The oxidation of H-cluster in gas phase, and in aqueous enzyme phase, has been investigated by means of quantum mechanics (QM) and combined quantum mechanics-molecular mechanics (QM/MM). Several potential reaction pathways (in the above mentioned chemical environments) have been studied, wherein only the aqueous enzyme phase has been found to lead to an inhibited hydroxylated cluster. Specifically, the inhibitory process occurs at the distal iron (Fed) of the catalytic H-cluster (which is also the atom involved in H2 synthesis). The processes involved in the H-cluster oxidative pathways are O2 binding, e- transfer, protonation, and H2O removal. We found that oxygen binding is non-spontaneous in gas phase, and spontaneous for aqueous enzyme phase where both Fe atoms have oxidation state II; however, it is spontaneous for the partially oxidized and reduced clusters in both phases. Hence, in the protein environment the hydroxylated H-cluster is obtained by means of completely exergonic reaction pathway starting with proton transfer. A unifying endeavor has been carried out for the purpose of understanding the thermodynamic results vis-à-vis several other performed electronic structural methods, such as frontier molecular orbitals (FMO), natural bond orbital partial charges (NBO), and H-cluster geometrical analysis. An interesting result of the FMO examination (for gas phase) is that an e- is transferred to LUMOα rather than to SOMOβ, which is unexpected because SOMOβ usually resides in a lower energy rather than LUMOα for open-shell clusters.

ABSTRACT:This work puts forth a reaction pathway for the reactivation of exogenous ligand inhibited H-cluster, the active site of Fe-only hydrogenases. The Hcluster is a dimetal complex, Fe-Fe, with the metal centers bridged by di(thiomethyl)amine. Exogenous ligands, H 2 O, and OH Ϫ , are bound to the distal iron (Fe d ). Density functional theory (DFT) calculations on the native and rutheniummodified H-cluster have been performed using the B3LYP functional with 6-31ϩG** and 6-311ϩG** basis sets. We have ascertained that there is a thermodynamically favorable pathway for the reactivation of the OH Ϫ inhibited H-cluster, which proceeds by an initial protonation of the Fe d -OH Ϫ complex. The proposed reaction pathway has all its intermediate reactions ensue exothermically.

[Fe-Fe]-hydrogenases are enzymes that reversibly catalyze the reaction of protons and electrons to molecular hydrogen, which occurs in anaerobic media. In living systems, [Fe-Fe]-hydrogenases are mostly used for H2 production. The [Fe-Fe]-hydrogenase H-cluster is the active site, which contains two iron atoms. The latest theoretical investigations1,2 advocate that the structure of di-iron air inhibited species are either FepII-FedII-O-H-, or FepII-FedII-O-O-H, thus O2 has to be prevented from binding to Fed in all di-iron subcluster oxidation states in order to retain a catalytically active enzyme. By performing residue mutations* on [Fe-Fe]-hydrogenases, we were able to weaken O2 binding to distal iron (Fed) of Desulfovibrio desulfuricans hydrogenase (DdH). Individual residue deletions were carried out in the 8 Å apoenzyme layer radial outward from Fed to determine what residue substitutions should be made to weaken O2 binding. Residue deletions and substitutions were performed for three di-iron subcluster oxidation states, FepII-FedII, FepII-FedI, and FepI-FedI of [Fe-Fe]-hydrogenase. Two deletions (ΔThr152 and ΔSer202) were found most effective in weakening O2 binding to Fed in FepII-FedI hydrogenase (ΔGQM/MM = +5.4 kcal/mol). An increase in Gibbs' energy (+2.2 kcal/mol and +4.4 kcal/mol) has also been found for FepII-FedII, and FepI-FedI hydrogenase respectively. π-backdonation considerations for frontier molecular orbital and geometrical analysis corroborate the Gibbs's energy results.

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 H2O1,2. 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, 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 (ΔGΔGlu374 = -1.6 kcal/mol), single substitution (ΔGGlu374His = -3.1 kcal/mol), and combined residue substitutions (ΔGArg111Glu;Thr145Val;Glu374His;Tyr375Phe = -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 (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.