Unraveling flavoenzyme reaction mechanisms using flavin analogues and linear free energy relationships

Thibodeaux, Christopher J.; Chang, Wei‐chen; Liu, Hung‐wen

doi:10.1016/bs.mie.2019.03.010

Cited by 4 publications

(2 citation statements)

References 42 publications

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“…Substitution of the methyl groups on the flavin C7 and C8 influences its redox potential as well as the basicity and nucleophilicity of its N5 group as predicted by inductive effects (Scheme ). ,− Linear free-energy correlations between such perturbations and enzyme turnover typically reveal key mechanistic constraints limiting catalysis. , If electron transfer from reduced flavin to the protonated substrate was rate determining, then catalytic efficiency should increase as the reducing power increases . Accordingly, the 7-chloro- and 8-methoxy-desmethylFMN (7-Cl-FMN, 8-MeO-FMN) were prepared by literature procedures , and used to reconstitute HhIYD with an on-column method described previously (Supporting Information).…”

Section: Resultsmentioning

confidence: 99%

Substrate Electronics Dominate the Rate of Reductive Dehalogenation Promoted by the Flavin-Dependent Iodotyrosine Deiodinase

et al. 2023

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Iodotyrosine deiodinase (IYD) is unusual in its reliance on flavin to promote reductive dehalogenation of halotyrosines under aerobic conditions. Applications of this activity can be envisioned for bioremediation, but expansion of its specificity requires an understanding of the mechanistic steps that limit the rate of turnover. Key processes capable of controlling steady-state turnover have now been evaluated and described in this study. While proton transfer is necessary for converting the electron-rich substrate into an electrophilic intermediate suitable for reduction, kinetic solvent deuterium isotope effects suggest that this process does not contribute to the overall efficiency of catalysis under neutral conditions. Similarly, reconstituting IYD with flavin analogues demonstrates that a change in reduction potential by as much as 132 mV affects k cat by less than 3-fold. Furthermore, k cat/K m does not correlate with reduction potential and indicates that electron transfer is also not rate determining. Catalytic efficiency is most sensitive to the electronic nature of its substrates. Electron-donating substituents on the ortho position of iodotyrosine stimulate catalysis and conversely electron-withdrawing substituents suppress catalysis. Effects on k cat and k cat/K m range from 22- to 100-fold and fit a linear free-energy correlation with a ρ ranging from −2.1 to −2.8 for human and bacterial IYD. These values are consistent with a rate-determining process of stabilizing the electrophilic and nonaromatic intermediate poised for reduction. Future engineering can now focus on efforts to stabilize this electrophilic intermediate over a broad series of phenolic substrates that are targeted for removal from our environment.

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Section: Resultsmentioning

confidence: 99%

Substrate Electronics Dominate the Rate of Reductive Dehalogenation Promoted by the Flavin-Dependent Iodotyrosine Deiodinase

et al. 2023

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“…Many flavins have been found covalently attached to amino acid side chains, as reviewed by Scrutton and Fraaije . Besides these, at the C8 position alone, replacements of the methyl with Cl, Br, F, COH, CN, OH, SH, and more have been reported. − Some of these occur in nature. ,, Many have valuable spectroscopic properties and therefore have been intentionally incorporated in enzymes. − The reported studies use them as spectroscopic probes to shed light on the reactivities produced by the protein site. − However, to understand what their shifts in absorption maxima or reactivities mean, at a fundamental and unifying level, we need to understand the electronic structure that underlies them. Conversely, the electronic spectra employed in experiments also provide invaluable opportunities to test the fidelity of the description provided by computation.…”

Section: Introductionmentioning

confidence: 99%

Tuning the Quantum Chemical Properties of Flavins via Modification at C8

et al. 2021

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Flavins are central to countless enzymes but display different reactivities depending on their environments. This is understood to reflect modulation of the flavin electronic structure. To understand changes in orbital natures, energies, and correlation over the ring system, we begin by comparing seven flavin variants differing at C8, exploiting their different electronic spectra to validate quantum chemical calculations. Ground state calculations replicate a Hammett trend and reveal the significance of the flavin π-system. Comparison of higher-level theories establishes CC2 and ACD(2) as methods of choice for characterization of electronic transitions. Charge transfer character and electron correlation prove responsive to the identity of the substituent at C8. Indeed, bond length alternation analysis demonstrates extensive conjugation and delocalization from the C8 position throughout the ring system. Moreover, we succeed in replicating a particularly challenging UV/Vis spectrum by implementing hybrid QM/MM in explicit solvents. Our calculations reveal that the presence of nonbonding lone pairs correlates with the change in the UV/Vis spectrum observed when the 8-methyl is replaced by NH2, OH, or SH. Thus, our computations offer routes to understanding the spectra of flavins with different modifications. This is a first step toward understanding how the same is accomplished by different binding environments.

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Probing the mechanism of flavin action in the oxidative decarboxylation catalyzed by salicylate hydroxylase

Brandão

Vieira

Araújo

et al. 2023

Methods in Enzymology

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Unraveling flavoenzyme reaction mechanisms using flavin analogues and linear free energy relationships

Cited by 4 publications

References 42 publications

Substrate Electronics Dominate the Rate of Reductive Dehalogenation Promoted by the Flavin-Dependent Iodotyrosine Deiodinase

Substrate Electronics Dominate the Rate of Reductive Dehalogenation Promoted by the Flavin-Dependent Iodotyrosine Deiodinase

Tuning the Quantum Chemical Properties of Flavins via Modification at C8

Probing the mechanism of flavin action in the oxidative decarboxylation catalyzed by salicylate hydroxylase

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