15N solid-state NMR as a probe of flavin H-bonding

Abstract

Flavins mediate a wide variety of chemical reactions in biology. To learn how one cofactor can be made to execute different reactions in different enzymes, we are developing solid-state NMR (SSNMR) to probe the flavin electronic structure, via the (15)N chemical shift tensor principal values (δ(ii)). We find that SSNMR has superior responsiveness to H-bonds, compared to solution NMR. H-bonding to a model of the flavodoxin active site produced an increase of 10 ppm in the δ(11) of N5, although none of the H-bonds directly engage N5, and solution NMR detected only a 4 ppm increase in the isotropic chemical shift (δ(iso)). Moreover SSNMR responded differently to different H-bonding environments, as H-bonding with water caused δ(11) to decrease by 6 ppm, whereas δ(iso) increased by less than 1 ppm. Our density functional theoretical (DFT) calculations reproduce the observations, validating the use of computed electronic structures to understand how H-bonds modulate the flavin's reactivity.

Figure 1

Optical spectrum of TPARF in benzene and effects of complexation with DBAP.

Figure 2

Solution NMR of ¹⁵N-N5-TPARF in benzene, alone or with added saturating DBAP or water (5 stoichiometric equivalents).

Figure 3

Comparison of the ¹⁵N CP-MAS spectra of dry [¹⁵N-N5] TPARF, [¹⁵N-N5] TPARF in benzene and [¹⁵N-N5] TPARF in benzene saturated with DBAP, all at a MAS speed of 3000 Hz. (Central peaks in the red box are enlarged for comparison of the δ_isos.)

Figure 4

Distribution of NPA electron density in LF (top) and LF complexed with DBAP (bottom). The total charge of LF changed from 0.01 to 0.04 upon complexation with DBAP. Structures are oriented as in Scheme 1, red indicates excess electron density, yellow indicates electron deficiency.

Figure 5

Changes in GIAO NMR CSPVs and δ_iso calculated from optimized LF•H₂O complexes. The configuration with a water H-bonding to N5 (Δ) yielded a large decrease in δ₁₁ whereas the configurations with water H-bonding to different positions (X, *, +) yielded increases in δ₁₁. Boltzmann-weighted averages of the shifts of single-water configurations are in filled circles, ●. The red □s denote the experimental changes in chemical shift upon addition of water and the associated experimental errors are used to generate error margins shown as dashes connected by lines. Calculated results that fall between these errors can be considered to be in agreement with experiment.

Scheme 1

Structure of TPARF (black) in complex with DBAP (grey), showing the numbering of the flavin N positions. The flavin variants discussed herein are distinguished only by their side chains: R = methyl for lumiflavin (LF); R = ribitylphosphate for FMN; R = ribityladenosine diphosphate for FAD, R = tetraacetylribityl for TARF, and R = tetraphenylacetylribityl for TPARF.