Kelly McIntosh - University of Windsor

Placement: Concordia University
Supervisor: Dr. Ann English

Probing the Role of Cytochrome C Peroxidase as a Peroxynitrite Reductase

Cytochrome C Peroxidase (CCP) is a 34-kDa heme protein found in the intermembrane space of yeast mitochondria. Its potential antioxidant role as a peroxynitrite [ONOO(H)] reductase was studied both in vitro and in vivo. It is essential for ONOO(H) scavengers to be highly efficient in order to effectively compete with scavengers such as CO₂ that generate toxic products. Using stopped-flow kinetics, the apparent second-order rate constant (k_app) for the reaction of CCP with peroxynitrite was analyzed as a function of proton concentration. We predicted that k_app would increase at low pH values (< 6.8) since protonated ligands are preferred by the CCP active site. The kinetics of peroxynitrite reduction were complemented by further studies in vivo to assess how well CCP offers protection against nitrosative stress. Using a mutant yeast strain with the CCP gene deleted, challenge experiments were performed with various concentrations of peroxynitrite-producing, 3-morpholino-sydnonimine (SIN-1). Assuming that CCP functions as a defense enzyme, a substantial decrease in viability of the CCP knockout yeast was predicted on exposure to SIN-1. Based on the combined in vivo and in vitro results, CCP’s role as a protective ONOO(H) scavenger will be evaluated..