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1. Building on a theme: The redox hierarchy of pyridine nucleotide-disulfide oxidoreductases

   https://doi.org/10.1016/j.abb.2024.109966  

   Smith, Madison M; Moran, Graham R (May 2024, Archives of Biochemistry and Biophysics)

   Jin, Jian-Ping; Forman, Henry (Ed.)

   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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2. A single hydrogen bond that tunes flavin redox reactivity and activates it for modification

   https://doi.org/10.1039/d4sc01642d  

   Das, Debarati; Miller, Anne-Frances (May 2024, Chemical Science)

   Electron bifurcation produces high-energy products based on less energetic reagents. This feat enables biological systems to exploit abundant mediocre fuel to drive vital but demanding reactions, including nitrogen fixation and CO2 capture. Thus there is great interest in understanding principles that can be portable to man-made devices. Bifurcating electron transfer flavoproteins (Bf ETFs) employ two flavins with contrasting reactivities to acquire pairs of electrons from the modest reductant, NADH. The flavins then disperse the electrons individually to high- and a low reduction midpoint potential (E°) acceptors, the latter of which captures most of the energy. Maximum efficiency requires that only one electron access the exergonic path that will 'pay for' production of the low-E° product. It is therefore critical that one of the flavins, the 'electron transfer' (ET) flavin, is tuned to execute single-electron (1e—) chemistry only. To learn how, and extract fundamental principles, we systematically altered interactions with the flavin O2 position. Removal of a single hydrogen bond (H-bond) disfavored formation of the flavin anionic semiquinone (ASQ) relative to the oxidized (OX) state, lowering E°ASQ/OX by 150 mV and retuning the flavin's tendency to 1e— vs. 2e— reactivity. This was achieved by replacing conserved His 290 with Phe while also replacing the supporting Tyr 279 with Ile. Although this variant binds oxidized FADs at 90% the WT level, the ASQ state of the ET flavin is not stable in the absence of H290's H-bond, and dissociates, contrary to WT. Removal of this H-bond also altered the ET flavin's covalent chemistry. Whereas the WT ETF accumulates modified flavins whose formation is believed to rely on an anionic paraquinone methide intermediate, the FADs of the H-bond lacking variant remain unchanged over weeks. Hence the variant that destabilizes the anionic semiquinone also suppresses the anionic intermediate in flavin modification, testifying to electronic similarities between these two species. These correlations suggest that the H-bond that stabilizes the crucial flavin ASQ also promotes flavin modification. The two effects may indeed be inseparable, as a Jekyll and Hydrogen bond. 

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3. Elucidation of the Catalytic Sequence of Dihydroorotate Dehydrogenase B from Lactoccocus lactis : Evidence for Accumulation of a Flavin Bisemiquinone State in Catalysis

   https://doi.org/10.1021/acs.biochem.4c00025  

   Smith, Corine O; Moran, Graham R (May 2024, Biochemistry)

   Schepartz, Alanna (Ed.)

   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. 

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4. Electrochemical Zero-Mode Waveguide Studies of Single Enzyme Reactions

   https://doi.org/10.1109/NANO.2018.8626402  

   Han, D.; Kwon, S.-R.; Fu, K.; Crouch, G.M.; Bohn, P.W. (July 2018, 2018 IEEE 18th International Conference on Nanotechnology (IEEE-NANO))

   null (Ed.)

   Because electron transfer reactions are fundamental to life processes, such as respiration, vision, and energy catabolism, it is critically important to understand the relationship between functional states of individual redox enzymes and the macroscopically observed phenotype, which results from averaging over all copies of the same enzyme. To address this problem, we have developed a new technology, based on a bifunctional nanoelectrochemical-nanophotonic architecture - the electrochemical zero mode waveguide (E-ZMW) - that can couple biological electron transfer reactions to luminescence, making it possible to observe single electron transfer events in redox enzymes. Here we describe E-ZMW architectures capable of supporting potential-controlled redox reactions with single copies of the oxidoreductase enzyme, glutathione reductase, GR, and extend these capabilities to electron transfer events where reactive oxygen species are synthesized within the  100 zL volume of the nanopore. 

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5. Noncovalent interactions that tune the reactivities of the flavins in bifurcating electron transferring flavoprotein

   https://doi.org/10.1016/j.jbc.2023.104762  

   González-Viegas, María; Kar, Rajiv K.; Miller, Anne-Frances; Mroginski, Maria-Andrea (June 2023, Journal of Biological Chemistry)

   Bifurcating electron transferring flavoproteins (Bf-ETFs) tune chemically identical flavins to two contrasting roles. To understand how, we used hybrid quantum mechanical molecular mechanical calculations to characterize non-covalent interactions applied to each flavin by the protein. Our computations replicated the differences between the reactivities of the flavins: the electron transferring flavin (ETflavin) was calculated to stabilize anionic semiquinone (ASQ) as needed to execute its single-electron transfers, whereas the Bf flavin (Bfflavin) was found to disfavor the ASQ state more than does free flavin and to be less susceptible to reduction. The stability of ETflavin ASQ was attributed in part to H-bond donation to the flavin O2 from a nearby His side chain, via comparison of models employing different tautomers of His. This H-bond between O2 and the ET site was uniquely strong in the ASQ state, whereas reduction of ETflavin to the anionic hydroquinone (AHQ) was associated with side chain reorientation, backbone displacement and reorganization of its H-bond network including a Tyr from the other domain and subunit of the ETF. The Bf site was less responsive overall, but formation of the Bfflavin AHQ allowed a nearby Arg side chain to adopt an alternative rotamer that can H-bond to the Bfflavin O4. This would stabilize the anionic Bfflavin and rationalize effects of mutation at this position. Thus, our computations provide insights on states and conformations that have not been possible to characterize experimentally, offering explanations for observed residue conservation and raising possibilities that can now be tested. 

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