Truncation of the COOH-terminal domain of the psbE gene product in Synechocystis sp. PCC 6803: requirements for photosytem II assembly and function

The COOH-terminal domain of the 80-residue cytochrome b559 alpha-subunit (psbE gene product) in Synechocystis sp. PCC 6803 was sequentially truncated in order to determine the minimum polypeptide length needed for function and assembly. A stop codon was introduced into the Arg-50, Arg-59, or Tyr-69 codons of the psbE gene, generating mutants truncated by 31, 22, and 12 residues, respectively. Removal of 12 residues caused a decrease of 20% in PSII function. Truncation of 22 or 31 residues caused a large decrease (60-85%) in the photoautotrophic growth rate, the rate of O2 evolution, and the amplitude of the 77 K 696-nm fluorescence, and a concomitant increase in the constant yield fraction (F0/Fmax) of the chlorophyll fluorescence. The level of residual activity in the Arg50-stop mutant was 10-20% of the wild type, which was reflected in a similar low level of immunochemically detected D2 polypeptide. Quantitation of the PSII reaction center stoichiometry of the Arg50-stop mutant by analysis of [14C]DCMU binding also showed a 5-fold decrease (1:910 Chl in wild type and 1:5480 Chl in R50) in the PSII reaction center concentration. However, the K(D) value for DCMU in the residual 15% of the complexes to which it bound was approximately equal to that (25 nM) of the wild type. Northern blot analysis showed no decrease in the b559 psbe mRNA level. Chemical difference spectral analysis of heme content indicated that the level of native cytochrome b559 heme in the Arg50-stop mutant (1:640 Chl) was 80% that of wild type (1:510 Chl). It was concluded that removal of 12 residues from approximately 38 in the lumen-side COOH-terminal domain of the cytochrome b559 alpha-subunit had only a small effect on function and assembly. Removal of 31 residues caused a decrease of 80-90% in reaction center function and in the content of assembled PSII reaction centers, without greatly affecting the assembly of cytochrome b559. The existence of a small fraction (ca. 15%) of active PSII complexes in the R50-stop mutant would imply that the COOH-terminal 31 residues of the alpha-subunit are not required for functions essential for O2 evolution such as the binding of manganese.