title: Expression, mutagenesis, and characterization of recombinant low-potential cytochrome c550 of photosystem II.
abstract: Cytochrome c(550) of the photosystem II complex of cyanobacteria is an unusual member of the large protein family of monoheme c-type cytochromes. Despite the fact that it shares considerable amino acid sequence similarity and has a protein fold similar to the other members of the family, Cyt.c(550) has a midpoint potential (E(m7) = -250 mV) that is much lower than the positive midpoint potentials characteristic (E(m7) = 100-300mV) of this cytochrome family. An E. coli heterologous expression system involving secretion of the recombinant protein from Synechocystis PCC6803 to the periplasm was utilized to allow production of wild-type and mutant forms of the cytochrome. For most of the variants studied, the yield of protein was significantly enhanced by growth at 28 degrees C and inclusion of sucrose and betaine, in addition to isopropyl-beta-d-thiogalactoside (IPTG), to the growth medium of the E. coli expression host. Analysis of the protein products revealed that the wild-type protein maintained the redox and visible spectroscopic characteristics of the authentic protein. Mutations in the residues engaging in hydrogen bond interactions with the heme propionate (Asn49) and the axial 6th ligand His92 (Pro93) resulted in small (12-20 mV), but reproducible, upshifts in midpoint redox potential. Substitution of the axial ligand His92 with Met produced no discernible changes in the optical spectrum relative to the wild-type despite the fact that in this mutant, unlike the others studied here, the thioether linkage either was not formed or was highly labile as evidenced by loss of the heme during SDS-PAGE. On the other hand, the midpoint potential of the C550-H92M mutant was upshifted by approximately 70 mV. This value is significantly less of a perturbation than that observed in a similar mutant that is natively expressed in Thermosynechoccocus but appears to have an intact thioether linkage between the heme and the polypeptide moiety.
authors.affiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
authors.forename: Heather
authors.initials: H
authors.lastname: Andrews
authors.forename: Zhaoliang
authors.initials: Z
authors.lastname: Li
authors.forename: Adriana
authors.initials: A
authors.lastname: Altuve-Blanco
authors.forename: Mario
authors.initials: M
authors.lastname: Rivera
authors.forename: Robert L
authors.initials: RL
authors.lastname: Burnap
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D001483:Base Sequence; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006418:Heme / Q000737:chemistry; D008564:Membrane Potentials; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D045332:Photosystem II Protein Complex / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013053:Spectrophotometry; D046939:Synechocystis / Q000737:chemistry* / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D003574:Cytochrome c Group; D004269:DNA, Bacterial; D045332:Photosystem II Protein Complex; D011994:Recombinant Proteins; D006418:Heme; C031732:cytochrome C-550