title: Binding of Yeast Cytochrome c to Forty-Four Charge-Reversal Mutants of Yeast Cytochrome c Peroxidase: Isothermal Titration Calorimetry.
abstract: Previously, we constructed, expressed, and purified 46 charge-reversal mutants of yeast cytochrome c peroxidase (CcP) and determined their electronic absorption spectra, their reaction with H2O2, and their steady-state catalytic properties [ Pearl , N. M. et al. (2008) Biochemistry 47 , 2766 - 2775 ]. Forty-four of the mutants involve the conversion of either an aspartate or glutamate residue to a lysine residue, while two are positive-to-negative mutations, R31E and K149D. In this paper, we report on a calorimetric study of the interaction of each charge-reversal mutant (excluding the internal mutants D76K and D235K) with recombinant yeast iso-1 ferricytochrome c(C102T) (yCc) under conditions where only one-to-one yCc/CcP complex formation is observed. Thirteen of the 44 surface-site charge-reversal mutants decrease the binding affinity for yCc by a factor of 2 or more. Eight of the 13 mutations (E32K, D33K, D34K, E35K, E118K, E201K, E290K, E291K) occur within, or on the immediate periphery, of the crystallographically defined yCc binding site [ Pelletier , H. and Kraut , J. (1992) Science 258 , 1748 - 1755 ], three of the mutations (D37K, E98K, E209K) are slightly removed from the crystallographic site, and two of the mutations (D165K, D241K) occur on the "back-side" of CcP. The current study is consistent with a model for yCc binding to CcP in which yCc binds predominantly near the region defined by crystallographic structure of the 1:1 yCc-CcP complex, whether as a stable electron-transfer active complex or as part of a dynamic encounter complex.
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois 60115, United States.
authors.forename: James E
authors.initials: JE
authors.lastname: Erman
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois 60115, United States.
authors.forename: Lidia B
authors.initials: LB
authors.lastname: Vitello
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois 60115, United States.
authors.forename: Naw May
authors.initials: NM
authors.lastname: Pearl
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois 60115, United States.
authors.forename: Timothy
authors.initials: T
authors.lastname: Jacobson
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois 60115, United States.
authors.forename: Meka
authors.initials: M
authors.lastname: Francis
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois 60115, United States.
authors.forename: Erik
authors.initials: E
authors.lastname: Alberts
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois 60115, United States.
authors.forename: Allen
authors.initials: A
authors.lastname: Kou
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois 60115, United States.
authors.forename: Kathy
authors.initials: K
authors.lastname: Bujarska
mesh_terms: D019943:Amino Acid Substitution; D018360:Crystallography, X-Ray; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D020125:Mutation, Missense; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c; D003578:Cytochrome-c Peroxidase