Studies on Dihydroorotate Dehydrogenase by Electron Paramagnetic Resonance Spectroscopy

When dihydroorotate dehydrogenase, from Zymobacterium oroticum, was reduced by DPNH or dihydroorotate and examined by electron paramagnetic resonance spectrometry, a relatively intense free radical signal and the asymmetric signal at g = 1.94, found in many nonheme iron proteins, were observed within a few milliseconds. Both shape and saturation with microwave power of the free radical signals varied with the specific electron-donating and -accepting substrates, the presence of an inhibitor, the pH, and the time of reaction. Optical reflectance spectra of frozen samples indicated formation of at least two intermediates, one typical for DPNH plus enzyme (λmax at 565 and at 610 to 620 mµ) and one for dihydroorotate (or DPNH plus orotate) and enzyme (λmax at 585 and 640 mµ). These optical and electron paramagnetic resonance observations point to a variety of intermediate species in the reactions catalyzed by this enzyme. The maximal free radical signals, obtained rapidly with DPNH at pH 6.5, were approximately 1.5 times those which appeared with dihydroorotate at pH 8.2, whereas the iron signals were more nearly comparable.