Architecture and dynamics of a supramolecular oxygen transport system in human homogentisate 1,2-Dioxygenase

Alkaptonuria (AKU) is an ultra-rare inherited metabolic disorder caused by impaired activity of homogentisate 1,2-dioxygenase (HGD), a Fe(II)-dependent enzyme that catalyzes the oxidative cleavage of homogentisic acid in the tyrosine degradation pathway. Although high-resolution structures of human HGD have been solved, a fundamental mechanistic question has remained unresolved: how molecular oxygen reaches the deeply buried catalytic iron required for catalysis. Here, we identify a previously unreported AKU-associated HGD variant, c.925G¿A (p.G309R), and use it as a mechanistic perturbation reference to dissect the structural determinants of oxygen access. By integrating replicated classical and steered molecular dynamics simulations with transient pocket detection, tunnel mapping, O 2 spatial-occupancy analysis, residue-level tunnel composition, and PCA/tICA-based dynamic validation, we identify a structurally accessible and dynamically supported O 2 -translocation architecture connecting the central pore of the hexameric enzyme to the non-heme Fe(II) active sites. This pathway is not intrinsic to a single subunit but emerges from a cooperative arrangement of residues contributed by three protomers, generating six symmetry-related O 2 -access routes per hexamer. The G309R substitution perturbs the architecture and continuity of this tunnel system, providing a mechanistic explanation for enzyme dysfunction without evidence of active-site structural perturbation or global destabilization. Together, our findings support oxygen-tunnel integrity as a previously unrecognized mechanistic requirement for human HGD activity and introduce disruption of oxygen trafficking as an additional pathogenic mode in AKU, with implications for structure-guided variant interpretation and precision-medicine strategies.