A photochemically induced dynamic nuclear polarisation (photo-CIDNP) study is presented of the amino acids that are polarisable with a flavin dye. These include derivatives of tryptophan, tyrosine, histidine, methylated lysines and methionine. The influence of pH, concentration and chemical modification on the magnitude of the CIDNP effect has been studied to obtain mechanistic information about the radical pair formation. The pH and concentration dependence of tyrosine and tryptophan polarisation could be accounted for quantitatively. The CIDNP evidence indicates that hydrogen-atom abstraction is important in generating radical pairs in the case of histidine and tyrosine, while electron transfer prevails in the case of tryptophan, the methylated lysines and methionine.

1. The aromatic proton resonances in the 360-MHz 'H nuclear magnetic resonance (NMR) spectrum of bovine pancreatic ribonuclease were divided into histidine, tyrosine and phenylalanine resonances by means of pH titrations and double resonance experiments.2. Photochemically induced dynamic nuclear polarization spectra showed that one histidine (His-1 19) and two tyrosines are accessible to photo-excited flavin. This permitted the identification of the C-4 proton resonance of His-119.3. The resonances of the ring protons of Tyr-25, Tyr-76 and Tyr-1 15 and the C-4 proton of His-12 were identified by comparison with subtilisin-modified and nitrated ribonucleases. Other resonances were assigned tentatively to 4. On addition of active-site inhibitors, all phenylalanine resonances broadened or disappeared. The resonance that was most affected was assigned tentatively to Phe-120.5. Four of the six tyrosines of bovine RNase, identified as Tyr-76, Tyr-115 and, tentatively, Tyr-73 and Tyr-92, are titratable above pH 9. The rings of Tyr-73 and Tyr-115 are rapidly rotating or flipping by 180" about their CP-Cc" bond and are accessible to flavin in photochemically induced dynamic nuclear polarization experiments. Tyr-25 is involved in a pH-dependent conformational transition, together with Asp-14 and His-48. A scheme for this transition is proposed.6. Binding of active-site inhibitors to bovine RNase only influences the active site and its immediate surroundings. These conformational changes are probably not connected with the pH-dependent transition in the region of 7. In NMR spectra of RNase A at elevated temperatures, no local unfolding below the temperature of the thermal denaturation was observed. NMR spectra of thermally unfolded RNase A indicated that the deviations from a random coil are small and might be caused by interactions between neighbouring residues.

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Upon light excitation of the flavin moiety in flavin adenine dinucleotide (FAD) a biradical is formed via electron transfer from the adenine part. The photo-CIDNP effects arising from this biradical are presented. At high magnetic field the polarization is due to T0-S mixing and shows a pronounced pH dependence similar to that of the fluorescence behavior. This is interpreted in terms of a shift of the stacking/destacking equilibrium of FAD toward the stacked form when going from acid to neutral pH. The complete absence of CIDNP at neutral pH indicates that the biradical is more completely stacked than the ground-state molecule. This gives rise to a strong exchange interaction that suppresses CIDNP at both high and low fields.