Modeling PHS Activity in Azurin Comparing Tert-Butyl Hydrogen Peroxide with Oxygen Gas as Oxidants

Abstract

Metalloproteins are common among living organisms, with one third to one half of all proteins requiring a metal ion for proper structure or function. They are associated with many key processes, such as signal transduction, nutrient storage and enzyme-mediated catalysis. One such metalloprotein, phenoxazinone synthase (PHS), is a hexameric, multicopper oxidase that creates aminophenoxazinone (APX) from ortho-aminophenol (oAP). APX is a major component of actinomycin D, an antibiotic. One way to examine the enzymatic activity of PHS is through the use of the protein azurin as a model system. Native azurin is a small copper metalloprotein, with a single Type 1 copper center, and has no PHS activity. However, it can model the reactivity of PHS through the addition of a Type 2 copper center via targeted mutagenesis. In our studies, the resulting models were then given additional mutations, in order to more accurately understand the effects that the proteins’ specific structure has on the kinetics and rate of the redox reaction. One of the original models, NiR3His-Azurin, has the added Type 2 copper center. This scaffold protein was found to be active in PHS enzyme assays and is the predecessor to a “second-generation” azurin mutant: Met121Leu, which was designed to alter the reduction potential and therefore reactivity of the copper centers in azurin. In the Met121Leu mutant, a methionine near the copper active site is replaced with a leucine. This leads to an increased reduction potential, and the hypothesis that the resulting PHS activity rate will be decreased, due to a decrease in the reoxidation of the protein, a rate limiting step in the catalysis. The differences between the PHS activity of the NiR3His and Met121Leu mutants were explored by using both oxygen gas (O2) and tert-butyl hydrogen peroxide (TBHP) as oxidants. It is known that TBHP is a stronger oxidant than O2, and should cause a faster turnover of the redox reaction by the protein, thereby giving a higher rate of activity. However, O2 allows for the study of the protein using the native oxidant. Preliminary trials indicate that the Met121Leu variant is not significantly than the observable 0.0118 mMol/min Vmax for Nir3His, using both TBHP and O2 as oxidants.