1H NMR studies of the imidazole complex of cytochrome c: Resonance assignment and structural characterization of the heme cavity

Abstract

Two-dimensional 1H 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 b5. © 1995 American Chemical Society.