PARAMAGNETIC H-1-NMR SPECTROSCOPY OF THE CYANIDE DERIVATIVE OF MET80ALA-ISO-1-CYTOCHROME-C

Mutation of the axial methionine residue (Met80) of iso-1-cytochrome c to an alanine gives a ligand-binding protein (Ala80cyt c) with spectroscopic features similar to those of myoglobin and peroxidases. H-1 NMR spectroscopy has been utilized to probe the active-site structure of the cyanide derivative of Ala80cyt c. The signals of the axial His18 protons have been assigned by using a superWEFT pulse sequence with variable, short recycle delays and a values as well as 1D NOE, 2D NOESY, and COSY experiments. The 1H NMR signals of the heme substituents also have been assigned; the similarity of the heme methyl shifts to those of wild-type ferricyt c-CN- indicates that the proximal His orientation is unchanged in the mutant. The large hyperfine shift and short T-1 of the signal attributable to the Tyr67 hydroxyl proton suggest that this proton is hydrogen-bonded to the ligated cyanide.