Open AccessX‐ray crystallography, CD and kinetic studies revealed the essence of the abnormal behaviors of the cytochrome b 5 Phe35→Tyr mutant
Author(s) -
Yao Ping,
Wu Jian,
Wang YunHua,
Sun BingYun,
Xia ZongXiang,
Huang ZhongXian
Publication year - 2002
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2002.03120.x
Subject(s) - heme , chemistry , mutant , van der waals force , stereochemistry , cytochrome , crystallography , hemeprotein , mutagenesis , wild type , denaturation (fissile materials) , biophysics , biochemistry , molecule , enzyme , biology , organic chemistry , nuclear chemistry , gene
Conserved phenylalanine 35 is one of the hydrophobic patch residues on the surface of cytochrome b 5 (cyt b 5 ). This patch is partially exposed on the surface of cyt b 5 while its buried face is in direct van der Waals' contact with heme b. Residues Phe35 and Phe/Tyr74 also form an aromatic channel with His39, which is one of the axial ligands of heme b. By site‐directed mutagenesis we have produced three mutants of cyt b 5 : Phe35→Tyr, Phe35→Leu, and Phe35→His. We found that of these three mutants, the Phe35→Tyr mutant displays abnormal properties. The redox potential of the Phe35→Tyr mutant is 66 mV more negative than that of the wild‐type cyt b 5 and the oxidized Phe35→Tyr mutant is more stable towards thermal and chemical denaturation than wild‐type cyt b 5 . In this study we studied the most interesting mutant, Phe35→Tyr, by X‐ray crystallography, thermal denaturation, CD and kinetic studies of heme dissociation to explore the origin of its unusual behaviors. Analysis of crystal structure of the Phe35→Tyr mutant shows that the overall structure of the mutant is basically the same as that of the wild‐type protein. However, the introduction of a hydroxyl group in the heme pocket, and the increased van der Waals' and electrostatic interactions between the side chain of Tyr35 and the heme probably result in enhancement of stability of the Phe35→Tyr mutant. The kinetic difference of the heme trapped by the heme pocket also supports this conclusion. The detailed conformational changes of the proteins in response to heat have been studied by CD for the first time, revealing the existence of the folding intermediate.