Fluorescence Properties of Flavin Semiquinone Radicals in Nitronate Monooxygenase

Su, Dan; Kabir, Mohammad Pabel; Orozco‐Gonzalez, Yoelvis; Gozem, Samer; Gadda, Giovanni

doi:10.1002/cbic.201900016

Cited by 23 publications

(35 citation statements)

References 80 publications

(38 reference statements)

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“…This result contradicts the previously proposed mechanism of PAO in which C4ahydroperoxyflavin was proposed to be involved in the process of oxidative decarboxylation (23). The mechanism of L-LOX/MOG proposed here is also different from the mechanism of oxidative denitrification in nitronate monooxygenase, in which an anionic flavosemiquinone and a radical superoxide anion are proposed to be involved in the oxidative denitrification (36,55). The H 2 O 2 in the L-LOX/MOG reaction must also be in the bound state because free H 2 O 2 could not react with imino acid to generate decarboxylated product (Scheme 2, E3 and E4).…”

Section: Discussioncontrasting

confidence: 99%

Mechanistic insights into the dual activities of the single active site of l-lysine oxidase/monooxygenase from Pseudomonas sp. AIU 813

Trisrivirat

Lawan

Chenprakhon

et al. 2020

Journal of Biological Chemistry

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L-Lysine oxidase/monooxygenase (L-LOX/MOG) from Pseudomonas sp. AIU 813 catalyzes the mixed bioconversion of L-amino acids, particularly L-lysine, yielding an amide and carbon dioxide by an oxidative decarboxylation (i.e. apparent monooxygenation), as well as oxidative deamination (hydrolysis of oxidized product), resulting in α-keto acid, hydrogen peroxide (H2O2), and ammonia. Here, using high-resolution MS and monitoring transient reaction kinetics with stopped-flow spectrophotometry, we identified the products from the reactions of L-lysine and L-ornithine, indicating that besides decarboxylating imino acids (i.e. 5-aminopentanamide from L-lysine), L-LOX/MOG also decarboxylates keto acids (5-aminopentanoic acid from L-lysine and 4-aminobutanoic acid from L-ornithine). The reaction of reduced enzyme and oxygen yielding an imino acid and H2O2, with no detectable C4a-hydroperoxyflavin. Single turnover reactions in which L-LOX/MOG was first reduced by L-lysine to form imino acid before mixing with various compounds revealed that under anaerobic conditions, only hydrolysis products are present. Similar results were obtained upon H2O2 addition after enzyme denaturation. H2O2 addition to active L-LOX/MOG resulted in formation of more 5-aminopentanoic acid, but not 5-aminopentamide, suggesting that H2O2 generated from L-LOX/MOG in situ can result in decarboxylation of the imino acid, yielding an amide product, and extra H2O2 resulted in decarboxylation only of keto acids. Molecular dynamics simulations and detection of charge transfer species suggested that interactions between the substrate and its binding site on L-LOX/MOG are important for imino acid decarboxylation. Structural analysis indicated that the flavoenzyme oxidases catalyzing decarboxylation of an imino acid all share a common plug loop configuration that may facilitate this decarboxylation.

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Section: Discussioncontrasting

confidence: 99%

Mechanistic insights into the dual activities of the single active site of l-lysine oxidase/monooxygenase from Pseudomonas sp. AIU 813

Trisrivirat

Lawan

Chenprakhon

et al. 2020

Journal of Biological Chemistry

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“…c, Partial reduction of oxidized NicA2 with sodium dithionite produced a species with an increased absorbance from 525–650 nm. The spectrum of the titration point with the highest absorbance in this region is most consistent with a mixed population of oxidized flavin, flavin hydroquinone, and neutral flavin semiquinone 19 . Further titration with sodium dithionite resulted in complete reduction to the hydroquinone (FADH 2 ) state.…”

Section: Extended Datamentioning

confidence: 64%

“…The use of dithionite as a reductant enabled us to study the oxidative half-reaction in the absence of nicotine reaction products. During the dithionite titration, NicA2’s FAD first populated a form with a UV-vis spectrum resembling a neutral flavin semiquinone 19 before reaching the fully reduced FADH 2 state ( Extended Data Fig. 1c ).…”

Section: Resultsmentioning

confidence: 99%

A cytochrome c is the natural electron acceptor for nicotine oxidoreductase

et al. 2021

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Nicotine oxidoreductase (NicA2), a member of the flavin-containing amine oxidase family, is of medical relevance as it shows potential as a therapeutic to aid cessation of smoking due to its ability to oxidize nicotine into a non-psychoactive metabolite. However, the use of NicA2 in this capacity is stymied by its dismal O 2 -dependent activity. Unlike other enzymes in the amine oxidase family, NicA2 reacts very slowly with O 2 , severely limiting its nicotine-degrading activity. Instead of using O 2 as an oxidant, we discovered that NicA2 donates electrons to a cytochrome c, which means that NicA2 is actually a dehydrogenase. This is surprising, as enzymes of the flavin-containing amine oxidase family were invariably thought to use O 2 as an electron acceptor. Our findings establish new perspectives for engineering this potentially useful therapeutic and prompt a reconsideration of the term “oxidase” in referring to members of the flavin-containing amine “oxidase” family.

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“…We note that TD-DFT with B3LYP exchange has been successfully used to simulate the UV-visible spectra of flavin in good agreement with experiments in prior studies. 18,[56][57][58][59] All the multiconfigurational QM/MM calculations were computed with the OpenMolcas-TINKER interface. 60,61 TD-DFT calculations in the presence of ASEC point charges were performed in Gaussian 03.…”

Section: 𝑌 =mentioning

confidence: 99%

Ionic Atmosphere Effect on the Absorption Spectrum of a Flavoprotein: A Reminder to Consider Solution Ions

Dratch

Orozco‐Gonzalez

Gadda

et al. 2021

J. Phys. Chem. Lett.

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Ionizable residues and monoatomic ions in solution modulate enzyme catalysis and the structural stability of proteins; however, the delicate interplay between these short-range charges and longrange charges, and their contributions to the electrostatic environment in a protein active site, is currently not fully understood. The study presented here utilizes the FMN-dependent NADH:quinone oxidoreductase from Pseudomonas aeruginosa PAO1 (NQO, EC 1.6.5.9, UniProtKB Q9I4V0) as a model system to study the effect of introducing an active site negative charge on the flavin absorption spectrum both in the absence and presence of a long-range electrostatic potential coming from solution ions. Using pH-dependent UV-visible spectroscopy, there were no observed changes in the flavin absorption spectrum when an active site tyrosine (Y277) deprotonated in vitro. These results could only be reproduced computationally using Average Solvent Electrostatic Configuration (ASEC) hybrid quantum mechanics / molecular mechanics (QM/MM) simulations that included both positive and negative solution ions. The same calculations performed with minimal ions to neutralize protein charges predicted that deprotonating Y277 would significantly affect the flavin absorption spectrum. Analyzing the distribution of solution ions from ASEC and radial distribution functions derived from molecular dynamics indicated that the solution ions reorganize around the protein surface upon Y277 deprotonation to cancel the effect of the tyrosinate on the flavin absorption spectrum. Biochemical experiments were performed to support this hypothesis. This work highlights the importance of salt ions, which are sometimes overlooked, since they can contribute a non-uniform and anisotropic long-range potential to the electrostatic environment of an active site.

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Fluorescence Properties of Flavin Semiquinone Radicals in Nitronate Monooxygenase

Cited by 23 publications

References 80 publications

Mechanistic insights into the dual activities of the single active site of l-lysine oxidase/monooxygenase from Pseudomonas sp. AIU 813

Mechanistic insights into the dual activities of the single active site of l-lysine oxidase/monooxygenase from Pseudomonas sp. AIU 813

A cytochrome c is the natural electron acceptor for nicotine oxidoreductase

Ionic Atmosphere Effect on the Absorption Spectrum of a Flavoprotein: A Reminder to Consider Solution Ions

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