Differences in functional cross-talk between loops C and D in two mitochondrial cytochromes

The heme in the protein cytochrome (cyt) c is surrounded by loops C and D that differ in their sequences across species. Mutations G41S and Y48H in human (hu) cyt c are associated with thrombocytopenia and have been extensively studied. Herein, we describe effects of the same mutations in horse heart (hh) cyt c and make comparisons to those in the hu protein. While wild-type (WT) hu and hh cyt c proteins have similar global stabilities, unfolding of the hh protein is less cooperative. The differences are further amplified in the mutants. Loop dynamics were probed computationally through MD simulations and experimentally by characterizing the alkaline transition. With G41S and Y48H mutations in hh cyt c, loop C made fewer contacts with loop D and the dynamics of loop D were enhanced. Only the Y48H mutation decreased the reduction potential in hh cyt c, but both G41S and Y48H mutations increased the intrinsic peroxidase activity. The rate constant k_f for the Met-to-Lys ligand switch in the alkaline transition differed in the hu and hh cyt c mutational series: this rate constant decreased upon mutations in the hu protein but increased with analogous mutations in the hh protein. The observed trends are rationalized based on the sequence variation in the two proteins, particularly the identity of residue 46 and differences in interactions it makes with loop D. Functional implications of the sequence difference for electron transfer and peroxidase activity are discussed.