The proposed research is designed to help illuminate the general strategies used by enzymes to activate molecular oxygen. Reductive activation of dioxygen occurs in a wide range of biological processes, from oxidative metabolism by cytochrome P450s and non-metal- containing flavoenzymes to activation of chemotherapeutic agents like bleomycin. Recently attention has turned to the mechanisms by which organic cofactors, like flavins and pterins, react with dioxygen. This work will focus on glucose oxidase (GO), a flavoenzyme that uses molecular oxygen to convert glucose to gluconolactone. The oxidative phase of GO catalysis is particularly amenable to study because it is kinetically uncomplicated, especially at high pH where the rate- controlling step is a single electron transfer. The microscopic steps involved in dioxygen activation will be examined including the parameters that control electron transfer rates. From kinetic studies the electron transfer distance will be determined and will be used, in conjunction with the available crystallographic data, to design site- directed mutant proteins. The mutagenesis studies will allow removal of positively charged amino acid residue(s) which are believed to communicate electrostatic stabilization to the incipient superoxide intermediate. Thus, the proposed mechanism of enzyme action will be tested. In addition, the oxygen-18 kinetic isotope effects (0-18 KIEs) associated with GO catalysis will be investigated. The driving-force dependence will be probed through a systematic study involving the use of apo enzyme reconstituted with synthetic flavins. The results will provide a frame of reference for the interpretation of 0-18 KIEs observed in other systems where single-electron transfer mechanisms are indicated. Attempts will also be made to study the temperature dependence of the 0- 18 KIE and, thereby, address the possibility of nuclear tunneling during the reduction process.