The tautomeric state of histidines in myoglobin

Abstract

1H-15N HMQC spectra were collected on 15N-labeled sperm whale myoglobin (Mb) to determine the tautomeric state of its histidines in the neutral form. By analyzing metaquoMb and metcyanoMb data sets collected at various pH values, cross-peaks were assigned to the imidazole rings and their patterns interpreted. Of the nine histidines not interacting with the heme in sperm whale myoglobin, it was found that seven (His-12, His-48, His-81, His-82, His-113, His-116, and His-119) are predominantly in the N epsilon2H form with varying degrees of contribution from the Ndelta1 H form. The eighth, His-24, is in the Ndelta1H state as expected from the solid state structure. 13C correlation spectra were collected to probe the state of the ninth residue (His-36). Tentative interpretation of the data through comparison with horse Mb suggested that this ring is predominantly in the Ndelta1H state. In addition, signals were observed from the histidines associated with the heme (His-64, His-93, and His-97) in the 1H-15N HMQC spectra of the metcyano form. In several cases, the tautomeric state of the imidazole ring could not be derived from inspection of the solid state structure. It was noted that hydrogen bonding of the ring was not unambiguously reflected in the nitrogen chemical shift. With the experimentally determined tautomeric state composition in solution, it will be possible to broaden the scope of other studies focused on the electrostatic contribution of histidines to the thermodynamic properties of myoglobin.

Structural Dynamics in the Active Site of Murine Neuroglobin and Its Effects on Ligand Binding

“…In water at 298 K, the imidazole side chain of histidine has a pK of 6.14 (39,40), with a preference of 4:1 for the proton at N ⑀2 instead of N ␦1 . In a protein, the pK values of protonating groups can vary drastically because of steric and charge interactions within their specific microenvironments.…”

Molecular Dynamics Simulations of CN− Dynamics and Spectroscopy in Myoglobin

“…However, the structural considerations above together with energetics from previously published QM/ MM calculations [15] strongly suggest that the His e 64 form is present under experimental conditions. We note that the protonation state of His64 is the subject of a long-standing discussion in the literature, but that support for the His e 64 assignment for MbCN has been provided by NMR studies [48] and from calculations of MbCO. [19,20] In one of the trajectories for FeÀCN/His e 64, one water molecule makes a transition from the Xe4 pocket to a region involved in the distal pathway leading to the solvent lined by residues Leu29, Phe33 and Phe43 (see Figure 5 c).…”

Electrostatic Effects in a Network of Polar and Ionizable Groups in Staphylococcal Nuclease

“…33 The tautomeric state of a histidine side chain in proteins is influenced by the properties of the local microenvironment. 34 Because the microenvironments of His121 and His124 were altered in the variant proteins, and because a change in the microenviron- Lines are intended to guide the eye between chemical shift correlations, to demonstrate that the pattern has the inverted L shape characteristic of the N ε2 tautomer. ment could lead to a change in the dominant tautomeric state, which would be reflected in a shift in the pK a value, it was necessary examine the tautomeric state of the histidines.…”