Proton magnetic resonance study of the histidines in hemerythrin and chemical identification of the nonligand histidines

Abstract

Three non-iron-liganded histidines were studied in methemerythrin azide monomers from Phascolopsis gouldii by 250 MHz proton correlation NMR spectroscopy. Four of the 7 histidines in the protein are not observed because of paramagnetic broadening by the coordinated Fe; neither are they observed as contact or pseudocontact shifted resonances. The NMR titration of the 3 free histidines establishes them as normal histidines with pK'' values of 7.00 .+-. 0.03 and Hill coefficients of 0.90, 0.81 and 0.81 .+-. 0.03. The chemical shift of the protonated and neutral histidines is normal and the bandwidth of the resonance absorption is 5 Hz. A pH-dependent reversible transition in the chemical shift of the histidine C(2)H occurs at pH 6.5; above this pH the 3 protons occur as a singlet but break into 3 singlets of different chemical shift at acid pH values. Two of the 3 free histidines were identified by their susceptibility to photooxidation as His-82 and His-34.