NMR reveals hydrogen bonds between oxygen and distal histidines in oxyhemoglobin

Compared with free heme. the proteins hemoglobin (Hb) and myoglobin (Mb) ex hibit greatly enhanced affinity for oxygen relative to carbon monoxide. Thi s physiologically vital property has been attributed to either steric hindr ance of CO or stabilization of O-2 binding by a hydrogen bond with the dist al histidine. We report here the first direct evidence of such a hydrogen b ond in both alpha- and beta-chains of oxyhemoglobin, as revealed by heteron uclear NMR spectra of chain-selectively labeled samples. Using these spectr a, we have assigned the imidazole ring H-1 and N-15 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 shift s with values predicted from x-ray structures suggests differences between the solution and crystal structures of oxy-Hb. The chemical shift discrepan cies could be accounted for by very small displacements of the proximal and distal histidines. This suggests that NMR could be used to obtain very hig h-resolution heme pocket structures of Hb, Mb, and other heme proteins.