MUTATION OF CHLOROPHYLL LIGANDS IN THE CHLOROPHYLL-BINDING CP47-PROTEIN AS STUDIED IN A SYNECHOCYSTIS SP PCC-6803 PHOTOSYSTEM-I-LESS BACKGROUND

Site-directed mutations have been introduced to replace conserved histidine residues in the chlorophyll-binding protein CP47 of photosystem II (PS II) in a PS I-less/apcE(-) background strain of the cyanobacterium Synechocystis sp. PCC 6803. In thylakoids isolated from such a system, the degree of loss of the 695-nm fluorescence emission maximum at 77 K compared to that at 685 nm generally was consistent with the decrease in oxygen evolution rates measured at saturating light intensity. Taking into account that in the absence of CP47 and PS I some chlorophyll remains detectable in cells, the relative 695-nm fluorescence emission and the rate of oxygen evolution also correlate with the relative amount of chlorophyll per cell and with the number of PS II reaction centers on a chlorophyll basis. Interestingly, the 77 K fluorescence excitation spectra monitoring 695-nm emission of thylakoids from the CP47 His-to-Tyr mutants in a photosystem I-less/apcE(-) background showed increases in the 413- and 531-nm absorption regions, compared to spectra of thylakoids from the background strain. These wavelengths coincide with absorption maxima of pheophytin. No increase in the 531-nm excitation band was observed in thylakoids from mutants lacking PS II or with a His-to-Asn mutation. These results are interpreted to indicate that replacement of conserved histidine residues by tyrosine in CP47 leads to the loss of Mg2+ from chlorophyll, resulting in the formation of pheophytin, or to the binding of pheophytin (rather than chlorophyll) at a particular pigment-binding site of CP47 during biogenesis and assembly of the protein. It was observed that the light-harvesting efficiency of CP47 His mutants was lower judging from the light intensity dependence of electron transport and analysis of fluorescence decay kinetics. This suggests that the presence of pheophytin in the antenna decreases antenna efficiency.