1H NMR studies of the effect of mutation at Valine45 on heme microenvironment of cytochrome b5

• Published in: Biochimie Vol. 85; no. 10; pp. 1007 - 1016
• Main Authors: Cao, Chunyang, Zhang, Qi, Wang, Zhi-Qiang, Wang, Yue-Feng, Wang, Yun-Hua, Wu, Houming, Huang, Zhong-Xian
• Format: Journal Article
• Language: English
• Published: France Elsevier Masson SAS 01.10.2003
• Subjects: Animals; Cattle; Cytochrome b5/mutant V45E, V45H, V45Y/ 1H NMR/structure-function relationship; Cytochromes b5 - chemistry; Cytochromes b5 - genetics; Enzyme Stability; Glutamine - chemistry; Heme - chemistry; Heme - genetics; Histidine - chemistry; Imidazoles - chemistry; Ligands; Magnetic Resonance Spectroscopy; Microsomes - enzymology; Molecular Structure; Mutation; Protein Conformation; Temperature; Tyrosine - chemistry; Valine - chemistry; NMR; TPPI; 2D; 1D; Cytochrome b 5/mutant V45E, V45H, V45Y/ 1H NMR/structure-function relationship; DSS; NOE; CCW; NOESY; WT
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2003.08.005

1D and 2D 1H NMR were employed to probe the effects on the heme microenvironment of cytochrome b 5 caused by the mutation from Val45 to Tyr45, His45 and Glu45. Compared with wild type (WT) cytochrome b 5, in all mutants the heme ring are CCW rotated relative to the imidazole planes of axial ligands and the angles β between two axial ligand imidazole planes are not changed, being in agreement with the temperature dependence of the shifts of the heme protons. The ratios of heme isomers (major to minor) are smaller than that in WT. The 4-vinyl group of the heme in V45Y assumes cis-orientation, being similar to that of WT, while in V45E and V45H, both cis and trans orientation are found. The relationships between the structure and biological function of the mutants are discussed in terms of the geometry of heme and axial ligands, the hydrophobicity of heme pocket and the electrostatic potential of the heme-exposed area.