Biochemistry of oxygenases: Mechanistic studies of a cofactor-independent, CO-formingm dioxygenase belonging to the a/ß-hydrolase fold family

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2-Methyl-3-hydroxy-4(1H)quinolone 2,4-dioxygenase (Hod) is a cofactor-independent dioxygenase with an α/β-hydrolase fold, catalyzing the cleavage of its organic substrate to carbon monoxide and N-acetylanthranilate. The overall goal of this project is to contribute to a comprehensive understanding of how cofactor-free oxygenases work, and to mechanistically compare cofactor-independent with metal- and flavin-dependent oxygenases. Transient kinetics studies will be performed to obtain insight into the time-dependent microscopic pathway of the Hod-catalyzed reaction. Combined with available structural data (Dr. R. Steiner, London), a molecular understanding of the reaction pathway is expected. The following issues will be addressed: (1) Determination of dissociation constants of Hod and Hod “muteins” for organic substrates. (2) Pre-steady state kinetics using stopped-flow spectrophotometry/spectrofluorometry: Detection of spectral intermediates and determination of rate constants for the formation and decay of transient species. (3) Kinetic analysis of the reaction catalyzed by Hod “muteins” to assess the role of active-site residues. (4) Analysis of the effects of pH on the kinetics, to deduce pKa values for intermediate species and/or for protonable groups of the enzyme, and to assess the role of individual active-site residues in proton transfer steps. (5) Detection and analysis of intermediate radicals by rapid-freeze EPR complementing the UV/Vis stopped-flow experiments, using isotope substituted substrates for detailed analysis of quenched radical species.