Three phases in pH dependent heme abstraction from myoglobin

Area of Science:

• Biochemistry
• Protein Chemistry
• Spectroscopy

Background:

• Myoglobin (Mb) is a crucial protein for oxygen transport.
• Heme extraction studies are vital for understanding protein stability and function.
• Methylethyl ketone is a solvent used to probe protein-heme interactions.

Purpose of the Study:

• To investigate the pH-dependent heme extraction from myoglobin using methylethyl ketone.
• To correlate pH-induced structural changes with specific protonation events.
• To elucidate the mechanism of heme dissociation and protein unfolding.

Main Methods:

• Heme extraction assay using methylethyl ketone.
• Spectroscopic analysis including resonance Raman (rR) and circular dichroism (CD).
• pH titration studies across a range of acidic conditions.

Main Results:

• Heme extraction exhibited three distinct pH-dependent phases.
• rR and CD spectroscopy identified three protonation steps corresponding to these phases.
• Phase 1 (pH 5-6): Protonation of a heme propionate disrupting H-bonding with Arg 45.
• Phase 2 (pH 5-4): Protonation of a second heme propionate H-bonding with Ser 92.
• Phase 3 (pH < 3.5): Dissociation of the Fe(II)-His bond leading to heme loss and unfolding.

Conclusions:

• The pH-dependent heme extraction mechanism involves sequential protonation events.
• Specific amino acid residues and heme propionates play critical roles in maintaining heme binding.
• Acidic conditions trigger a cascade leading to myoglobin unfolding and loss of function.