Effects of naturally occurring S47F/A mutations on the structure and function of human cytochrome c

The sequence and structure of human cytochrome c ( h Cyt c ) exhibit evolutionary conservations, with only a limited number of naturally occurring mutations in humans. Herein, we investigated the effects of the naturally occurring S47F/A mutations on the structure and function of h Cyt c in the oxidized form. Although the naturally occurring S47F/A mutations did not largely alter the protein structure, the S47F and S47A variants exhibited a small fraction of high-spin species. Kinetic studies showed that the peroxidase activity of the variants was enhanced by ∼2.5-fold under neutral pH conditions, as well as for the rate in reaction with H 2 O 2 , when compared to those of wild-type h Cyt c . In addition, we evaluated the interaction between h Cyt c and human neuroglobin ( h Ngb) by isothermal titration calorimetry (ITC) studies, which revealed that the binding constant was reduced by ∼8-fold as result of the mutation of the hydrophilic Ser to the hydrophobic Phe/Ala. These findings provide valuable insights into the role of Ser47 in Ω-loop C in sustaining the structure and function of h Cyt c .