Allen Orville 's research interests explained in more detail:

Choline Oxidase (CHO)
The accumulation of glycine betaine (N,N,N-(CH3)3-glycine) in many pathogens is an essential factor in their stress response toward hyperosmotic environments. The metabolite is most commonly generated by either a flavin-dependent dehydrogenase or an oxidase. Choline oxidase (CHO) from Arthrobacter globiformis catalyzes the four electron oxidation of choline to glycine betaine, with betaine aldehyde as a two-electron oxidized intermediate. In each of the two oxidative half-reactions, a molecule of O2 is converted into a H2O2 molecule. The ~130 mV midpoint reduction potential of CHO for the two-electron transfer in the catalytically competent enzyme-product complex is the highest determined for flavoenzymes to date. We have very recently solved the 1.8 Å resolution crystal structure of choline oxidase. The electron density maps clearly resolve a covalent linkage between the His99(N epsilon 2) and FAD(C8M) atoms. Moreover, the electron density maps for the FAD also reveal an unusually distorted isoalloxazine ring system. The electron density is consistent with an sp3 hybridized C4a flavin adduct, which is currently modeled as either a C4a-OO(H) or C4a-O(H) complex. Our recent density functional theory (DFT) calculations (B3LYP/6-31G(d,p)) define the electronic structure of the flavin adduct, and the contributions of the active site residues that stabilize the observed structure. We hypothesize that the C4a-OO(H) complex is generated in situ in a two step process. First, the FAD is photo-reduced to FADH- by the synchrotron x-ray irradiation. Then O2 from the aerobic crystal binds to the C4a position of the FADH-. The complex does not release H2O2 because the cryogenic x-ray data collection methods do not establish the appropriate proton inventory on the surrounding residues. Rather, these same residues further stabilize the peroxy species with several H-bonds. In contrast, the proper H+ inventory is established by choline oxidation, which provides two protons and two electrons in the first reductive half-reaction. In the oxidative half-reaction, O2 accepts two electrons through an FAD C4a interaction and the delivery of two protons yields H2O2, which readily dissociates. Therefore, photo-reduction of the FAD does not increase the proton count on the active site histidine residues and/or flavin. Consequently, the C4a-complex may not be protonated and it does not break down. The C4a-OO(H) or C4a-O(H) reactive intermediates have been proposed for many flavoenzymes, but have only rarely been detected by transient kinetics and spectroscopy. Thus we are the first to directly observe such a species in any flavoenzyme, despite decades of effort by many researchers in the field and numerous mechanistic proposals that invoke such a species!

Last Modified: April 10, 2009
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