THE INFLUENCE OF MUTATION AT GLU44 AND GLU56 OF CYTOCHROME B(5) ON THE PROTEINS STABILIZATION AND INTERACTION BETWEEN CYTOCHROME-C AND CYTOCHROME B(5)

To characterize the roles played by Glu44 and Glu56 of cytochrome b(5) in the formation of the electrostatic complex between cytochrome c an d cytochrome b(5), the Glu44, Glu56, or both sites were changed to ala nine by site-directed mutagenesis. The influence of these two residues on the protein stability was probed by investigating the kinetic beha viors of protein denaturation in urea or upon heating and the heme-tra nsfer reactions between apo-myoglobin and the variants of cytochrome b (5). It has been found that when the Glu44 and/or Glu56 are mutated to alanine, the protein stability increases slightly due to the fact tha t the hydrophilic residue is changed to a hydrophobic one, resulting i n the two pairs of helices surrounding the heme taking a more compact conformation. The difference in voltammetric behavior of cytochrome c, cytochrome b(5), and its three mutants, Cyt b(5) E44A, E56A, and E44/ 56A, alone and in 1:1 protein complexes demonstrates that both Glu44 a nd Glu56 of cytochrome b(5) take part in the electrostatic interaction with cytochrome c. The entropy changes, Delta S(rc)degrees and enthal py changes, Delta H degrees, derived from the temperature dependence o f the formal reduction potentials of each protein in different protein systems suggest that, because of the mutual interaction with cytochro me c, cytochrome b(5) mutants, especially the E44A-containing mutants, in the protein complexes suffer greater conformational changes upon r eduction than that of the wild type. The variation of these thermodyna mic parameters indicates that the strength of mutual interactions betw een cytochrome c and cytochrome b(5) or its mutants has the following order: Cyt c/Cyt b(5) > Cyt c/Cyt b(5) E56A > Cyt c/Cyt b(5) E44A > Cy t c/Cyt b(5) E44/56A.