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Dihydroorotate dehydrogenases (DHODs) are common to all life and catalyze the oxidation of dihydroorotate (DHO) to orotate the precursor of all pyrimidine nucleotides. The core structure of all DHODs has a TIM-barrel topology (the PyrD subunit or domain) that harbors an FMN cofactor that interacts with DHO. There are two classes of DHOD enzymes. Each has unique structures and oxidant substrates that conserve part of the energy available by coupling the reaction to ATP synthesis. The class 1 enzymes are soluble and divided into classes 1A and 1B. Class 1A has fumarate as the electron acceptor forming succinate and is the simplest form of DHOD, successively binding DHO and fumarate at the same active site locale. Class 1B uses NAD+ as the oxidant and this form of DHOD is heterodimeric having, in addition to the PyrD subunit, a subunit (PyrK) whose structure is like those of ferredoxin reductases. PyrK adds a second active site with a bound FAD that interacts with the NAD+ substrate and includes an Fe2S2 center that resides at the interface of the subunits, forming a conduit for electrons. Class 2 DHODs have ubiquinone (UQ) as the electron acceptor. This form of DHOD is membrane associated via an N-terminal domain that also forms a quinone binding site end-on to the FMN xylene moiety. This arrangement uses the flavin to mediate between the substrates and as a redox partition between water-soluble NAD+ and lipid soluble UQ10. In this review, we summarize the structure and mechanism of DHOD enzymes. 

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. 

The physiological role of dihydroorotate dehydrogenase (DHOD) enzymes is to catalyze the oxidation of dihydroorotate to orotate in pyrimidine biosynthesis. DHOD enzymes are structurally diverse existing as both soluble and membrane-associated forms. The Family 1 enzymes are soluble and act either as conventional single subunit flavin-dependent dehydrogenases known as Class 1A (DHODA) or as unusual heterodimeric enzymes known as Class 1B (DHODB). DHODBs possess two active sites separated by ∼20 Å, each with a noncovalently bound flavin cofactor. NAD is thought to interact at the FAD containing site, and the pyrimidine substrate is known to bind at the FMN containing site. At the approximate center of the protein is a single Fe2S2 center that is assumed to act as a conduit, facilitating one-electron transfers between the flavins. We present anaerobic transient state analysis of a DHODB enzyme from Lactoccocus lactis. The data presented primarily report the exothermic reaction that reduces orotate to dihydroorotate. The reductive half reaction reveals rapid two-electron reduction that is followed by the accumulation of a four-electron reduced state when NADH is added in excess, suggesting that the initial two electrons acquired reside on the FMN cofactor. Concomitant with the first reduction is the accumulation of a long-wavelength absorption feature consistent with the blue form of a flavin semiquinone. Spectral deconvolution and fitting to a model that includes reversibility for the second electron transfer reveals equilibrium accumulation of a flavin bisemiquinone state that has features of both red and blue semiquinones. Single turnover reactions with limiting NADH and excess orotate reveal that the flavin bisemiquinone accumulates with reduction of the enzyme by NADH and decays with reduction of the pyrimidine substrate, establishing the bisemiquinone as a fractional state of the two-electron reduced intermediate observed.