Abstract
CP47 is a chlorophyll-binding protein of the proximal antenna system of Photosystem II containing 16 chlorophyll molecules. Twelve histidine residues found within the transmembrane α-helices I-VI of CP47 serve as axial ligands for the magnesium ion of these chlorophyll molecules and a role for histidyl ligation of chlorophyll has been supported by various site-directed mutagenesis studies. Successful substitution of these histidine residues with either Asn or Gln within the transmembrane helices I-VI has been observed but replacement of His-469 with a glutamine residue within the helix VI of the CP47 protein resulted in a major reduction in the amount of assembled PS II reaction centres and photoautotrophic growth was retarded. A pseudorevertant for the strain carrying the His to Gln substitution (H469Qrev) was able to grow photoautotrophically at a rate similar to wild type but the secondary mutation was not present in the psbB gene that encodes CP47. Moreover, the introduction of Ala, Lys, Pro and Tyr at the 469 position resulted in obligate heterotrophic strains with reduced photoautotrophic growth and unassembled PS II centres. The current study was carried out to understand the role of the His-469 residue in CP47 and also to identify the gene responsible for the secondary mutation in the H469Qrev pseudorevertant. All strains carrying substitutions for His-469 (H469A, H469Arev, H469K, H469Krev, H469P, H469Prev, H469Q, H469Qrev, H469Y and H469Yrev) were studied using a range of physiological and biochemical measurements. Mutants created in the wild-type background exhibited loss of photoautotrophic growth with concomitant reduction in the oxygen evolution rates and reduced number of assembled PS II centres. However, physiological characterisation of the strains in the genetic background of the H469Qrev pseudorevertant resulted in a range of phenotypes showing varied levels of recovery when compared to their corresponding mutants in the wild-type background. Furthermore, the search for secondary mutations in the H469Qrev pseudorevertant in turn revealed seven unique gene mutations. Introduction of these mutations into the very impaired H469Y cells did not transform this strain but during these studies, a new pseudorevertant was isolated (H469Yrev+6). The new pseudorevertant did not contain any of the mutations identified in the H469Qrev strain but exhibited increased photoautotrophic growth and oxygen evolution rates at a comparable level to the wild-type strain. Additionally, the new suppressor present in the H469Yrev+6 strain was able to restore PS II activity to H469A, H469K and H469P cells but not the H469Q and H469Y strains.