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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.
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Building on a theme: The redox hierarchy of pyridine nucleotide-disulfide oxidoreductases
Flavin disulfide reductases (FDRs) are FAD-dependent enzymes that transmit electrons from NAD(P)H to reduce specific oxidant substrate disulfides. These enzymes have been studied extensively, most particularly the paradigm examples: glutathione reductase and thioredoxin reductase. The common, though not universal, traits of the family include a tyrosine- or phenylalanine-gated binding pocket for NAD(P) nicotinamides adjacent to the FAD isoalloxazine re-face, and a disulfide stacked against the si-face of the isoalloxazine whose dithiol form is activated for subsequent exchange reactions by a nearby histidine acting as a base. This arrangement promotes transduction of the reducing equivalents for disulfide exchange relay reactions. From an observational standpoint the proximal parallel stacking of three redox moieties induces up to three opportunities for unique charge transfer interactions (NAD(P)H FAD, NAD(P)+•FADH2, and FAD•thiolate). In transient state, the charge transfer transitions provide discrete signals to assign reaction sequences. This review summarizes the lineage of observations for the FDR enzymes that have been extensively studied. Where applicable and in order to chart a consistent interpretation of the record, only data derived from studies that used anaerobic methods are cited. These data reveal a recurring theme for catalysis that is elaborated with specific additional functionalities for each oxidant substrate.
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- Award ID(s):
- 2203593
- PAR ID:
- 10517102
- Editor(s):
- Jin, Jian-Ping; Forman, Henry
- Publisher / Repository:
- Elsivier
- Date Published:
- Journal Name:
- Archives of Biochemistry and Biophysics
- Volume:
- 755
- Issue:
- C
- ISSN:
- 0003-9861
- Page Range / eLocation ID:
- 109966
- Subject(s) / Keyword(s):
- flavoprotein transient kinetics disulfide, reductase, thioredoxin glutathione
- Format(s):
- Medium: X Size: N/A Other: N/A
- Size(s):
- N/A
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1016/j.abb.2024.109966
