Finding gas diffusion pathways in proteins: application to O2 and H2 transport in CpI [FeFe]-hydrogenase and the role of packing defects.

We report on a computational investigation of the passive transport of H2 and O2 between the external solution and the hydrogen-producing active site of CpI [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 CpI's static crystal structure. Our results suggest that small hydrophobic molecules, such as H2 and O2, diffusing inside CpI, 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 H2 and O2, which in our model are differentiated only by their size.