Binding of Yeast Cytochrome c to Forty-Four Charge-Reversal Mutants of Yeast Cytochrome c Peroxidase: Isothermal Titration Calorimetry
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- 作者：James E. Erman ; Lidia B. Vitello ; Naw May Pearl ; Timothy Jacobson ; Meka Francis ; Erik Alberts ; Allen Kou ; Kathy Bujarska
- 刊名：Biochemistry
- 出版年：2015
- 出版时间：August 11, 2015
- 年：2015
- 卷：54
- 期：31
- 页码：4845-4854
- 全文大小：385K
- ISSN：1520-4995
文摘
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) Biochemistry47, 2766鈭?775]. 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) Science258, 1748鈭?755], three of the mutations (D37K, E98K, E209K) are slightly removed from the crystallographic site, and two of the mutations (D165K, D241K) occur on the 鈥渂ack-side鈥?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鈥揅cP complex, whether as a stable electron-transfer active complex or as part of a dynamic encounter complex.