Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive

Area of Science:

• Biochemistry
• Enzymology
• Spectroscopy

Background:

• APO is a heme-thiolate peroxygenase from Agrocybe aegerita.
• Ferryl intermediates are key catalytic species in peroxygenase activity.

Purpose of the Study:

• To kinetically and spectroscopically characterize the ferryl intermediate (APO-II) of APO.
• To elucidate the role of the axial cysteine ligand in ferryl intermediate reactivity.

Main Methods:

• Generation of APO-II using metachloroperoxybenzoic acid and nitroxyl radicals.
• UV-visible spectroscopy and rapid-mixing stopped-flow techniques.
• pH-jump spectrophotometry for pKa determination.

Main Results:

• APO-II exhibits a split Soret UV-vis spectrum indicative of thiolate ligation.
• A basic pKa of 10.0 for the Fe(IV)-O-H in APO-II was determined.
• APO-II demonstrates high reactivity towards benzylic C-H and phenolic substrates (10-10^7 M^-1 s^-1).

Conclusions:

• The axial cysteine ligand enhances the proton affinity of the ferryl oxygen.
• This increased proton affinity provides driving force for C-H and O-H bond scission.
• The findings highlight the crucial role of the heme-thiolate moiety in peroxygenase catalysis.