ELECTRON-NUCLEAR DOUBLE RESONANCE FROM FLAVIN RADICALS IN LIQUID AND POLYCRYSTALLINE PHASE AND CONJUGATED TO PROTEIN

Electron-nuclear double resonance (ENDOR) has been observed from flavin (isoalloxazine) anionic radicals and radical chelates (Zn2+ and Cd2+) in N,N-dimethyl-formamide solution. Hyperfine couplings to strongly coupled methyl protons could be determined and assigned by the aid of isotopically and chemically substituted compounds. No signals from other protons could be detected in the liquid phase. The methyl couplings of the anionic radical and the radical ligand of the chelates are not identical. No difference between the methyl couplings of the zinc and cadmium chelates was found. Large frozen samples showed powder ENDOR signals from rotating methyl groups. Weakly coupled protons were observed in the vicinity of the matrix ENDOR signal from the dimethylformamide protons. The low-temperature technique was extended to a flavoprotein, NADPH dehydrogenase (EC 1.6.99.1). The spectrum of prosthetic FMN radicals was similar to that from anionic radicals in polycrystalline dimethylformamide. The strongly coupled methyl group is rotating rapidly at - 160°. Indication of a preferential orientation of the ribityl chain was found. A strong matrix ENDOR signal was observed, which decreased by about 40% when 2H2O was used as solvent. This figure should be correlated to the percent of exchangeable protons in the immediate environment of the active site. This finding together with the strength and temperature behaviour of the matrix signal indicates that the active sites are inside the protein. © 1969.