tailieunhanh - Báo cáo khoa học: X-ray crystallography, CD and kinetic studies revealed the essence of the abnormal behaviors of the cytochrome b5 Phe35fiTyr mutant

Conservedphenylalanine 35 is one of the hydrophobic patch residues on the surface of cytochromeb5 (cytb5). This patch is partially exposed on the surface of cyt b5while its buried face is in direct van der Waals’contact with heme b. Resi-dues Phe35 and Phe/Tyr74 also form an aromatic channel with His39, which is one of the axial ligands of heme b. By site-directedmutagenesiswehaveproduced threemutantsof cytb5:Phe35fiTyr, Phe35fiLeu, and Phe35fiHis. | Eur. J. Biochem. 269 4287-4296 2002 FEBS 2002 doi X-ray crystallography CD and kinetic studies revealed the essence of the abnormal behaviors of the cytochrome b5 Phe35fiTyr mutant Ping Yao1 Jian Wu2 Yun-Hua Wang1 Bing-Yun Sun1 Zong-Xiang Xia2 and Zhong-Xian Huang1 Chemical Biology Laboratory Department of Chemistry Fudan University Shanghai People s Republic of China 2State Key Laboratory of Bio-organic and Natural Products Chemistry Shanghai Institute of Organic Chemistry Chinese Academy of Sciences Shanghai People s Republic of China 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 15. Reii-dues 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 Phe35fiTyr Phe35fiLeu and Phe35fiHis. We found that of these three mutants the Phe35fiTyr mutant displays abnormal properties. The redox potential of the Phe35fiTyr mutant is 66 mV more negative than that of the wild-type cyt b5 and the oxidized Phe35fiTyr mutant is more stable towards thermal and chemical denaturation than wild-type cyt b5. In this study we studied the most interesting mutant Phe35fiTyr 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 Phe35fiTyr 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 Phe35fiTyr mutant. The kinetic difference of the heme trapped by the heme

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