The pentaammineruthenium(III)-histidine complex in ribonuclease A as an optical probe of conformational change

The formation of stable transition metal complexes between pentaammineruthenium(III) and the imidazole moiety of histidine residues in ribonuclease A is described. Studies of the kinetics of incorporation suggest that three of the four histidines are reactive and ion-exchange chromatography affords the isolation of three derivatives containing a single ruthenium-histidine complex. Equilibrium studies of the thermal- and urea-induced unfolding of one of these derivatives show that the ligand-to-metal change-transfer absorptions above 300 nm can be used to follow the conformational changes in the vicinity of the complex. For the urea-induced unfolding of this derivative, the coincidence of the transition curves obtained from the change-transfer absorptions and from difference ultraviolet spectroscopy at 286 nm, which measures the exposure of buried tyrosine residues to solvent, suggest that only two conformations, native and unfolded, are present at equilibrium in significant concentrations.