Phenoxazinone synthase Activity of Azurin Variants

Abstract

Phenoxazinone synthase (PHS) is a multicopper oxidase that catalyzes the oxidation of ortho-aminophenol to aminophenoxazinone, by coupling the 6 e- oxidation with the reduction of O2. To study this reaction, an azurin protein scaffold model was used. Our models contain the naturally occurring T1 copper center and an additional designed surface T2 copper center. The T2 copper centers in these variants were modeled after 2 other naturally occurring copper oxidoreductases, nitrite reductase (NiR) and peptidylglycine α-hydroxylating monooxygenase (PHM). A PHS activity assay was adapted to investigate the activities of PHM-Azurin and NiR-Azurin using an already 2 e- reduced peroxide shunt pathway as well as the native O2 as oxidants. The assay was optimized with a 20 mM potassium phosphate buffer at pH 7.5 and a 10 mM tert- butylhydroperoxide shunt at a final enzyme concentration of 1 μM. Second generation variants in which the T1 copper center's reduction potential was decreased and an electron transfer tryptophan mutation was incorporated were assayed and found to increase turnover number for almost all surface T2 copper variants.