Conformational and mechanical stability of the isolated large subunit of membrane-bound [NiFe]-hydrogenase from Cupriavidus necator

Abstract

Comprising at least a bipartite architecture, the large subunit of [NiFe]-hydrogenase harbors the catalytic nickel–iron site while the small subunit houses an array of electron-transferring Fe-S clusters. Recently, some [NiFe]-hydrogenase large subunits have been isolated showing an intact and redox active catalytic cofactor. In this computational study we have investigated one of these metalloproteins, namely the large subunit HoxG of the membrane-bound hydrogenase from Cupriavidus necator (CnMBH), targeting its conformational and mechanical stability using molecular modelling and long all-atom Gaussian accelerated molecular dynamics (GaMD). Our simulations predict that isolated HoxG is stable in aqueous solution and preserves a large portion of its mechanical properties, but loses rigidity in regions around the active site, in contrast to the MBH heterodimer. Inspired by biochemical data showing dimerization of the HoxG protein and IR measurements revealing an increased stability of the [NiFe] cofactor in protein preparations with higher dimer content, corresponding simulations of homodimeric forms were also undertaken. While the monomeric subunit contains several flexible regions, our data predicts a regained rigidity in homodimer models. Furthermore, we computed the electrostatic properties of models obtained by enhanced sampling with GaMD, which displays a significant amount of positive charge at the protein surface, especially in solvent-exposed former dimer interfaces. These data offer novel insights on the way the [NiFe] core is protected from de-assembly and provide hints for enzyme anchoring to surfaces, which is essential information for further investigations on these minimal enzymes.

Comprehensive structural, infrared spectroscopic and kinetic investigations of the roles of the active-site arginine in bidirectional hydrogen activation by the [NiFe]-hydrogenase ‘Hyd-2’ from Escherichia coli

“…Helix 4, spanning residues 94–113, is also rotated by 2.5° about its C-terminus relative to its position in Hyd-2, increasing the distance between the carboxylate group of D103 and residue 479. These two shifts likely result from a loss of hydrogen bonds to the guanidinium head group of R479 in the native enzyme, a residue important for maintaining the rigidity of the protein near the active site, 99 with a cumulative effect of expanding the active site. This movement necessitates that K479 adopt a rarely observed conformation to form a single hydrogen bond with each of the carboxylate groups of D103 and D544, (see Fig.…”