NMR Spectroscopy on Flavins and Flavoproteins

Abstract

(1)H-, (11)B-, (13)C-, (15)N-, (17)O-, (19)F-, and (31)P-NMR chemical shifts of flavocoenzymes and derivatives of it, as well as of alloxazines and isoalloxazinium salts, from NMR experiments performed under various experimental conditions (e.g., dependence of the chemical shifts on temperature, concentration, solvent polarity, and pH) are reported. Also solid-state (13)C- and (15)N-NMR experiments are described revealing the anisotropic values of corresponding chemical shifts. These data, in combination with a number of coupling constants, led to a detailed description of the electronic structure of oxidized and reduced flavins. The data also demonstrate that the structure of oxidized flavin can assume a configuration deviating from coplanarity, depending on substitutions in the isoalloxazine ring, while that of reduced flavin exhibits several configurations, from almost planar to quite bended. The complexes formed between oxidized flavin and metal ions or organic molecules revealed three coordination sites with metal ions (depending on the chemical nature of the ion), and specific interactions between the pyrimidine moiety of flavin and organic molecules, mimicking specific interactions between apoflavoproteins and their coenzymes. Most NMR studies on flavoproteins were performed using (13)C- and (15)N-substituted coenzymes, either specifically enriched in the pterin moiety of flavin or uniformly labeled flavins. The chemical shifts of free flavins are used as a guide in the interpretation of the chemical shifts observed in flavoproteins. Although the hydrogen-bonding pattern in oxidized and reduced flavoproteins varies considerably, no correlation is obvious between these patterns and the corresponding redox potentials. In all reduced flavoproteins the N(1)H group of the flavocoenzyme is deprotonated, an exception is thioredoxin reductase. Three-dimensional structures of only a few flavoproteins, mostly belonging to the family of flavodoxins, have been solved. Also the kinetics of unfolding and refolding of flavodoxins has been investigated by NMR techniques. In addition, (31)P-NMR data of all so far studied flavoproteins and some (19)F-NMR spectra are discussed.

Spectroscopic Properties of Lumiflavin: A Quantum Chemical Study

“…The change in water is by 13.44 ppm and in cyclohexane by 4.86 ppm. In summary, based on the assignment of the NMR shieldings we confirm the experimental findings that identified N 5 as the most favorable nucleophilic reaction center (87,88). However, we find the shielding of N 1 to be the most sensitive to the polarization of the solvent environment.…”

Proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor

“…Riboflavin auxotrophic E. coli BSV11 strain was used as expression host for production of apo-EE. This strain is defective in riboflavin synthesis and was obtained by Tn5 transposon mutagenesis 13 . For enzyme production, a 10 mL pre-culture inoculated with cells harbouring the pBAD-EE plasmid was grown o/n at 37 °C in Luria-Bertani (LB) medium containing 100 μg/mL ampicillin and 50 μM riboflavin.…”

“…The majority (90%) of flavoproteins bind their cofactor non-covalently 7 . Flavin dissociation provides the opportunity of studying the properties of the apoenzyme 11 12 , and allows for reconstituting the holoprotein with isotopically enriched 13 or artificial 14 flavins. Furthermore, apoflavoenzymes can be selectively immobilised by anchoring to a flavin-containing carrier 15 .…”

Site-Selective Synthesis of 15N- and 13C-Enriched Flavin Mononucleotide Coenzyme Isotopologues

“…Flavins uniformly enriched with 15 N and 13 C are employed to improve the NMR signal sensitivity, particularly when the cofactors are bound to proteins. Selective 15 N and 13 C enrichment is used to enhance signals for specific atoms of interest in an unlabeled background and for individual peak assignments . Site-specifically 15 N and 13 C enriched flavins are highly useful for unequivocal band assignments in IR and Raman spectroscopy …”