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Article

Mapping the Electron Transfer Interface between Cytochrome b₅ and Cytochrome c^†

Yi Ren,^‡^§ Wen-Hu Wang,^‡ Yun-Hua Wang,^‡ Martin Case,^§ Wen Qian,^‡ George McLendon,*^§ and Zhong-Xian Huang*^‡

Chemical Biology Laboratory, Department of Chemistry, Fudan University, Shanghai 200433, China, and Department of Chemistry, Princeton University, Princeton, New Jersey 08544

Biochemistry, 2004, 43 (12), pp 3527–3536

DOI: 10.1021/bi036078k

Publication Date (Web): March 5, 2004

†

This project was supported by the National Science Foundation of China.

‡

Fudan University.

Princeton University.

To whom correspondence should be addressed. Z.-X.H.: telephone, +86-21-65643973; fax, +86-21-65641740; e-mail, zxhuang@fudan. edu.cn. G.M.: telephone, (609) 258-6808; e-mail, GLM@princeton.edu.

Abstract

To characterize the cytochrome b₅ (Cyt b₅)−cytochrome c (Cyt c) interactions during electron transfer, variants of Cyt b₅ have been employed to assess the contributions of electrostatic interactions (substitution of surface charged residues Glu44, Glu48, Glu56, and Asp60 and heme propionate), hydrophobic interactions, and the thermodynamic driving forces (substitutions for hydrophobic residues in heme pocket residues Phe35, Pro40, Val45, Phe58, and Val61). The electrostatic interactions play an important role in maintaining the stability and specificity of the Cyt b₅−Cyt c complex that is formed. There is no essential effect on the intraprotein complex electron transfer even if most of the involved negatively charged residues on the surface of Cyt b₅ have been removed. The results support a dynamic docking paradigm for Cyt b₅−Cyt c interactions. The orientation that is optimal for binding may not be optimal form for electron transfer. Substitution of hydrophobic residues does not have a significant effect on the binding between Cyt b₅ and Cyt c; rather, it regulates the electron transfer rates via changes in the driving force. Combining the electron transfer studies of the Cyt b₅−Cyt c system and the Cyt b₅−Zn−Cyt c system, we obtain the reorganization energy (0.6 eV) at an ionic strength of 150 mM.