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.
Highlights
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 photolyasemediated DNA repair, magnetoreception of migratory birds, and the blue-light driven phototropism in plants
Proteins with flavin cofactors are of particular interest because their functions are widespread: Since their discovery in the 1 930s1 flavins have been recognized as nearly ubiquitous cofactors that are involved in the catalysis of a wide range of biological redox p rocesses[2,3]
Starting in the early 1980s, considerable effort was invested in solving the puzzle of how flavin cofactors are modulated to optimize their properties for the catalysis of a specific r eaction[15]
Summary
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 photolyasemediated DNA repair, magnetoreception of migratory birds, and the blue-light driven phototropism in plants. Mapping the unpaired electron-spin density by directly measuring 13C hfcs using ENDOR is much more promising, but has been hampered by (i) the low natural abundance of 13C, (ii) the limited availability of 13C flavin isotopologs, and (iii) the strong overlap of 13C hyperfine resonances with those arising from other magnetic nuclei, predominantly nitrogens, in ENDOR spectra recorded with EPR frequencies around 10 GHz, so that an unambiguous signal assignment could not be achieved.
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