Effects of charged amino-acid mutation on the solution structure of cytochrome b(5) and binding between cytochrome b(5) and cytochrome c

The solution structure of oxidized bovine microsomal cytochrome b(5) mutant (E48, E56/A, D60/A) has been determined through 1524 meaningful nuclear Ov erhauser effect constraints together with 190 pseudocontact shift constrain ts. The final family of 35 conformers has rmsd values with respect to the m ean structure of 0.045 +/-0.009 nm and 0.088 +/-0.011 nm for backbone and h eavy atoms, respectively. A characteristic of this mutant is that of having no significant changes in the whole folding and secondary structure compar ed with the X-ray and solution structures of wild-type cytochrome b(5). The binding of different surface mutants of cytochrome b(5) with cytochrome c shows that electrostatic interactions play an important role in maintaining the stability and specificity of the protein complex formed. The differenc es in association constants demonstrate the electrostatic contributions of cytochrome b(5), surface negatively charged residues, which were suggested to be involved in complex formation in the Northrup and Salemme models, hav e cumulative effect on the stability of cyt c-cyt b(5) complex, and the con tribution of Glu48 is a little higher than that of Glu44. Moreover, our res ult suggests that the docking geometry proposed by Northrup, which is invol ved in the participation of Glu48, Glu56, Asp60, and heme propionate of cyt ochrome b(5), do occur in the association between cytochrome b(5) and cytoc hrome c.