Assignment of the heme c resonances in the 360 MHz 1H NMR spectra of cytochrome c

Abstract

In the 360 MHz 1H NMR spectra of horse heart ferrocytochrome c recorded after suitable digital resolution enhancement, the resonances of all the heme c protons with the exception of those of the propionic acid side chains were observed as well resolved lines. From spin decoupling and nuclear Overhauser effects in homonuclear double resonance experiments, all these resonances were assigned to their respective positions in heme c. With saturation transfer experiments in solutions of partially reduced cytochrome c, individual assignments were further obtained for the six heme c methyl resonances in ferricytochrome c. The present experiments add individual assignments to the earlier identifications of the heme c ring methyl and meso-proton resonances, and show that the earlier identifications of the thioether bridge methyl resonances must be revised. These data provide a basis for more detailed descriptions of the electronic structure of heme c and its possible relations with the pathway of the electron transfer in and out of the cytochrome c molecule. Furthermore, the pseudocontact shifts of the thioether bridge methyl resonances could be related to the electronic g-tensor measured by EPR in ferricytochrome c single crystals at low temperature. From this it will now be possible without chemical modification of the protein, to compare in detail the solution conformations near the heme c in reduced and oxidized cytochrome c and thus hopefully to obtain additional insights into the mechanism of the biological redox reaction of this protein.