Finding gas diffusion pathways in proteins: Application to O-2 and H-2 transport in Cpl [FeFe]-hydrogenase and the role of packing defects

We report on a computational investigation of the passive transport of H-2 and O-2 between the external solution and the hydrogen-producing active site of Cpl [FeFe]-hydrogenase from Clostridium pasteurianum. Two distinct methodologies for studying gas access are discussed and applied: (1) temperature-controlled locally enhanced sampling, and (2) volumetric solvent accessibility maps, providing consistent results. Both methodologies confirm the existence and function of a previously hypothesized pathway and reveal a second major pathway that had not been detected by previous analyses of Cpl's static cry tal structure. Our results suggest that small hydrophobic molecules, such as H-2 and O-2, diffusing inside Cpl, take advantage of well-defined preexisting packing defects that are not always apparent from the protein's static structure, but that can be predicted from the protein's dynamical motion. Finally, we describe two contrasting modes of intraprotein transport for H-2 and O-2, which in our model are differentiated only by their size.