Random mutagenesis in the large extrinsic loop E and transmembrane alpha-helix VI of the CP 47 protein of Photosystem II

The intrinsic chlorophyll-protein CP 47 is a component of Photosystem II wh ich functions in both light-harvesting and oxygen evolution. Using the Esch erichia cell mutator strain XL-I Red, we introduced mutations at 14 sites i n the large extrinsic loop E of CP 47 and its adjacent transmembrane cu-hel ix VI. Four mutant cell lines were recovered in which the histidyl residues H-455, H-466 and H-469 were altered. The cell lines H455T, H455Y, H469Y, a nd the double mutant F432L,H466R exhibited phenotypes that supported the id entification of the histidyl residues H-455, H-466 and H-469 as chlorophyll ligands. Four additional mutant cell lines were recovered which contained mutations at positions R-448 in the large extrinsic loop of CP 47. These mu tants, R448K, R338Q, R448S, and R448W, exhibited variable phenotypes rangin g from moderate alteration of photoautotrophic growth and oxygen evolution rates to a complete inhibition of these parameters. Those mutants exhibitin g photoautotrophic growth and oxygen evolution capability under standard co nditions were unable to grow photoautotrophically or evolve oxygen when gro wn at low chloride concentrations. Finally, a mutant cell line exhibiting a substitution at position (342)G was recovered. The mutant G342D exhibited moderate alterations of photoautotrophic growth and oxygen evolution. In ad dition to these alterations, mutants were recovered in which deletions and insertions (leading to frame shifts) and stop codons were introduced. These mutants uniformly lacked the ability to either grow photoautotrophically o r evolve oxygen.