title: Site directed mutagenesis of the heme axial ligands of cytochrome b559 affects the stability of the photosystem II complex.
abstract: Cytochrome (cyt) b559, an integral membrane protein, is an essential component of the photosystem II (PSII) complex in the thylakoid membranes of oxygenic photosynthetic organisms. Cyt b559 has two subunits, alpha and beta, each with one predicted membrane spanning alpha-helical domain. The heme cofactor of this cytochrome is coordinated between two histidine residues. Each of the two subunit polypeptides of cyt b559 has one His residue. To investigate the influence of these His residues on the structure of cyt b559 and the PSII complex, we used a site directed mutagenesis approach to replace each His residue with a Leu residue. Introduction of these missense mutations in the transformable unicellular cyanobacterium, Synechocystis 6803, resulted in complete loss of PSII activity. Northern blot analysis showed that these mutations did not affect the stability of the polycistronic mRNA that encompasses both the psbE and the psbF genes, encoding the alpha and the beta subunits, respectively. Moreover, both of the single His mutants showed the presence of the alpha subunit which was 1.5 kd smaller than the same polypeptide in wild type cells. A secondary effect of such a structural change was that D1 and D2, two proteins that form the catalytic core (reaction center) of PSII, were also destabilized. Our results demonstrate that proper axial coordination of the heme cofactor in cyt b559 is important for the structural integrity of the reaction center of PSII.
authors.affiliation: Department of Biology, Washington University, St Louis, MO 63130.
authors.forename: H B
authors.initials: HB
authors.lastname: Pakrasi
authors.forename: P
authors.initials: P
authors.lastname: De Ciechi
authors.forename: J
authors.initials: J
authors.lastname: Whitmarsh
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D002736:Chloroplasts / Q000737:chemistry / Q000201:enzymology*; D000458:Cyanobacteria / Q000201:enzymology / Q000235:genetics*; D003573:Cytochrome b Group / Q000235:genetics*; D004789:Enzyme Activation; D004795:Enzyme Stability; D006418:Heme / Q000235:genetics*; D006639:Histidine / Q000235:genetics; D008024:Ligands; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics*; D045332:Photosystem II Protein Complex; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D003573:Cytochrome b Group; D008024:Ligands; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D012333:RNA, Messenger; D006418:Heme; D006639:Histidine; C019313:cytochrome b559