NMR reveals hydrogen bonds between oxygen and distal histidines in oxyhemoglobin

Abstract

Compared with free heme, the proteins hemoglobin (Hb) and myoglobin (Mb) exhibit greatly enhanced affinity for oxygen relative to carbon monoxide. This physiologically vital property has been attributed to either steric hindrance of CO or stabilization of O(2) binding by a hydrogen bond with the distal histidine. We report here the first direct evidence of such a hydrogen bond in both alpha- and beta-chains of oxyhemoglobin, as revealed by heteronuclear NMR spectra of chain-selectively labeled samples. Using these spectra, we have assigned the imidazole ring (1)H and (15)N chemical shifts of the proximal and distal histidines in both carbonmonoxy- and oxy-Hb. Because of their proximity to the heme, these chemical shifts are extremely sensitive to the heme pocket conformation. Comparison of the measured chemical shifts with values predicted from x-ray structures suggests differences between the solution and crystal structures of oxy-Hb. The chemical shift discrepancies could be accounted for by very small displacements of the proximal and distal histidines. This suggests that NMR could be used to obtain very high-resolution heme pocket structures of Hb, Mb, and other heme proteins.

Figure 1

Histidine region of the 600-MHz (¹H, ¹⁵N) echo anti-echo HMQC spectra of chain-selectively and fully ¹⁵N-labeled HbCO A and HbO₂ A in water and D₂O at 29°C. Cross-peaks in blue, violet, green, and orange come from spectra of (15)N-α-labeled HbCO A, (15)N-β-labeled HbCO A, (15)N-α-labeled HbO₂ A, and (15)N-β-labeled HbO₂ A, respectively. These experiments used a large spectral width in ¹⁵N and a short refocusing delay (see Materials and Methods for details). The spectra were acquired without ¹⁵N decoupling; therefore doublets are observed for directly bonded (15)N-¹H pairs. Also shown in green is a spectrum of (15)N-α-labeled HbO₂ A with a smaller ¹⁵N spectral width and a longer refocusing delay, set to eliminate the α58His (Hɛ₂, Nɛ₂) doublet, which overlaps the Hɛ₁ cross-peak at 5.64 ppm. Cross-peaks in black are those of fully ¹⁵N-labeled HbO₂ A in D₂O solution, which used a narrow ¹⁵N spectral width and a short refocusing delay. Cross-peaks originating from the same residue are connected by lines. Other cross-peaks originate from the solvent-exposed and interfacial histidyl residues not discussed in this paper (see ref. for details).

Figure 2

Proximal and distal histidyls of the HbO₂ A x-ray crystal structure (PDB entry 1HHO; ref. 8), with the hemes of the α- and β-subunits superimposed. The coordinates of hydrogen atoms were calculated from those of heavy atoms, using standard bond lengths and angles. The α- and β-subunits are shown in lighter and darker colors, respectively. This structure suggests that the distal histidine in the α-subunit is better disposed to form a H-bond with the O₂ ligand than is its counterpart in the β-subunit.