Insight into the Chemistry of Flavin Reduction and Oxidation inEscherichia coliDihydroorotate Dehydrogenase Obtained by Rapid Reaction Studies

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Abstract
Dihydroorotate dehydrogenase (DHOD) oxidizes dihydroorotate (DHO) to orotate in the only redox reaction of pyrimidine biosynthesis. The enzyme from Escherichiacoli is a membrane-bound FMN-containing enzyme that is thought to use ubiquinone as the oxidizing substrate. The chemistry of the reduction of the flavin in DHOD from E. coli by the substrate dihydroorotate (DHO) was studied at 4 °C in anaerobic stopped-flow experiments conducted over a broad range of pH values. A Michaelis complex that was characterized by a ∼20 nm red-shift of the oxidized flavin absorbance formed within the dead-time of the stopped-flow instrument (∼1 ms) upon mixing with DHO. The flavin of the intermediate was reduced by DHO, forming a reduced flavin−orotate charge-transfer complex. The rate constant for the flavin reduction reaction increased with pH, from a value of 1 s-1 at pH 6.5 to ∼360 s-1 at pH values greater than an observed pKa of 9.5 which was ascribed to Ser175, the active-site base. At all pH values, the reduced flavin−orotate charge-transfer complex dissociated too slowly to be catalytically relevant. Therefore, the oxidizing quinone substrate must bind to the reduced enzyme−orotate complex at a site distinct from the substrate binding site, in agreement with steady-state kinetic studies [Björnberg, O., Grüner, A.-C., Roepstorff, P., and Jensen, K. F. (1999) Biochemistry38, 2899−2908]. Menadione was used as a model quinone substrate to oxidize dithionite-reduced DHOD. The reduced enzyme−orotate complex reacted rapidly with menadione (180 s-1), demonstrating that the reduced enzyme−orotate complex is a catalytically competent intermediate.