Solution structure of cytochrome b 5 mutant (E44/48/56A/D60A) and its interaction with cytochrome c

Using 1617 meaningful NOEs with 188 pseudocontact shifts, a family of 35 conformers of oxidized bovine microsomal cytochrome b 5 mutant (E44/48/56A/D60A) has been obtained and is characterized by good resolution (rmsd to the mean structure are 0.047 ± 0.007 nm and 0.095 ± 0.008 nm for backbone and heavy atoms, respectively). The solution structure of the mutant, when compared with the X‐ray structure of wild‐type cytochrome b 5 , has no significant changes in the whole folding and secondary structure. The binding between cytochrome b 5 and cytochrome c shows that the association constant of the mutant–cytochrome c complex is much lower than the one for wild‐type complex (2.2 × 10 4   m −1 vs. 5.1 × 10 3   m −1 ). The result suggests the four acidic residues have substantial effects on the formation of the complex between cytochrome b 5 and cytochrome c , and therefore it is concluded reasonably that the electrostatic interaction plays an important role in maintaining the stability and specificity of the complex formed. The competition between the ferricytochrome b 5 mutant and [Cr(oxalate) 3 ] 3– for ferricytochrome  c shows that site III of cytochrome c , which is a strong binding site to wild‐type cytochrome b 5 , still binds to the mutant with relatively weaker strength. Our results indicate that certain bonding geometries do occur in the interaction between the present mutant and cytochrome c and these geometries, which should be quite different from the ones of the Salemme and Northrup models.