title: The role of a disulfide bridge in the stability and folding kinetics of Arabidopsis thaliana cytochrome c(6A).
abstract: Cytochrome c(6A) is a eukaryotic member of the Class I cytochrome c family possessing a high structural homology with photosynthetic cytochrome c(6) from cyanobacteria, but structurally and functionally distinct through the presence of a disulfide bond and a heme mid-point redox potential of +71mV (vs normal hydrogen electrode). The disulfide bond is part of a loop insertion peptide that forms a cap-like structure on top of the core α-helical fold. We have investigated the contribution of the disulfide bond to thermodynamic stability and (un)folding kinetics in cytochrome c(6A) from Arabidopsis thaliana by making comparison with a photosynthetic cytochrome c(6) from Phormidium laminosum and through a mutant in which the Cys residues have been replaced with Ser residues (C67/73S). We find that the disulfide bond makes a significant contribution to overall stability in both the ferric and ferrous heme states. Both cytochromes c(6A) and c(6) fold rapidly at neutral pH through an on-pathway intermediate. The unfolding rate for the C67/73S variant is significantly increased indicating that the formation of this region occurs late in the folding pathway. We conclude that the disulfide bridge in cytochrome c(6A) acts as a conformational restraint in both the folding intermediate and native state of the protein and that it likely serves a structural rather than a previously proposed catalytic role.
authors.affiliation: Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, UK.
authors.forename: Jody M
authors.initials: JM
authors.lastname: Mason
authors.forename: Derek S
authors.initials: DS
authors.lastname: Bendall
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Howe
authors.forename: Jonathan A R
authors.initials: JA
authors.lastname: Worrall
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry; D000458:Cyanobacteria / Q000737:chemistry; D003545:Cysteine / Q000378:metabolism; D045365:Cytochromes c6 / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012694:Serine / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D045365:Cytochromes c6; D004220:Disulfides; D006418:Heme; D012694:Serine; D003545:Cysteine