Oxygen Pathways and Allostery in Monomeric Sarcosine Oxidase via Single-Sweep Free-Energy Reconstruction

Monomeric sarcosine oxidase (MSOX) is a flavoprotein D-amino acid oxidase with reported sarcosine and oxygen activation sites on the re and si faces of the flavin ring, respectively. O2 transport routes to the catalytic interior are not well understood and are difficult to ascertain solely from MSOX crystal structures. A composite free-energy method known as single-sweep is used to map and thermodynamically characterize oxygen sites and routes leading to the catalytically active Lys265 from the protein surface. The result is a network of pathways and free energies within MSOX illustrating that oxygen can access two free-energy minima on the re face of the reduced flavin from four separate solvent portals. No such minimum is observed on the si face. The pathways are geometrically similar for three major states of the enzyme: (1) apo with a closed flavin cleft, (2) apo with an open flavin cleft, and (3) inhibitor-bound with a closed flavin cleft. Interestingly, free energies along these transport pathways display significantly deeper minima when the substrate-mimicking inhibitor 2-furoic acid is bound at the sarcosine site, even at locations far from this site. This suggests a substrate-dependent allosteric modulation of the kinetics of O2 transport from the solvent to the active site.