Cytochrome B5 Mutant V61h V45h, V45y, V45e And P40v Nmr Study

This thesis concerns the solution structures and the relationship between the structure and function of cytb5 mutants. Cytb5, as a main component of respiratory chain in living system, is a low spin electron-transfer hemoprotein that carries out a variety of important physiological functions and has been extensively studied by biochemical and biophysical methods. However, many questions concerning the roles of specific amino acids in the electron transfer and other properties remain. Coordinated with site-directed mutagenesis, NMR is a promising method to answer those questions. In this thesis, the solution structure of cytb5 mutant V61H was calculated by modern 1D and 2D NMR technology and molecular modeling, and heme environment of cytb5 mutant V45H, V45E, V45Y and P40V was investigated.In the first part of this thesis, the structural studies about cytb5 focusing on NMR structures in the recent years were reviewed. The aim of the studies in this thesis was presented as well. To date, a number of studies concerning the influences of orientation of axial ligand plane on the spread and shift pattern of heme proton resonances and reduction potential as well as other spectroscopic properties using theoretical approach and model compounds have been reported. However, except the mutant A67V, there are very few examples where the mutant was successively designed to disturb the orientation of axial ligand. In cytb5, the residue Pro⁴⁰, Val⁴⁵ and Val⁶¹ are close to axial ligand His³⁹ and His⁶³, respectively. Therefore, the NMR studies of cytb5 mutants V61H, V45H, V45Y, V45E and P40V may provide further evidences for the roles of those specific residues and the relationship between the structure and function in cytb5. Another purpose of this study is to investigate the effect of the hydrophobicity of heme pocket on the reduction potential and stability...