¹⁵N Solid-State NMR as a Probe of Flavin H-Bonding

Abstract

Flavins mediate a wide variety of different 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 15N chemical shift tensor principal values (δii). 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 δ11 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 (δiso). Moreover SSNMR responded differently to different H-bonding environments as H-bonding with water caused δ11 to decrease by 6 ppm whereas δiso 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.

Mutants of Cytochrome P450 Reductase Lacking Either Gly-141 or Gly-143 Destabilize Its FMN Semiquinone

“…A staggering array of versatile redox reactions is catalyzed by flavin-containing oxidases, oxygenases, dehydrogenases, and electron transfer proteins that occur in essential biochemical systems and pathways, including but not limited to photosynthesis, respiration, light-activated signal transduction, bacterial cell wall synthesis, DNA synthesis and repair, magnetic sensing, chemiluminescence, photomorphogenesis, and regulation of circadian rhythms (1)(2)(3)(4). The extraordinary chemical versatility of flavins provides metabolic economy, which means that only a few different cofactors are necessary to accomplish the biochemical reactions required for life (5,6).…”

“…Flavoproteins employ hydrogen bonding, microscopic dielectric constants, polarity, placement of local charges, and aromatic stacking interactions (which are mediated by dispersion and electrostatic forces) with the flavin to selectively stabilize a particular oxidation state and thereby provide the enzyme with the ability to control the cofactor redox potential and ultimately enzymatic function (12). In addition to tuning the thermodynamics of the reaction, flavoproteins also kinetically regulate electron transfer by influencing the rate of proton transfer (3).…”

Manipulating reduction potentials in an artificial safranin cofactor

“…The combination of natural and designed proteins with artificial cofactors is a rapidly expanding focus of modern enzyme design efforts [1–4]. The anticipated benefit of this combination is that a cofactor energetically and structurally optimized to perform the catalytic task at hand will aid the enzyme design or re-engineering effort by removing the necessity for the enzyme to “tune” the cofactor reactivity for the desired task[5–7].…”

“…The safraninemauveine was the first synthetic dye, created by Perkin in 1856 [1]. It was synthesized by the oxidation of crude aniline, itself derived from the nitration and reduction of a benzene-toluene mixture, and purified in a 5% yield from a mixture of products containing a variety of oligoanilines[24].…”

“…Given the large number of commercially available aniline derivatives, we hoped such a synthetic route may lead to a similarly large scope of safranine products. Furthermore, given our recent demonstration that the chemical shift tensor of the isoalloxazine N(5) nitrogen in flavin compounds is very informative as to the chemical reactivity imparted upon flavins by their environments[1, 26], we desired to created a route which enables the ready and inexpensive incorporation of isotopically labeled nitrogen at the equivalent N(5) position in the phenazine ring of these analogues.…”