Coupled Methyl Group Rotation in FMN Radicals Revealed by Selective Deuterium Labeling

Brosi, Richard; Illarionov, Boris; Heidinger, Lorenz; Kim, Ryu-Ryun; Fischer, Markus; Weber, Stefan; Bacher, Adelbert; Bittl, Robert; Schleicher, Erik

doi:10.1021/acs.jpcb.9b11331

Cited by 2 publications

(1 citation statement)

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“…Quite surprisingly, however, rather large hfcs, and hence, high spin densities, are also observed for C1′, C8 and C9. This finding could not be obtained from previous experimental spin density characterizations 22 , 42 , 43 due to the fact that hyperfine data on H6 or H5 only inadequately probe the spin densities at C8 or C9. In contrast to that, rather low electron spin density is detected on C2, C6, C7α, C8α and C9a, which serve as probes in the pyrimidine ring and the xylene ring.…”

Section: Resultsmentioning

confidence: 67%

Selective 13C labelling reveals the electronic structure of flavocoenzyme radicals

Schleicher

Rein

Illarionov

et al. 2021

Sci Rep

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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.

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Section: Resultsmentioning

confidence: 67%

Selective 13C labelling reveals the electronic structure of flavocoenzyme radicals

Schleicher

Rein

Illarionov

et al. 2021

Sci Rep

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Quantum Spin Resonance in Engineered Magneto-Sensitive Fluorescent Proteins Enables Multi-Modal Sensing in Living Cells

Abrahams,

Spreng,

Štuhec

et al. 2024

Preprint

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Quantum mechanical phenomena have been identified as fundamentally significant to an increasing number of biological processes. Simultaneously, quantum sensing is emerging as a cutting-edge technology for precision biosensing. However, biological based candidates for quantum-sensors have thus far been limited toin vitrosystems, are prone to light induced degradation, and require sophisticated experimental setups making high-throughput studies prohibitively complex. We recently created a new class of magneto-sensitive fluorescent proteins (MFPs) [1], which we now show overcome these challenges and represent the first biological quantum-based sensor that functions at physiological conditions and in living cells. Through directed evolution, we demonstrate the possibility of engineering these proteins to alter properties of their response to magnetic fields and radio frequencies. These effects are explained in terms of the spin correlated radical pair (SCRP) mechanism, involving the protein backbone and a bound flavin cofactor. Using this engineered system we demonstrate the first observation of a fluorescent protein exhibiting Optically Detected Magnetic Resonance (ODMR) in living bacterial cells at room temperature, at sufficiently high signal-to-noise to be detected in a single cell, paving the way for development of a new class ofin vivobiosensors. Magnetic resonance measurements using fluorescent proteins enable unprecedented technologies, for instance 3D spatial localisation of the fluorescence using gradient fields (i.e. Magnetic Resonance Imaging using an endogenous probe). We further demonstrate the use of multiple variants of MFPs for multiplexing or lock-in amplification of fluorescence signals, opening a new approach to combining or extracting multiple signals from a biological measurement. Taken together, our results represent a new intersection of imaging and perhaps actuation modalities for engineered biological systems, based on and designed around understanding the quantum mechanical properties of MFPs. [1] doi:10.5281/zenodo.8137174

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Coupled Methyl Group Rotation in FMN Radicals Revealed by Selective Deuterium Labeling

Abstract: Flavin semiquinones are common intermediate redox states in flavoproteins, and thus, knowledge of their electronic structure is essential for fully understanding their chemistry and chemical versatility. In this contribution, we use a combination of high-field ENDOR

Cited by 2 publications

References 73 publications

Selective 13C labelling reveals the electronic structure of flavocoenzyme radicals

Selective 13C labelling reveals the electronic structure of flavocoenzyme radicals

Quantum Spin Resonance in Engineered Magneto-Sensitive Fluorescent Proteins Enables Multi-Modal Sensing in Living Cells

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