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

Abstract

The intrinsic chlorophyll-protein CP 47 is a component of Photosystem II which functions in both light-harvesting and oxygen evolution. Using the Escherichia coli mutator strain XL-1 Red, we introduced mutations at 14 sites in the large extrinsic loop E of CP 47 and its adjacent transmembrane alpha-helix VI. Four mutant cell lines were recovered in which the histidyl residues 455H, 466H and 469H were altered. The cell lines H455T, H455Y, H469Y, and the double mutant F432L,H466R exhibited phenotypes that supported the identification of the histidyl residues 455H, 466H and 469H as chlorophyll ligands. Four additional mutant cell lines were recovered which contained mutations at positions 448R in the large extrinsic loop of CP 47. These mutants, R448K, R448Q, R448S, and R448W, exhibited variable phenotypes ranging from moderate alteration of photoautotrophic growth and oxygen evolution rates to a complete inhibition of these parameters. Those mutants exhibiting photoautotrophic growth and oxygen evolution capability under standard conditions were unable to grow photoautotrophically or evolve oxygen when grown at low chloride concentrations. Finally, a mutant cell line exhibiting a substitution at position 342G was recovered. The mutant G342D exhibited moderate alterations of photoautotrophic growth and oxygen evolution. In addition 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 or evolve oxygen.