Symmetric ligand binding pathways and dual-state bottleneck in NiFe hydrogenases from unbiased molecular dynamics

NiFe hydrogenases make up a family of enzymes that can be used to produce biofuel, thus making them important for industrial applications. In this work, we utilized unbiased molecular dynamics simulations to capture binding and unbinding events of the substrate, H2, to and from the NiFe hydrogenases from two different organisms. We obtained multiple (un)binding events and reproduced experimental association rate constants. We observed symmetry between the binding and unbinding pathways used by H2 to access and leave the catalytic site. Moreover, we found that the main bottleneck for ligand binding, the distance between residues V74 and L122, can shift between two states with different bottleneck widths, a feature which can be exploited to modulate the access of small molecules to the catalytic site. The pathway probabilities presented here can be used to benchmark enhanced sampling methods which investigate protein–ligand binding.