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Flavoprotein quinone reductases regenerate quinols which serve metabolic and antioxidant roles. These enzymes catalyze the two-electron oxidation of substrates and the subsequent two electron reduction of quinones. Despite the net two electron transfer between substrates, the binding mode of quinones is typically end-on to the flavin, rather than stacked, dictating that the oxidative half reaction cannot proceed via hydride transfer and must instead occur by two successive single electron transfers. Here we present a review of six of the most well-studied flavoprotein quinone reductases to establish a framework for discussing this positional orientation for the quinone oxidant. There are two non-mutually exclusive rationalizations for this binding mode where the flavin isoalloxazine acts as a redox partition. The first is that energetics of the single electron transfer pathway create a kinetic barrier to the reverse reaction, trapping electrons in the quinone pool and countering the high ratio of quinol to quinone present in the membrane. The second is that the end-on binding allows the enzymes to utilize different binding sites for cytosolic and membrane associated substrates, avoiding the need to desorb substrates. These effects may be additive and serve to funnel electrons into the quinone pool as efficiently as possible. 

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. 

Human ferroptosis suppressor protein 1 (HsFSP1) is an NAD(P)H:quinone oxidoreductase with broad substrate specificity that has been widely implicated in aiding malignant neoplastic cell survival. FSP1 is myristoylated and associated with membranes, where it regenerates the reduced forms of quinones using electrons from NADPH. The quinol products intercept reactive oxygen species and ameliorate lipid peroxidation, preventing ferroptosis, a form of regulated cell death. While FSP1 enzymes have been reported to have 6-OH-FAD as an active cofactor, aerobic titration of the enzyme with NADPH in the presence and absence of ubiquinone (UQ) reveals that this is more likely an artifact and that the native form of HsFSP1 has unmodified FAD as the cofactor. Moreover, HsFSP1 suppresses the reaction of the reduced FAD with molecular oxygen three-fold which, from a kinetic standpoint, severely limits the opportunity for cofactor modification. The isolated form of the enzyme has NADP+ bound and the rate of release of this product limits the observed rate of reduction by NAD(P)H molecules. The reduction of substrate quinones occurs rapidly (≥2000 s–1), dictating that the rate of turnover is wholly defined by the rate of release of NADP+ from the HsFSP1·NADP+ complex. Given that HsFSP1 does not distinguish ubiquinone from ubiquinol by significant differences in binding affinity, this pronounced catalytic commitment to quinone reduction serves to overcome presumed kinetic limitations imposed by the abundance of ubiquinol relative to ubiquinone in the membrane. This characteristic also maintains the enzyme ostensibly fully in the oxidized state under turnover conditions, preventing significant futile reduction of dioxygen. 

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. 

Thioredoxin/glutathione reductase from Schistosoma mansoni (SmTGR) is a multifunctional enzyme that catalyzes the reduction of glutathione (GSSG) and thioredoxin, as well as the deglutathionylation of peptide and non-peptide substrates. SmTGR structurally resembles known glutathione reductases (GR) and thioredoxin reductases (TrxR) but with an appended N-terminal domain that has a typical glutaredoxin (Grx) fold. Despite structural homology with known GRs, the site of GSSG reduction has frequently been reported as the Grx domain, based primarily on aerobic, steady-state kinetic measurements and x-ray crystallography. Here, we present an anaerobic characterization of a series of variant SmTGRs to establish the site of GSSG reduction as the cysteine pair most proximal to the FAD, Cys154/Cys159, equivalent to the site of GSSG reduction in GRs. Anaerobic steady-state analysis of U597C, U597S, U597C + C31S, and I592STOP SmTGR demonstrate that the Grx domain is not involved in the catalytic reduction of GSSG, as redox silencing of the C-terminus results in no modulation of the observed turnover number (∼0.025 s−1) and redox silencing of the Grx domain results in an increased observed turnover number (∼0.08 s−1). Transient-state single turnover analysis of these variants corroborates this, as the slowest rate observed titrates hyperbolically with GSSG concentration and approaches a limit that coincides with the respective steady-state turnover number for each variant. Numerical integration fitting of the transient state data can only account for the observed trends when competitive binding of the C-terminus is included, indicating that the partitioning of electrons to either substrate occurs at the Cys154/Cys159 disulfide rather than the previously proposed Cys596/Sec597 sulfide/selenide. Paradoxically, truncating the C-terminus at Ile592 results in a loss of GR activity, indicating a crucial non-redox role for the C-terminus. 

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. 

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. 

Thioredoxin/glutathione reductase (TGR) from the platyhelminthic parasitic worms has recently been identified as a drug target for the treatment of schistosomiasis. Schistosomes lack catalase, and so are heavily reliant on the regeneration of reduced thioredoxin (Trx) and glutathione (GSH) to reduce peroxiredoxins that ameliorate oxidative damage from hydrogen peroxide generated by the host immune response. This study focuses on the characterization of the catalytic mechanism ofSchistosoma mansoniTGR (SmTGR). Variant forms of SmTGR were studied to assign the function of residues that participate in the electron distribution chain within the enzyme. Using anaerobic transient state spectrophotometric methods, redox changes for the FAD and NADPH were observed and the function of specific residues was defined from observation of charge transfer absorption transitions that are indicative of specific complexations and redox states. The C159S variant prevented distribution of electrons beyond the flavin and as such did not accumulate thiolate-FAD charge transfer absorption. The lack of this absorption facilitated observation of a new charge transfer absorption consistent with proximity of NADPH and FAD. The C159S variant was used to confine electrons from NADPH at the flavin, and it was shown that NADPH and FAD exchange hydride in both directions and come to an equilibrium that yields only fractional FAD reduction, suggesting that both have similar reduction potentials. Mutation of U597 to serine resulted in sustained thiolate-FAD charge transfer absorption and loss of the ability to reduce Trx, indicating that the C596-U597 disulfide functions in the catalytic sequence to receive electrons from the C154 C159 pair and distribute them to Trx. No kinetic evidence for a loss or change in function associated with the distal C28-C31 disulfide was observed when the C31S variant reductive half-reaction was observed. The Y296A variant was shown to slow the rate of but increase extent of reduction of the flavin, and the dissociation of NADP⁺. The H571 residue was confirmed to be the residue responsible for the deprotonation of the C159 thiol, increasing its reactivity and generating the prominent thiolate-FAD charge transfer absorption that accumulates with oxidation of the flavin. 

Dihydropyrimidine dehydrogenase (DPD) is an enzyme that uses an elaborate architecture to catalyze a simple net reaction: the reduction of the vinylic bond of uracil and thymine. Known DPDs have two active sites separated by approximately 60 Å. One active site has an FAD cofactor and binds NAD(P) and the other has an FMN cofactor and binds pyrimidines. The intervening distance is spanned by four Fe4S4 centers that act as an electron conduit. Recent advancements with porcine DPD have revealed unexpected chemical sequences where the enzyme undergoes reductive activation by transferring two electrons from NADPH to the FMN via the FAD such that the active form has the cofactor set FAD•4(Fe4S4)•FMNH2. Here we describe the first comprehensive kinetic investigation of a bacterial form of DPD. Using primarily transient state methods, DPD from E. coli (EcDPD) was shown to have a similar mechanism to that observed with the mammalian form in that EcDPD is observed to undergo reductive activation before pyrimidine reduction and displays half-of-sites activity. However, two distinct aspects of the EcDPD reaction relative to the mammalian enzyme were observed that relate to the effector roles for substrates: (i) the enzyme will rapidly take up electrons from NADH, reducing a flavin in the absence of pyrimidine substrate, and (ii) the activated form of the enzyme can become fully oxidized by transferring electrons to pyrimidine substrates in the absence of NADH. 

Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, has been shown to play an essential role in the metabolic reprogramming and progression of hepatocellular carcinoma (HCC). HCC accounts for approximately 75% of primary liver cancers and is within the top three causes of cancer death worldwide. As a result of treatment limitations, the overall 5-year survival rate for all patients with HCC is under 20%. The prevalence of HCC necessitates continued development of novel and effective treatment methods. In recent years, the therapeutic potential of selective inactivation of hOAT has been demonstrated for the treatment of HCC. Inspired by previous increased selectivity for hOAT by the expansion of the cyclopentene ring scaffold to a cyclohexene, we designed, synthesized, and evaluated a series of novel fluorinated cyclohexene analogues and identified (R)-3-amino-5,5-difluorocyclohex-1-ene-1-carboxylic acid as a time-dependent inhibitor of hOAT. Structural and mechanistic studies have elucidated the mechanism of inactivation of hOAT by 5, resulting in a PLP-inactivator adduct tightly bound to the active site of the enzyme. Intact protein mass spectrometry, 19F NMR spectroscopy, transient state kinetic studies, and X-ray crystallography were used to determine the structure of the final adduct and elucidate the mechanisms of inactivation. Interestingly, despite the highly electrophilic intermediate species conferred by fluorine and structural evidence of solvent accessibility in the hOAT active site, Lys292 and water did not participate in nucleophilic addition during the inactivation mechanism of hOAT by 5. Instead, rapid aromatization to yield the final adduct was favored.