Direct Detection of a Histidine−Histidine Side Chain Hydrogen Bond Important for Folding of Apomyoglobin

Sperm whale myoglobin in which the heme group has been removed (apomyoglobin) unfolds to an equilibrium intermediate form at pH 4 and can be completely unfolded at acid pH and low salt conditions. The titration of a pair of partially buried histidine side chains, His24 and His119, is particularly important for the acid-induced formation of the intermediate form from native apomyoglobin. Modifying a recently introduced 1 H− 15 N HNN-COSY nuclear magnetic resonance (NMR) experiment (Dingley, A. J.; Grzesiek, S. J. Am. Chem. Soc. 1998, 120, 8293−8297) allowed us to detect a 2 J NN scalar coupling between imidazole NH nitrogen of His119 and the unsubstituted imidazole nitrogen of His24. These measurements directly verify the existence of a previously proposed side chain−side chain hydrogen bond important for the folding mechanism of apomyoglobin.