Truncation of the COOH-terminal domain of the psbE gene product in Synechocystis sp. PCC 6803: requirements for photosystem 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 KD 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).(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Cramer
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D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003062:Codon; D000458:Cyanobacteria / Q000235:genetics / Q000378:metabolism*; D003573:Cytochrome b Group / Q000737:chemistry* / Q000235:genetics; D004579:Electron Transport; D045342:Light-Harvesting Protein Complexes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D045322:Photosynthetic Reaction Center Complex Proteins / Q000378:metabolism*; D045332:Photosystem II Protein Complex; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D003062:Codon; D003573:Cytochrome b Group; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; C019313:cytochrome b559