Studies on Dihydroorotate Dehydrogenase by Electron Paramagnetic Resonance Spectroscopy: II. Kinetic Studies by Rapid Freezing

Abstract

Abstract The kinetics of the reactions catalyzed by dihydroorotate dehydrogenase from Zymobacterium oroticum have been studied by means of a rapid mixing-freezing technique and observation by electron paramagnetic resonance and optical reflectance spectroscopy at low temperature. With both DPNH and dihydroorotate as reducing substrate, within the limits of error, the kinetics of appearance or disappearance of the radical signal and the signal at g = 1.94 were identical. The reduction of the flavin to the fully reduced state is not a catalytically significant reaction. Reduction of the enzyme components by DPNH is a fast step with a half-time of a few milliseconds, whereas reoxidation by orotate or oxygen is relatively slow and limiting in turnover. In the steady state, with DPNH as substrate, the electron carriers in the enzyme are largely reduced even in the presence of excess orotate or oxygen. The turnover numbers calculated from the present experiments agree with those derived from the kinetics of over-all catalysis under catalytic assay conditions. The formation of ferrous phenanthroline is a much slower reaction than the appearance of the g = 1.94 signal, even when enzyme previously reduced by DPNH is mixed with the chelator, and is not a suitable indicator of catalytically significant changes in the enzyme. It is concluded that formation of flavin semiquinone and electron uptake by a protein-bound iron complex of unknown structure are both catalytically significant reaction steps in all presently known reactions of the enzyme. In the presence of a mercurial, an electron path to oxygen not involving the iron complex with electron paramagnetic resonance signal at g = 1.94 may be established, while all other catalytic functions are severely impaired.