High resolution EPR studies of the fine structure of heme proteins. Third harmonic detection approach

When third harmonic detection is applied in EPR studies of the superhyperfine structure of nitrosyl derivatives of a number of human hemoglobin variants, significant resolution enhancement is obtained. This has allowed a detailed analysis of the number of superhyperfine lines, their g-values and their splittings, and has led to a more complete understanding of the interaction between the axial ligands of the heme iron. Studies of the effect of the modulation amplitude on EPR line-shapes revealed that the amplitude required to resolve fine structure in the third harmonic mode is 3-10 time larger than that used to record an undistorted first derivative spectrum. The application of this approach for other systems is discussed, and practical guidelines for its use are given.