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. 17 for details).