The Examination of Compound II in Cytochrome P450s

Open Access
- Author:
- Lin, Leon
- Area of Honors:
- Chemistry
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Michael Thomas Green, Thesis Supervisor
Pshemak Maslak, Thesis Honors Advisor
Thomas E Maslak, Faculty Reader - Keywords:
- Cytochrome
P450s
Protein
C-H
Bond
Acivation
ferryl
pka
compound
II
Reactive
Intermediates - Abstract:
- Cytochrome P450’s are cysteine ligated heme enzymes found in a large variety of organisms and are able to oxidize C-H bonds, which are typically unreactive, and create functional substituents. The ability of P450 to catalyze oxidations on unreactive substrates is quite remarkable and is used in many processes such as metabolism of pharmaceuticals. The power of P450s to oxidize C-H bonds lies in their reactive intermediates compound I and compound II. The specific P450 being examined, CYP158A1 from Streptomyces coelicolor, yields significant amounts of compound II after site-directed mutagenesis. The electron reduction potential of compound I and the pKa of compound II plays a crucial role in the driving force for C-H hydroxylation. In order to examine compound II, CYP158A1 must be manipulated to increase the yield and stability of compound II. This is done through the changing of buffer environments and residues around the heme. Site-directed mutagenesis is used to replace tyrosine residues with phenylalanine residues which allow a high yield of compound II to be produced via peroxide shunt. The determination of the third P450 ferryl pKa of 12.615 is also done through the use of a pH jump experiment in conjunction with stopped flow spectroscopy.