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1. 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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2. The energetics and evolution of oxidoreductases in deep time

   https://doi.org/10.1002/prot.26563  

   McGuinness, Kenneth N.; Fehon, Nolan; Feehan, Ryan; Miller, Michelle; Mutter, Andrew C.; Rybak, Laryssa A.; Nam, Justin; AbuSalim, Jenna E.; Atkinson, Joshua T.; Heidari, Hirbod; et al (August 2023, Proteins: Structure, Function, and Bioinformatics)

   Abstract The core metabolic reactions of life drive electrons through a class of redox protein enzymes, the oxidoreductases. The energetics of electron flow is determined by the redox potentials of organic and inorganic cofactors as tuned by the protein environment. Understanding how protein structure affects oxidation–reduction energetics is crucial for studying metabolism, creating bioelectronic systems, and tracing the history of biological energy utilization on Earth. We constructed ProtReDox (https://protein-redox-potential.web.app), a manually curated database of experimentally determined redox potentials. With over 500 measurements, we can begin to identify how proteins modulate oxidation–reduction energetics across the tree of life. By mapping redox potentials onto networks of oxidoreductase fold evolution, we can infer the evolution of electron transfer energetics over deep time. ProtReDox is designed to include user‐contributed submissions with the intention of making it a valuable resource for researchers in this field. 

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3. Single occupancy spectroelectrochemistry of freely diffusing flavin mononucleotide in zero-dimensional nanophotonic structures

   https://doi.org/10.1039/c5fd00072f  

   Zaino, Lawrence P.; Grismer, Dane A.; Han, Donghoon; Crouch, Garrison M.; Bohn, Paul W. (January 2015, Faraday Discussions)

   Zero-mode waveguides (ZMW) have the potential to be powerful confinement tools for studying electron transfer dynamics at single molecule occupancy conditions. Flavin mononucleotide contains an isoalloxazine chromophore, which is fluorescent in the oxidized state (FMN) while the reduced state (FMNH 2 ) exhibits dramatically lower light emission, i.e. a dark-state. This allows fluorescence emission to report the redox state of single FMN molecules, an observation that has been used previously to study single electron transfer events in surface-immobilized flavins and flavoenzymes, e.g. sarcosine oxidase, by direct wide-field imaging of ZMW arrays. Single molecule electron transfer dynamics have now been extended to the study of freely diffusing molecules using fluorescence measurements of Au ZMWs under single occupancy conditions. The Au in the ZMW serves both as an optical cladding layer and as the working electrode for potential control, thereby accessing single molecule electron transfer dynamics at μM concentrations. Consistent with expectations, the probability of observing single reduced molecules increases as the potential is scanned negative, E appl < E eq , and the probability of observing emitting oxidized molecules increases at E appl > E eq . Different single molecules exhibit different electron transfer properties as reflected in the position of E eq and the distribution of E eq among a population of FMN molecules. Two types of actively-controlled electroluminescence experiments were used: chronofluorometry experiments, in which the potential is alternately stepped between oxidizing and reducing potentials, and cyclic potential sweep fluorescence experiments, analogous to cyclic voltammetry, these latter experiments exhibiting a dramatic scan rate dependence with the slowest scan rates showing distinct intermediate states that are stable over a range of potentials. These states are assigned to flavosemiquinone species that are stabilized in the special environment of the ZMW nanopore. 

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4. Reduction Midpoint Potentials of Bifurcating Electron Transfer Flavoproteins

   https://doi.org/10.1016/bs.mie.2019.03.017  

   Miller, A.-F.; Duan, H. D.; Varner, T. A.; Mohamed-Raseek, N. (January 2019, Methods in enzymology)

   Recently, a variety of enzymes have been found to accept electrons from NAD(P)H yet reduce lower-potential carriers such as ferredoxin and flavodoxin semiquinone, in apparent violation of thermodynamics. The reaction is favorable overall, however, because these enzymes couple the foregoing endergonic one-electron transfer to exergonic transfer of the other electron from each NAD(P)H, in a process called 'flavin-based electron bifurcation'. The reduction midpoint potentials (E°s) of the multiple flavins in these enzymes are critical to their mechanisms. We describe methods we have found to be useful for measuring each of the E°s of each of the flavins in bifurcating electron transfer flavoproteins. 

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5. Non-covalent assembly of proton donors and p- benzoquinone anions for co-electrocatalytic reduction of dioxygen

   https://doi.org/10.1039/D1SC01271A  

   Hooe, Shelby L.; Cook, Emma N.; Reid, Amelia G.; Machan, Charles W. (July 2021, Chemical Science)

   The two-electron and two-proton p -hydroquinone/ p -benzoquinone (H 2 Q/BQ) redox couple has mechanistic parallels to the function of ubiquinone in the electron transport chain. This proton-dependent redox behavior has shown applicability in catalytic aerobic oxidation reactions, redox flow batteries, and co-electrocatalytic oxygen reduction. Under nominally aprotic conditions in non-aqueous solvents, BQ can be reduced by up to two electrons in separate electrochemically reversible reactions. With weak acids (AH) at high concentrations, potential inversion can occur due to favorable hydrogen-bonding interactions with the intermediate monoanion [BQ(AH) m ]˙ − . The solvation shell created by these interactions can mediate a second one-electron reduction coupled to proton transfer at more positive potentials ([BQ(AH) m ]˙ − + n AH + e − ⇌ [HQ(AH) (m+n)−1 (A)] 2− ), resulting in an overall two electron reduction at a single potential at intermediate acid concentrations. Here we show that hydrogen-bonded adducts of reduced quinones and the proton donor 2,2,2-trifluoroethanol (TFEOH) can mediate the transfer of electrons to a Mn-based complex during the electrocatalytic reduction of dioxygen (O 2 ). The Mn electrocatalyst is selective for H 2 O 2 with only TFEOH and O 2 present, however, with BQ present under sufficient concentrations of TFEOH, an electrogenerated [H 2 Q(AH) 3 (A) 2 ] 2− adduct (where AH = TFEOH) alters product selectivity to 96(±0.5)% H 2 O in a co-electrocatalytic fashion. These results suggest that hydrogen-bonded quinone anions can function in an analogous co-electrocatalytic manner to H 2 Q. 

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