Protonation behavior of histidine 24 and histidine 119 in forming the pH 4 folding intermediate of apomyoglobin

Heteronuclear NMR methods are used to study the protonation of histidine and aspartate residues in the acid-induced unfolding of recombinant sperm whale apomyoglobin. The results are combined with fluorescence and circular dichroism measurements of acid-induced unfolding of wild-type and double mutant (H24V/H119F) proteins. They are consistent with a simple model in which the failure to protonate a single buried histidine, H24, is largely responsible for the partial unfolding of native (N) wild-type apomyoglobin to the pH 4 folding intermediate (T), H24 is known to form an unusual interaction in which its side chain is buried and hydrogen-bonded to the side chain of H119, Two-dimensional H-1-N-15 heteronuclear NMR spectra indicate that H24 is present in the rare delta tautomeric farm and remains neutral until N unfolds to I, while H119 becomes protonated before the N --> I reaction occurs. In the H24V/H119F double mutant, all histidines are protonated in N and the N --> I reaction occurs at lower pH. Therefore, the protonation of aspartate and/or glutamate residues must provide an additional driving force for the N to I reaction, Two-dimensional H-1-C-13 NMR experiments are used to measure the protonation of aspartates in selectively C-13-labeled apomyoglobin; the results indicate that none of the aspartate residues has a strongly depressed pK(a) in N, as would be expected if it forms a stabilizing salt bridge.