Multicopper manganese oxidase accessory proteins bind Cu and heme

Area of Science:

• Biochemistry
• Metalloprotein characterization
• Enzymology

Background:

• Multicopper oxidases (MCOs) utilize four copper ions to catalyze substrate oxidation via electron transfer to O2.
• The MnxG enzyme is a unique MCO involved in MnO2 mineralization, requiring accessory proteins MnxE and MnxF.
• The functions of MnxE and MnxF, despite sequence similarities to other Bacillus proteins, remain largely unknown.

Purpose of the Study:

• To investigate the biochemical properties and oligomeric state of MnxE and MnxF.
• To characterize the metal and heme binding capabilities of MnxE and MnxF.
• To elucidate the role of MnxE and MnxF in the MnxG-catalyzed manganese oxidation.

Main Methods:

• Co-expression of MnxE and MnxF independent of MnxG.
• Oligomerization analysis.
• Copper and heme binding characterization using Electron Paramagnetic Resonance (EPR), X-ray Absorption Spectroscopy (XAS), and UV-visible (UV-vis) spectrophotometry.

Main Results:

• MnxE and MnxF co-expressed proteins oligomerize into a likely hexameric complex.
• These proteins bind both copper and heme.
• Copper ions are located in two distinct Type 2 (T2) centers, one potentially novel, and heme is bound as a low-spin species.
• MnxE and MnxF are essential for MnxG's manganese oxidation activity.

Conclusions:

• MnxE and MnxF are the first identified accessory proteins essential for a multicopper oxidase.
• The binding of copper and heme by MnxE and MnxF suggests roles in electron transfer or copper trafficking within the MnxG complex.