Cofactor-apoprotein hydrogen bonding in oxidized and fully reduced flavodoxin monitored by trans-hydrogen-bond scalar couplings |
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Authors: | Löhr Frank Yalloway Gary N Mayhew Stephen G Rüterjans Heinz |
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Affiliation: | Institut für Biophysikalische Chemie, Zentrum für Biomolekulare Magnetische Resonanz, Johann Wolfgang Goethe-Universit?t, Marie Curie-Strasse 9, 60439 Frankfurt am Main, Germany. |
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Abstract: | Hydrogen bonding plays a key role in the tight binding of the FMN cofactor and the regulation of its redox properties in flavodoxins. Hydrogen bonding interactions can be directly observed in solution by multidimensional heteronuclear NMR spectroscopy through the scalar couplings between donor and acceptor nuclei. Here we report on the detection of intermolecular trans-hydrogen-bond couplings ((h)J) between the flavin ring system and the backbone of Desulfovibrio vulgaris flavodoxin in the oxidized and the two-electron reduced states. For this purpose, experiments are adapted from pulse sequences previously applied to determining (h)J coupling constants in nucleic acid-base pairs and proteins. The resulting (h2)J(N,N), (h4)J(N,N), (h3)J(C,N), and (h1)J(H,N) couplings involve the (15)N(1), (13)C(2), and (15)N(3) nuclei of the pyrimidine moiety of FMN, whereas no such interactions are detectable for (13)C(4) and (15)N(5). Several long-range (15)N-(15)N, (13)C-(15)N, and (1)H-(15)N J-coupling constants within the flavin are obtained as "by-products". The magnitudes of both (h)J and regular J couplings are found to be dependent on the redox state. In general, good correlations between (h)J coupling constants and donor-group (1)H chemical shifts and also crystallographic donor-acceptor distances are observed. |
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Keywords: | flavin NMR spectroscopy protein structures quantitative J correlation TROSY |
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