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Figure 1: 15N-PASS spectrum of {N1,N3,N5}-15N-labeled tetracetyl-riboflavin, collected at 2.5kHz MAS.
 

Figure 2: NBO-electron densities based on NMR-validated DFT calculations are also shown, note the significantly greater excess negative charge at N5 than at N1, which is consistent with observed differences in reactivity.

Electron redistribution in flavins as a means of protein control over flavin reactivity.

Flavins.

Flavins are the essence of the reactivity of a wide variety of crucial enzymes. These mediate extremely diverse reactions including oxidation/reduction, oxygenation, isomerization, hydroxylation and electron transfer, all based on this one versatile cofactor*. Thus, interactions with the protein modify the reactivity of the bound flavin. In addition to allowing only select substrates access to the flavin, the protein tunes the flavin reactivity to favour certain reactions and suppress others. This is accomplished at least in part via redistribution of the valence electron density within the highly delocalized flavin π system and modulation of the flavin valence orbital energies, via the non-covalent interactions usually used to bind the flavin. Thus, although redox activity is concentrated between N5 and N1, it is modulated by interactions with remote functionalities.

*Some enzyme-bound flavins are also chemically modified.

              We are determining how the frontier orbitals change in nature and energy in response to H bonds and distortion of the flavin ring system, using the synergistic combination of spectroscopy and quantum chemical computation [44,45]. We have employed SS-NMR (solid-state NMR) to obtain atom-specific information to test and refine our computational methodologies. The 'paramagnetic' contribution to NMR shielding reflects the energy separating the HOMO from low-lying unoccupied MOs, as well as the MOs' orbital angular momenta, and thus their natures (MO= molecular orbital). Moreover the three different principal values of the shielding tensor tend to reflect different orbitals. Thus, NMR shielding principal values obtained from SS-NMR are fundamentally related to frontier orbital natures and energy separations, which in turn underlie reactivity [55]. We have measured the three principal values of the NMR shielding tensor of N5 for flavins engaged in different H bonding interactions producing different reactivities, and related these to DFT calculations, to learn how flavin frontier electron distribution changes in response to hydrogen bonding [69]. Thus SS-NMR is a new and highly-sensitive tool for probing flavin electronics in proteins and understanding variations in flavin reactivity at a fundamental level.

Note that the semiquinone state of the flavin can also be used to greatly amplify the NMR signal of the protein (x15, so far), via Dynamic Nuclear Polarization [73].

New studies

We are also exploiting the superior sensitivity of resonance Raman spectroscopy and its ability to interrogate interactions with individual functionalities within the flavin. Thus we have two exceedingly perceptive tools to learn how the gentle but precise interactions with a protein site alter fundamental flavin electronics and modulate reactivity.

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Updated: Oct. 2017                                                                                               

Copyright 2017 A.-F. Miller     

Comments: A.-F. Miller