Nuclear magnetic resonance study of exchangeable protons in ferrocytochrome c

Five well-resolved exchangeable proton resonances have been observed in horse ferrocytochrome c in the low-field region between ?10.0 and ?12.0 ppm. A resonance at ?11.6 ppm is assigned to an amino proton of His 26 on the basis of its magnetic field position, pH dependence and its correlation with histidine at this position in eight species. A resonance at ?10.9 ppm observed in horse and donkey ferrocytochrome c is assigned to a hydrogen-bonded ?-amino proton of Lys 60. This resonance shifts upfield with increasing salt and decreasing pH, with the shifts increasing as one goes from chloride to bromide to iodide. With less assurance, a resonance at ?10.6 ppm is assigned to the indole amino of Trp 59 and a resonance at ?10.3 ppm to the amino proton of the His 18. These resonances have been used to study the binding of small anions to ferrocytochrome c, the results of which show that inorganic phosphate, ADP and ATP all bind in the immediate vicinity of His 26, and that the inorganic phosphate has the greatest effect upon the imidazole amino exchange time.