The loss of oxygen evolution, variable Chl fluorescence, and QA- accumulation indicate that electron transfer in the D1-L210H mutant is perturbed. This is partially attributed to the reduced yield of radical pair P680+Pheo- implied by transient absorption spectra. The energy transfer process also is affected in D1-L210H mutant. The conserved D1-L210A mutant exhibits the same phenotype as WT.;To investigate the dynamics of early electron transfer events in the PS II RCs, we mutated the D2-L210 with His. Our results indicate that D2-L210H mutation has substantially altered energy and electron transfer properties compared to WT. The conservative mutant D2-L210A also has a reduced capacity to carry out charge transfer. These results suggest that the amino acid substitutions at D2-L210 have significant effects on electron transfer efficiency of PS II.