Spectroscopic and kinetic studies of bovine xanthine oxidase and Rhodobacter capsulatus xanthine dehydrogenase

 
 
 
 

Spectroscopic and kinetic studies of bovine xanthine oxidase and Rhodobacter capsulatus xanthine dehydrogenase

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Title: Spectroscopic and kinetic studies of bovine xanthine oxidase and Rhodobacter capsulatus xanthine dehydrogenase
Author: Stockert, Amy L.
Description: Xanthine oxidase (XO) and dehydrogenase (XDH) are molybdenum enzymes that catalyze the conversion of hypoxanthine to xanthine and xanthine to uric acid. The enzymes contain two 2Fe/2S centers, a FAD and a molybdopterin. Research has indicated similar structure and mechanism, thus study of both enzymes prove useful in understanding mechanism. Resonance Raman (rR) studies of reduced enzyme in complex with violapterin (a true catalytic intermediate) allow enzymatic mechanism to be inferred by comparison of calculated shifts to observed shifts. The proposed mechanism utilizes a two-electron reduction from Mo (VI) to Mo (IV) in one step, however, two one-electron steps have also been proposed. To test this alternate mechanism, the one-electron reduction potentials of several substrates were determined and compared to kinetic parameters for each of these substrates. No correlation is observed between these two parameters ruling out the one-electron mechanism. Recombinant wild-type and several variants of RcXDH were used to study residues involved in catalysis. Kinetics for the E232A mutant show slower reaction rates for both the xanthine and hydroxyl-methylpurine (HMP). EPR studies of this mutant with HMP give a signal similar to the very rapid signal seen with the bovine XO with small modifications and remain unchanged in D2O. Kinetic studies on E730 mutants indicate substantial rate decreases as this residue is proposed to act as the active site base initiating catalysis. Wild-type EPR experiments with HMP have also been performed and a signal such as the rapid type I seen with the bovine XO is observed. The signal indicates two inequivalent protons that disappear upon reacting in D2O. In conclusion, resonance Raman studies have supported the computational model of the enzyme-reduced product complex predicted based on our proposed mechanism. Examination of the first step in the reductive half-reaction indicates a single two-electron reduction. Mutational studies of the active site residues of XDH identify E730 as an essential residue, possibly acting as the active site base initiating catalysis.
Permanent Link: http://rave.ohiolink.edu/etdc/view?acc_num=osu1089910515
http://hdl.handle.net/2374.OX/5026
Date: 2004

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