Proline-40 is Essential to Maintaining Cytochrome b_5's Stability and Its Electron Transfer with Cytochrome c

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Proline-40 is Essential to Maintaining Cytochrome b_5's Stability and Its Electron Transfer with Cytochrome c

In order to illustrate the roles played by Pro40 in the structure, properties and functions of Cytochrome b₅, three mutated genes, P40V, P40Y, P40G were constructed in this work. Only the P40V gene was successfully expressed into holoprotein in E. coli JM83. According to the results of X‐ray crystallographic analysis and various kinds of spectroscopy studies, it is evident that substituting valine for Pro40 does not result in significant alterations in the protein′s overall structure; however, local conformational perturbations in the proximity of the heme do occur. The redox potential of the P40V mutant is 40 mV lower than that of the wild type protein. Its stability towards heat, urea, acid and ethanol were significantly decreased. The mutation leads to a decrease in the hydrophobicity of the heme pocket, which is probably the major factor contributing to the above changes. Binding constants and electron transfer rates between cytochrome b_s and cytochrome c were determined using UV‐visible spectroscopy and stopped‐flow techniques for both the wild type and the mutant. The results showed that the substitution of Pro40 by valine does not influence the binding constant of cytochrome b₅ to cytochrome c; however, the electron transfer rate between them decreased significantly. This indicates that proline‐40 is essential to maintaining cytochrome b_ss stability and its electron transfer with cytochrome c. These studies also provided a good example that property and functional changes of a protein do not necessarily require large overall structural alterations; in most cases, only perturbations on the local conformations are sufficient to induce significant changes in protein′s properties and functions.