1M20

Crystal Structure of F35Y Mutant of Trypsin-solubilized Fragment of Cytochrome b5

Summary for 1M20

• Entry DOI: 10.2210/pdb1m20/pdb
• Related: 1EHB 1ES1 1LQX 1LR6 1M2I 1M2M
• Descriptor: Cytochrome b5, PROTOPORPHYRIN IX CONTAINING FE (3 entities in total)
• Functional Keywords: cytochrome b5, trypsin-cleaved fragment, mutant f35y, electron transport
• Biological source: Bos taurus (cattle)
• Cellular location: Endoplasmic reticulum membrane; Single-pass membrane protein; Cytoplasmic side: P00171
• Total number of polymer chains: 1
• Total formula weight: 10107.87

Authors

Yao, P.,Wu, J.,Wang, Y.-H.,Sun, B.-Y.,Xia, Z.-X.,Huang, Z.-X. (deposition date: 2002-06-20, release date: 2002-09-11, Last modification date: 2023-10-25)

Primary citation

Yao, P.,Wu, J.,Wang, Y.H.,Sun, B.Y.,Xia, Z.X.,Huang, Z.X.
X-ray crystallography, CD and kinetic studies revealed the essence of the abnormal behaviors of the cytochrome b5 Phe35-->Tyr mutant.
Eur.J.Biochem., 269:4287-4296, 2002

PubMed Abstract: Conserved phenylalanine 35 is one of the hydrophobic patch residues on the surface of cytochrome b5 (cyt b5). This patch is partially exposed on the surface of cyt b5 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 b5: 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 b5 and the oxidized Phe35-->Tyr mutant is more stable towards thermal and chemical denaturation than wild-type cyt b5. 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.

PubMed: 12199707
DOI: 10.1046/j.1432-1033.2002.03120.x

Experimental method

X-RAY DIFFRACTION (1.8 Å)