Capturing Fast Gas Migration in Proteins.

Small gases pose an unusual problem for studies of molecular transport in proteins. O2, CO, H2, and NO can cross short-lived internal spaces opened by protein fluctuations, often faster than experiments can follow continuous migration. Time-resolved crystallography can localize sufficiently populated intermediates, whereas spectroscopy, isotope exchange, and kinetic measurements report molecular exchange over their respective timescales without resolving the complete route. Pressurized noble-gas structures expose internal accommodation sites but rely on surrogate molecules whose size and interactions differ from those of physiological gases. Geometry-based tunnel searches identify available space, while molecular dynamics follows explicit movement through a fluctuating protein. Free-energy and enhanced-sampling approaches can access states or transitions that remain undersampled in direct trajectories. These techniques resolve different quantities rather than progressively more accurate estimates of gas transport. In this Perspective, we argue that gas-migration pathways should be evaluated by the physical consistency of independent observables, with each method interpreted according to the quantity it resolves. This distinction explains why a cavity visible crystallographically may not carry substantial flux, why a rapidly crossed route can remain structurally inconspicuous, and why static narrowing can alter diffusion without predicting its magnitude. Agreement among methods can support a transport assignment when the quantities they resolve are physically consistent with the same mechanism; apparent disagreement may instead reflect differences among occupancy, accessibility, residence, energetic preference, and molecular traffic.