Low activation barriers characterize intramolecular electron transfer in ascorbate oxidase

Area of Science:

• Biochemistry
• Enzymology
• Electron Transfer Mechanisms

Background:

• Ascorbate oxidase (AO) is a copper-containing enzyme crucial for plant defense and cell wall metabolism.
• Understanding the electron transfer kinetics within AO is vital for elucidating its catalytic mechanism.
• Previous studies have focused on substrate reduction, but internal electron transfer rates remain less defined.

Purpose of the Study:

• To investigate the anaerobic reduction kinetics of zucchini squash ascorbate oxidase (AO) by CO2- radical ions.
• To determine the rate constants and activation parameters for electron transfer within the enzyme.
• To identify the rate-determining step in the AO catalytic cycle.

Main Methods:

• Pulse radiolysis was employed to generate CO2- radical ions for anaerobic reduction of AO.
• Spectroscopic monitoring of type 1 [Cu(II)] at 610 nm and type 3 [Cu(II)] at 330 nm.
• Kinetic analysis was performed across varying reactant concentrations, pH, and temperatures (275–308 K).

Main Results:

• Direct bimolecular reduction of type 1 [Cu(II)] was observed, followed by unimolecular reoxidation in three phases.
• Intramolecular electron transfer from type 1 [Cu(I)] to type 3 [Cu(II)] was identified as the reoxidation pathway.
• Low activation enthalpies (9.1–6.8 kJ/mol) and negative activation entropies were calculated, indicating optimized electron transfer.

Conclusions:

• Intramolecular electron transfer is the rate-determining step in ascorbate oxidase activity.
• The enzyme possesses highly optimized electron transfer pathways, evidenced by exceptionally low enthalpy barriers.
• These findings provide critical insights into the catalytic mechanism and efficiency of ascorbate oxidase.