31P-NMR-CIDNP (CIDNP = chemically induced dynamic nuclear polarization) spectroscopy was applied for the first time to investigate the formation and reaction of phosphorus-centered radicals obtained from phosphorus-containing photoinitiators. 13C-NMR- and 1H-NMR-CIDNP and ESR spectroscopies were used as complementary experimental techniques for the elucidation of the photochemistry of these compounds. The large hyperfine coupling constants of the 31P-nucleus results in a violation of Kaptein's rules, which is the only observation of this kind in 13C-NMR-CIDNP spectra reported so far. Interpretation of the CIDNP spectra, using a modification of Kaptein's rules for the 13C-NMR- and 1H-NMR-CIDNP, consistently shows that all compounds investigated undergo a photoinduced cleavage of the carbonyl−phosphinoyl bond from a triplet state. The fate of the primary radicals is discussed, and it is unambiguously shown by trapping experiments that the novel bisacylphosphine oxide photoinitiators give four radicals in a stepwise process.

The interaction of yeast iso-1-cytochrome c with its physiological redox partner cytochrome c peroxidase has been investigated using heteronuclear NMR techniques. Chemical shift perturbations for both 15N and 1H nuclei arising from the interaction of isotopically enriched 15N cytochrome c with cytochrome c peroxidase have been observed. For the diamagnetic, ferrous cytochrome c, 34 amides are affected by binding, corresponding to residues at the front face of the protein and in agreement with the interface observed in the 1:1 crystal structure of the complex. In contrast, for the paramagnetic, ferric protein, 56 amides are affected, corresponding to residues both at the front and toward the rear of the protein. In addition, the chemical shift perturbations were larger for the ferric protein. Using experimentally observed pseudocontact shifts the magnetic susceptibility tensor of yeast iso-1-cytochrome c in both the free and bound forms has been calculated with HN nuclei as inputs. In contrast to an earlier study, the results indicate that there is no change in the geometry of the magnetic axes for cytochrome c upon binding to cytochrome c peroxidase. This leads us to conclude that the additional effects observed for the ferric protein arise either from a difference in binding mode or from the more flexible overall structure causing a transmittance effect upon binding.

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Solution 1H NMR spectroscopy has been used to characterize the cyanomet myoglobin complexes of a variety of chemically modified hemins in order to elucidate the importance of hemin peripheral electronic, relative to axial His imidazole-induced, rhombic perturbations in raising the orbital degeneracy of the π-bonding d xz ,d yz orbitals. Variation of the hemin 2- and/or 4-position substituents among hydrogen, ethyl, vinyl, acetyl, and formyl groups leads to conserved molecular structure of the heme pocket and orientation of the major magnetic axis for the heme iron, but systematically perturbed heme methyl contact shift patterns. Two strongly rhombically perturbed hemins with single acetyl groups on either pyrrole I or II exhibit heme methyl contact shift patterns and characteristic deviations from Curie law that are very similar to that induced in pseudosymmetric hemins upon incorporation into metMbCN in the alternate orientations about the α,γ-meso axis. The perturbation due to the 4-acetyl group and the axial His bond leads to increased contact shift spread and stronger deviations from Curie behavior compared to WT, indicative of an increased d xz /d yz spacing relative to WT. In contrast, the perturbation due to the 2-acetyl group and axial His nearly cancel, leading to a highly compressed methyl contact shift spread and weaker deviations from Curie behavior than WT. It is shown, moreover, that the larger d xz /d yz splitting with 4-acetylhemin, and the smaller splitting with 2-acetylhemin, relative to WT, result in the expected increase and decrease, respectively, for the axial His contact shift relative to WT. Comparison of the methyl shifts for 16 peripherally modified hemins as model compounds and incorporated into metMbCN shows that the rhombic influences are additive in each of the complexes. Thus, the present results show that chemical functionality of the heme periphery contributes to raising the orbital degeneracy of the heme iron and that such influences can account for orbital ground states that are not necessarily aligned with the axial His orientation. The range of variant 2- and/or 4-substitutions have led to equilibrium heme orientations that are largely the same as found in WT Mb, except for a 4-ethyl group, which favors the reversed heme orientation by 2:1.