A 360-MHz 1H CIDNP investigation of the photoreaction of β-NADH (nicotinamide adenine dinucleotide) with a substituted lumiflavin (7,8,10-trimethyl-3-carboxymethylisoalloxazine) has been performed with the recent technique of laser flash photolysis NMR. Nuclear spin polarization is observed in both NADH itself and the reaction product, NAD+. In the presence of oxygen, CIDNP spectra are consistent with a primary electron-transfer step from NADH to triplet-state flavin. Back electron transfer within the geminate radical ion pair generates polarized NADH, while the escaping NADH+· cation radicals deprotonate and react with dissolved oxygen to give NAD+. The lack of CIDNP time dependence during the period 1 µs-100 ms after flash photolysis agrees with this oxidation being diffusion controlled. In oxygen-free solution, however, the NAD+ polarization is seen, by comparison with INDO calculated hyperfine coupling constants, to be characteristic of the neutral radical NAD·. CIDNP results show that NADH+· is an oxidant while NAD· is a reductant toward flavosemiquinone radicals. The slow time dependence of the NAD+ polarization found in degassed solution probably reflects the slow oxidation of NAD· by residual oxygen of flavin. CIDNP spectrum simulations of the strong multiplet effect seen for the C-4 methylene protons in NADH gives information on the hyperfine couplings and g value of NADH+·. © 1982, American Chemical Society. All rights reserved.
