Chemistry PhD Final Defense – Michael Martinez
About the event
Title: Thermodynamic Driving Forces in Cytochrome P450 Enzyme and Cytochrome P450 Reductase
Abstract: For cytochrome P450s, the lipid bilayer is vital for simulating a native environment which in turn affects the metabolic activity of this family proteins. Though dissociations constants have been measured on solubilized proteins, the effects of the lipids cannot be disregarded. Furthermore, the donation of electrons from cytochrome P450 reductase (CPR) is required to continue the catalytic cycle once that ligand has bound, which has previously been shown to interact with a lipid bilayer biomimetic differently upon reduction by NADPH. It has been previously shown that P450 luminesces weakly when solubilized, and that interaction of CPR with a lipid bilayer is redox dependent. This work shows that (1) that the composition of the endoplasmic reticulum biomimetic affects the ligand affinity via the native luminescence of P4503A4, and (2) from temperature dependent fluorescence correlation spectroscopy and single particle tracking the standard state free energies, enthalpies, and entropies of the NAPDH dependent CPR insertion processes were all measured. Revealing that the partitioning of oxidized CPR into the ER is an exothermic process with a small positive change in entropy, while reduced CPR partitioning is endothermic with a large positive change in entropy. Both resulted in negative free energies and strong association to the ER, but the Kd of CPRox insertion is measurably smaller than that of CPRred.