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Selective ¹³C labelling reveals the electronic structure of flavocoenzyme radicals.

ABSTRACT: Flavocoenzymes are nearly ubiquitous cofactors that are involved in the catalysis and regulation of a wide range of biological processes including some light-induced ones, such as the photolyase-mediated DNA repair, magnetoreception of migratory birds, and the blue-light driven phototropism in plants. One of the factors that enable versatile flavin-coenzyme biochemistry and biophysics is the fine-tuning of the cofactor's frontier orbital by interactions with the protein environment. Probing the singly-occupied molecular orbital (SOMO) of the intermediate radical state of flavins is therefore a prerequisite for a thorough understanding of the diverse functions of the flavoprotein family. This may be ultimately achieved by unravelling the hyperfine structure of a flavin by electron paramagnetic resonance. In this contribution we present a rigorous approach to obtaining a hyperfine map of the flavin's chromophoric 7,8-dimethyl isoalloxazine unit at an as yet unprecedented level of resolution and accuracy. We combine powerful high-microwave-frequency/high-magnetic-field electron-nuclear double resonance (ENDOR) with ¹³C isotopologue editing as well as spectral simulations and density functional theory calculations to measure and analyse ¹³C hyperfine couplings of the flavin cofactor in DNA photolyase. Our data will provide the basis for electronic structure considerations for a number of flavin radical intermediates occurring in blue-light photoreceptor proteins.

SUBMITTER: Schleicher E

PROVIDER: S-EPMC8440535 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Selective <sup>13</sup>C labelling reveals the electronic structure of flavocoenzyme radicals.

Schleicher Erik E Schleicher Erik E Rein Stephan S Illarionov Boris B Lehmann Ariane A Al Said Tarek T Kacprzak Sylwia S Bittl Robert R Bacher Adelbert A Fischer Markus M Weber Stefan S

Scientific reports 20210914 1

Flavocoenzymes are nearly ubiquitous cofactors that are involved in the catalysis and regulation of a wide range of biological processes including some light-induced ones, such as the photolyase-mediated DNA repair, magnetoreception of migratory birds, and the blue-light driven phototropism in plants. One of the factors that enable versatile flavin-coenzyme biochemistry and biophysics is the fine-tuning of the cofactor's frontier orbital by interactions with the protein environment. Probing the ...[more]

PMID: 34521887

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Project description:Determining the accurate chemical structures of synthesized compounds is essential for biomedical studies and computer-assisted drug design. The unequivocal determination of N-adamantylation or N-arylation site(s) in nitrogen-rich heterocycles, characterized by a low density of hydrogen atoms, using NMR methods at natural isotopic abundance is difficult. In these compounds, the heterocyclic moiety is covalently attached to the carbon atom of the substituent group that has no bound hydrogen atoms, and the connection between the two moieties of the compound cannot always be established via conventional 1H-1H and 1H-13C NMR correlation experiments (COSY and HMBC, respectively) or nuclear Overhauser effect spectroscopy (NOESY or ROESY). The selective incorporation of 15N-labelled atoms in different positions of the heterocyclic core allowed for the use of 1H-15N (JHN) and 13C-15N (JCN) coupling constants for the structure determinations of N-alkylated nitrogen-containing heterocycles in solution. This method was tested on the N-adamantylated products in a series of azolo-1,2,4-triazines and 1,2,4-triazolo[1,5-a]pyrimidine. The syntheses of adamantylated azolo-azines were based on the interactions of azolo-azines and 1-adamatanol in TFA solution. For azolo-1,2,4-triazinones, the formation of mixtures of N-adamantyl derivatives was observed. The JHN and JCN values were measured using amplitude-modulated 1D 1H spin-echo experiments with the selective inversion of the 15N nuclei and line-shape analysis in the 1D 13С spectra acquired with selective 15N decoupling, respectively. Additional spin-spin interactions were detected in the 15N-HMBC spectra. NMR data and DFT (density functional theory) calculations permitted to suggest a possible mechanism of isomerization for the adamantylated products of the azolo-1,2,4-triazines. The combined analysis of the JHN and JCN couplings in 15N-labelled compounds provides an efficient method for the structure determination of N-alkylated azolo-azines even in the case of isomer formation. The isomerization of adamantylated tetrazolo[1,5-b][1,2,4]triazin-7-ones in acidic conditions occurs through the formation of the adamantyl cation.

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Selective ¹³C labelling reveals the electronic structure of flavocoenzyme radicals.

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Selective <sup>13</sup>C labelling reveals the electronic structure of flavocoenzyme radicals.

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Selective 13C labelling reveals the electronic structure of flavocoenzyme radicals.

Publications

Selective <sup>13</sup>C labelling reveals the electronic structure of flavocoenzyme radicals.

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OmicsDI is part of the ELIXIR infrastructure

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Selective ¹³C labelling reveals the electronic structure of flavocoenzyme radicals.