Binding Interface and Electron Transfer Between Nicotine Oxidoreductase and Its Cytochrome c Electron Acceptor

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Nicotine oxidoreductase (NicA2) is a flavoprotein amine oxidase.
• Unlike other family members, NicA2 uses cytochrome c (CycN) as an oxidant instead of dioxygen.
• Previous rigid body docking identified a potential CycN binding site on NicA2.

Purpose of the Study:

• To experimentally validate the predicted CycN binding interface on NicA2.
• To investigate the interaction mechanism between NicA2 and CycN.
• To elucidate the electron transfer pathway in the NicA2-CycN complex.

Main Methods:

• Unnatural amino acid incorporation to probe the NicA2-CycN binding interface.
• Protein-protein docking and AlphaFold for structural modeling.
• Site-directed mutagenesis of potentially redox-active residues in NicA2.

Main Results:

• Experimental data confirmed the predicted binding site for CycN on NicA2.
• Results align with structural models from protein-protein docking and AlphaFold.
• Mutagenesis studies suggest direct electron transfer from NicA2's flavin to CycN's heme.

Conclusions:

• The study experimentally validates the CycN binding site on NicA2.
• The validated binding site supports existing structural predictions.
• A model for direct electron transfer without a protein-derived wire is proposed.