Multiscale simulation reveals multiple pathways for H2 and O2 transport in a [NiFe]-hydrogenase

Hydrogenases are enzymes that catalyze the reversible conversion of hydrogen molecules to protons and electrons. The mechanism by which the gas molecules reach the active site is important for understanding the function of the enzyme and may play a role in the selectivity for hydrogen over inhibitor molecules. Here, we develop a general multiscale molecular simulation approach for the calculation of diffusion rates and determination of pathways by which substrate or inhibitor gases can reach the protein active site. Combining kinetic data from both equilibrium simulations and enhanced sampling, we construct a master equation describing the movement of gas molecules within the enzyme. We find that the time-dependent gas population of the active site can be fit to the same phenomenological rate law used to interpret experiments, with corresponding diffusion rates in very good agreement with experimental data. However, in contrast to the conventional picture, in which the gases follow a well-defined hydrophobic tunnel, we find that there is a diverse network of accessible pathways by which the gas molecules can reach the active site. The previously identified tunnel accounts for only about 60% of the total flux. Our results suggest that the dramatic decrease in the diffusion rate for mutations involving the residue Val74 could be in part due to the narrowing of the passage Val74-Arg476, immediately adjacent to the binding site, explaining why mutations of Leu122 had only a negligible effect in experiment. Our method is not specific to the [NiFe]-hydrogenase and should be generally applicable to the transport of small molecules in proteins.