H-1-NMR STUDIES OF THE IMIDAZOLE COMPLEX OF CYTOCHROME-C - RESONANCE ASSIGNMENT AND STRUCTURAL CHARACTERIZATION OF THE HEME CAVITY

Two-dimensional H-1 nuclear magnetic resonance exchange correlated spectroscopy (2D EXSY), 1D saturation transfer, and nuclear Overhauser effect (NOE) experiments have been utilized in conjunction with isotope labeling to allow a comprehensive assignment of the hyperfine shifted resonances in the low spin imidazole complex of cytochrome c (Im-cyt c). Various two-dimensional phase-sensitive experiments (EXSY, DQF-COSY, TOCSY) are combined to assign the majority of the heme pocket side-chain proton signals. 1D NOE and 2D NOESY spectra of Im-cyt c are presented. Qualitative interpretation of NOE data indicates the structure in the heme pocket of Im-cyt c has changed relative to cyt c. Large conformational changes are apparent on the methionine side of the heme in the environment of the axial-ligand. On the basis of the NOEs observed, the nearly parallel orientation of the bound imidazole to the plane of His18 imidazole has also been obtained, which agrees with the characteristic EPR g values similar to that of cytochrome b(5).