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.