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 ¹H−¹⁵N HNN-COSY nuclear magnetic resonance (NMR) experiment (Dingley, A. J.; Grzesiek, S. <i>J.</i> <i>Am.</i> <i>Chem.</i> <i>Soc.</i> <b>1998</b>, <i>120</i>, 8293−8297) allowed us to detect a ²<i>J</i>_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.