[Fe-Fe]-hydrogenase Reactivated by Residue Mutations as Bridging Carbonyl Rearranges: A QM/MM Study

In the current work, we found aqueous enzyme phase reaction pathways for the reactivation of the exogenously inhibited [Fe-Fe]-hydrogenases by O₂, or OH^-, which metabolizes to H₂O^1,2. We used the hybrid quantum mechanics/molecular mechanics (QM/MM) method to study the reactivation pathways of the exogenously inhibited enzyme matrix. The ONIOM calculations performed on the enzyme agree with experimental results³, i.e., wild-type [Fe-Fe]-hydrogenase H-cluster is inhibited by oxygen metabolites. An enzyme spherical region with a radius of 8 Å (from the distal iron, Fe_d) has been screened for residues that prevent H₂O from leaving the catalytic site and reactivate the [Fe-Fe]-hydrogenase H-cluster. In the screening process, polar residues were removed, one at a time, and frequency calculations provided the change in the Gibbs' energy for the dissociation of water (due to their deletion). When residue deletion resulted in significant Gibbs' energy decrease, further residue substitutions have been carried out. Following each substitution, geometry optimization and frequency calculations have been performed to assess the change in the Gibbs' energy for the elimination H₂O. Favorable thermodynamic results have been obtained for both single residue removal (ΔG_ΔGlu³⁷⁴ = -1.6 kcal/mol), single substitution (ΔG_Glu³⁷⁴_His = -3.1 kcal/mol), and combined residue substitutions (ΔG_Arg¹¹¹_Glu;Thr¹⁴⁵_Val;Glu³⁷⁴_His;Tyr³⁷⁵_Phe = -7.5 kcal/mol). Because the wild-type enzyme has only an endergonic step to overcome, i.e., for H₂O removal, by eliminating several residues, one at a time, the endergonic step was made to proceed spontaneously. Thus, the most promising residue deletions which enhance H₂O elimination are ΔArg¹¹¹, ΔThr¹⁴⁵, ΔSer¹⁷⁷, ΔGlu²⁴⁰, ΔGlu³⁷⁴, and ΔTyr³⁷⁵. The thermodynamics and electronic structure analyses show that the bridging carbonyl (CO_b) of the H-cluster plays a concomitant role in the enzyme inhibition/reactivation. In gas phase, CO_b shifts towards Fe_d to compensate for the electron density donated to oxygen upon the elimination of H₂O. However, this is not possible in the wild-type enzyme because the protein matrix hinders the displacement of CO_b towards Fe_d, which leads to enzyme inhibition. However, enzyme reactivation can be achieved by means of appropriate amino acid substitutions.