ABSTRACT: 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 results(3), 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, Fed) 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 (?G?Glu(374) = -1.6 kcal/mol), single substitution (?GGlu(374)His = -3.1 kcal/mol), and combined residue substitutions (?GArg(111)Glu;Thr(145)Val;Glu(374)His;Tyr(375)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 ?Arg(111), ?Thr(145), ?Ser(177), ?Glu(240), ?Glu(374), and ?Tyr(375). The thermodynamics and electronic structure analyses show that the bridging carbonyl (COb) of the H-cluster plays a concomitant role in the enzyme inhibition/reactivation. In gas phase, COb shifts towards Fed 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 COb towards Fed, which leads to enzyme inhibition. However, enzyme reactivation can be achieved by means of appropriate amino acid substitutions.
