Helix-Capping Histidines: Diversity of N–H···N Hydrogen Bond Strength Revealed by ^2h<i>J</i>_NN Scalar Couplings

In addition to its well-known roles as an electrophile and general acid, the side chain of histidine often serves as a hydrogen bond (H-bond) acceptor. These H-bonds provide a convenient pH-dependent switch for local structure and functional motifs. In hundreds of instances, a histidine caps the N-terminus of α- and 3₁₀-helices by forming a backbone NH···Nδ1 H-bond. To characterize the resilience and dynamics of the histidine cap, we measured the <i>trans</i> H-bond scalar coupling constant, ^2h<i>J</i>_NN, in several forms of Group 1 truncated hemoglobins and cytochrome <i>b</i>₅. The set of 19 measured ^2h<i>J</i>_NN values were between 4.0 and 5.4 Hz, generally smaller than in nucleic acids (∼6–10 Hz) and indicative of longer, weaker bonds in the studied proteins. A positive linear correlation between ^2h<i>J</i>_NN and the difference in imidazole ring ¹⁵N chemical shift (Δ¹⁵N = |δ¹⁵Nδ1 – δ¹⁵Nε2|) was found to be consistent with variable H-bond length and variable cap population related to the ionization of histidine in the capping and noncapping states. The relative ease of ^2h<i>J</i>_NN detection suggests that this parameter can become part of the standard arsenal for describing histidines in helix caps and other key structural and catalytic elements involving NH···N H-bonds. The combined nucleic acid and protein data extend the utility of ^2h<i>J</i>_NN as a sensitive marker of local structural, dynamic, and thermodynamic properties in biomolecules