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

Abstract

Dihydroorotate dehydrogenase (DHOD) oxidizes dihydroorotate (DHO) to orotate in the only redox reaction of pyrimidine biosynthesis. The enzyme from Escherichia coli 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 degrees C in anaerobic stopped-flow experiments conducted over a broad range of pH values. A Michaelis complex that was characterized by a approximately 20 nm red-shift of the oxidized flavin absorbance formed within the dead-time of the stopped-flow instrument ( approximately 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 approximately 360 s(-1) at pH values greater than an observed pK(a) 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) Biochemistry 38, 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.

Lys-D48 Is Required for Charge Stabilization, Rapid Flavin Reduction, and Internal Electron Transfer in the Catalytic Cycle of Dihydroorotate Dehydrogenase B of Lactococcus lactis

“…4, C and D). With the membrane-bound dihydroorotate dehydrogenase of E. coli, binding of DHO in the dead time of the stopped-flow instrument gives rise to a ϳ20 nm red-shift in the spectrum of the enzyme-bound FMN (18). With DHODB, no such spectral shift was observed and the first spectrum recorded in the diode array dataset was essentially identical to that of the oxidized pure enzyme.…”

Lactococcus lactis Dihydroorotate Dehydrogenase A Mutants Reveal Important Facets of the Enzymatic Function

“…the N193A, P56A, and N127A proteins, concentrations up to 12 mM of orotate were used. To determine the dissociation constants (K D ) for the enzyme-orotate complexes, the data were fitted to Equation 1, which assumes a uniform affinity of the ligand at all binding sites and a binding stoichiometry of 1:1 between the ligand and the number of binding sites (21),…”

“…The earliest studies on DHOD from bovine liver (18) and Crithidia fasciculata (19) together with more recent studies on DHODB from Enterococcus faecalis (20) and on DHODC from E. coli (21) describe the stereospecific oxidation of (S)-dihydroorotate to orotate as mediated by an active site base. The active site base abstracts the C5-S proton of dihydroorotate (DHO) followed by hydride transfer from the C6 position of DHO to the N5 of FMN.…”

Oxygen Reactivity in Flavoenzymes: Context Matters

“…If O 2 reacts elsewhere in DHOD, then the Lys66Met substitution should have little affect in the Class 2 enzyme, as observed. The physiological oxidizing substrate of Class 2 DHODs is ubiquinone, which binds near the methyls of the isoalloxazine of the flavin; the reaction occurs by two single-electron transfers 25 . Therefore, Class 2 DHODs have evolved to transiently stabilize the flavin semiquinone.…”