Symmetric Ligand Binding Pathways and Dual-State Bottleneck in [NiFe] Hydrogenases from Unbiased Molecular Dynamics

Abstract

[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.

1

Aligned crystallographic structures of Df hydrogenase and Mdg hydrogenase (PDB ID 1YQW and 1YQ9, respectively) show the high similarity of the secondary structures of the two hydrogenases. The small subunits of Df hydrogenase and Mdg hydrogenase are shown in blue and green, and the large subunits are in yellow and magenta, respectively. The catalytic site and the position of the residues of the bottleneck for ligand binding, V74 and L122, are shown in the inset.

2

Tunnels identified in the crystallographic structures of A) Df hydrogenase (Df H₂ase, PDB ID 1YWQ ) and B) Mdg hydrogenase (Mdg H₂ase, PDB ID 1YQ9 ) using the CAVER 3.0 plugin in Pymol. , Nine tunnels (T1-T9) and eight tunnels (T1-T8) were identified in Df and Mdg hydrogenase, respectively. The secondary structures are named according to order of appearance in the primary structure and subunits (S for small and L for large). Example: α2S, second α-helix from the small subunit.

3

Pathway probabilities are symmetric for H₂ binding and unbinding in A) Df hydrogenase (Df H₂ase) and B) Mdg hydrogenase (Mdg H₂ase). The colors of each pathway match the colors of the associated tunnels in Figure . The standard error for each bar comes from bootstrapping. The p-values obtained from the chi-square test were 0.053 and 0.121 for Df and Mdg hydrogenase, respectively, indicating that there is no significant difference between the binding and unbinding pathway probabilities (considering a threshold of 0.05).

4

Dual-state bottleneck in Df hydrogenase (Df H₂ase) observed in unbiased molecular dynamics simulations. A) Probability density of lowest distance values between residues V74 and L122 in Df hydrogenase and Mdg hydrogenase (Mdg Df H₂ase). The distance values in the crystallographic structures are shown as traced lines. B) Free energy landscape of the V74-L122 bottleneck in Df hydrogenase computed using a Markov state model (details in the ). Two main states were identified: open and closed. C) Representative snapshots of Df hydrogenase in the open (cyan residues) and closed (green residues) states of the V74-L122 bottleneck. The location of the snapshots in the free energy landscape in panel B is indicated with green and cyan stars.