Three phases in pH dependent heme abstraction from myoglobin.

The extent of heme extraction from myoglobin (Mb) by methylethyl ketone is found to be pH dependent and show three distinct phases. Parallel investigations of the protein using resonance Raman (rR) and circular dichroism (CD) across these pH regions indicate that these phases correspond to three different protonation steps in holoMb as the pH of the solution changed. The first transition occurs between pH5–6 and is due to the protonation of one of the heme propionate groups which disrupts its H-bonding with Arg 45 in the loop. The 2nd phase (pH5–4) likely involves the protonation of the 2nd propionate which H-bonds to Ser 92 in the F-helix. The third phase (pH<3.5) involves dissociation of the FeIIHis bond which eventually leads to complete heme dissociation and unfolding.