Three non-iron-liganded histidines have been studied in methemerythrin azide monomers from Pascolopsis gouldii by 250-MHz proton correlation nuclear magnetic resonance (NMR) spectroscopy. Four of the seven histidines in the protein are not observed because of paramagnetic broadening by the coordinated iron; neither are they observed as contact or pseudocontact shifted resonances. The NMR titration of the three 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 three protons occur as a singlet but break into three singlets of different chemical shift at acid pH values. Two of the three "free" histidines have been identified by their susceptibility to photooxidation as His-82 and His-34.
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