Nitrogen-15 and carbon-13 nuclear magnetic resonance of reduced flavins. Comparative study with oxidized flavins

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Abstract
[Elucidation of the electronic and structural properties reduced flavin compared to oxidized flavin are essential in understanding the redox reactions of flavoenzymes.] 15N and 13C NMR spectra of the fully reduced form of flavin were studied with riboflavin tetrabutyrate (RBUT), an organic solvent-soluble derivative of riboflavin. For the measurement of 15N resonances, 99% enriched [1,3-15N]RBUT and [1,3,5-15N]RBUT were synthesized. In order to assign the 13C resonances, 90% enriched [2-13C]RBUT, [4a-13C]RBUT, [4,10a-13C]RBUT and [8-2H3]RBUT were employed. The upfield shift of N(5) resonance upon reduction was remarkable (286 ppm), while the N(1) signal moved only by 79 ppm. The 1-bond 15N-H spin-spin coupling constant 1J[15N(5)-H] of the reduced RBUT was smaller than its 1J[15N(1)-H] and 1J[15N(3)-H]. These observations indicate that N(5) changed into sp3 hybridization upon reduction and lost the character of planar N. Most of the 13C nuclei of the reduced form resonated at higher field than did those of the oxidized form, which is well explained by the increase in .pi.-electron densities. Among the 13C resonances, the upfield shift of C(4a) was remarkable (32 ppm), which explains the reactivity of C(4a) in O2 flavoprotein complexation. 13C-15N spin-spin coupling constants were obtained from the measurements of 13C magnetic resonance of 15N-enriched RBUT. The values of the 1-bond 13C-15N coupling constants increased markedly with protonation at N(1) and N(5) upon reduction.

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