Abstract

Cyc2 is a transmembrane c-type cytochrome central to Extracellular Electron Transfer (EET) in Acidithiobacillus ferrooxidans, an acidophilic bacterium widely used in bioleaching to target metal extraction. Despite its major functional role, and its importance as a potential target for modification, Cyc2 structure remains unresolved. This study presents a computational characterization of Cyc2 using AlphaFold3, I- TASSER, and DiffDock, revealing a fused porin–cytochrome architecture with conserved heme-binding motifs, solvent-accessible Fe²⁺ docking pockets, and aromatic π- stacking networks facilitating electron tunnelling. Electrostatic mapping confirms a negatively charged barrel lumen, supporting cation selectivity. Docking simulations with rusticyanin validate redox coupling interfaces. These findings provide mechanistic insight into microbial iron oxidation and inform future applications in bio electrochemical systems and synthetic biology.

Keywords:Cyc2; Extracellular electron transfer; Iron oxidation; AlphaFold; DiffDock; Acidophiles; Porin-cytochrome fusion; Redox modelling