Rapid freeze-quench ENDOR study of chloroperoxidase compound I: the site of the radical

Area of Science:

• Biochemistry
• Spectroscopy
• Enzymology

Background:

• Heme-monooxygenase enzymes utilize Compound I (Cpd-I) as a key active intermediate.
• Cpd-I contains a ferryl heme and an active-site radical, with its electronic structure debated.
• Previous theoretical studies on Cpd-I models with thiolato ligands yielded conflicting results regarding radical localization.

Purpose of the Study:

• To resolve the electronic structure of Compound I (Cpd-I) in chloroperoxidase.
• To determine the precise location of the active-site radical in Cpd-I.
• To investigate whether the radical resides on the porphyrin ring or the cysteinyl sulfur ligand.

Main Methods:

• Electron-nuclear double resonance (ENDOR) spectroscopy was employed.
• The study focused on Cpd-I of the enzyme chloroperoxidase.
• Analysis involved determining spin density distribution.

Main Results:

• ENDOR spectroscopy provided definitive evidence for the radical's location.
• The active-site radical in chloroperoxidase Cpd-I is predominantly on the porphyrin.
• Spin density on the sulfur ligand was found to have an upper bound of approximately 0.23.

Conclusions:

• The radical in chloroperoxidase Cpd-I is primarily a porphyrin pi-cation radical.
• This finding suggests that Cpd-I in cytochromes P450 likely shares a similar electronic structure.
• Clarifies the fundamental nature of reactive intermediates in heme-dependent oxygenation reactions.