Pyrazole Cytochrome C Complexes

Cytochrome (cyt) c is well known to perform cellular functions unrelated to its role in respiratory electron flow, including signaling in the apoptosis pathway and peroxidation of lipids. These appear to involve a significant conformational change that allows access to the heme cleft. We have investigated the possible facilitation of such changes by small polar molecules that bind to the heme with displacement of the methionine. Among these, we found pyrazole (Prz) to bind weakly to ferricytochrome c with a strongly sigmoidal concentration dependence, with a midpoint ≈750mM and a Hill coefficient of 2.5, as measured in UV-Vis Spectroscopy at pH 6.5. Stopped-flow experiments show biphasic kinetics for binding of Prz and cyt c. Tentatively we model this behavior with a two-step binding reaction where Prz initially binds rapidly but weakly to displace the methionine ligand, with kfast ≈ 30s−1. The equilibrium is pulled over by a slower process, with kslow ≈ 0.05s−1, that is poorly understood. The slow phase contribution is small at pH 6.5, but increases at more alkaline pH. CW X-band EPR studies suggest the existence of two low-spin Prz-cyt c complexes, with g-values at gx = 1.55, gy = 2.28, gz = 2.83 and gx = 1.60, gy = 2.28, gz = 2.73, respectively. The ratio of one complex to the other as seen in EPR is highly dependent upon environment conditions including, but not limited to, method of freezing, glycerol concentration, and pH. Small Angle X-Ray Scattering experiments are planned to determine if significant structural changes are apparent on forming Prz-cyt c.