More Pieces of the Puzzle: Transient State Analysis of Dihydroorotate Dehydrogenase B from Lactoccocus lactis.

Abstract

Dihydroorotate dehydrogenases (DHODs) catalyze the transfer of electrons between dihydroorotate and specific oxidant substrates. Class 1B DHODs (DHODBs) use NAD⁺ as the oxidant substrate and have a heterodimeric structure that incorporates two active sites, each with a flavin cofactor. One Fe₂S₂ center lies roughly equidistant between the flavin isoalloxazine rings. This arrangement allows for simultaneous association of reductant and oxidant substrates. Here we describe a series of experiments designed to reveal sequences and contingencies in DHODB chemistry. From these data it was concluded that the resting state of the enzyme is FAD•Fe₂S₂•FMN. Reduction by either NADH or DHO results in two electrons residing on the FMN cofactor that has a 47 mV higher reduction potential than the FAD. The FAD•Fe₂S₂•FMNH₂ state accumulates with a bisemiquinone state that is an equilibrium accumulation formed from a partial transfer of one electron to the FAD. Pyrimidine reduction is reliant on the availability of the Cys135 proton, as the C135S variant slows orotate reduction by ∼40-fold. The rate of pyrimidine reduction is modulated by occupancy of the FAD site; NADH•FAD•Fe₂S₂•FMNH₂•orotate complex can reduce the pyrimidine at 16 s^-1, while NAD⁺•FAD•Fe₂S₂•FMNH₂•orotate complex reduces the pyrimidine at 5.4 s^-1 and the FAD•Fe₂S₂•FMNH₂•orotate complex at 0.6 s^-1. This set of effector states account for the apparent discrepancy in the slowest rate observed in transient state single turnover reactions with limiting NADH and the limiting rate observed in steady state.