A Theoretical Study for the Reactivation of O2 Inhibited [Fe-Fe]-Hydrogenase

The current investigation presents a reactivation pathway of the exogenously inhibited H-cluster (viz., by O2, or OH-, which metabolizes to H2O), for both vacuum and aqueous enzyme phase. The H-cluster is the catalytic site of [Fe-Fe]-hydrogenase, with the latter extracted from Desulfovibrio desulfuricans (Dd) bacteria. It consists of proximal iron, Fep, and distal Fed subunit, [Fep-Fed], which is bridged by di(thiomethyl)amine (DTMA) ligand, and a proximal cubane subunit, [Fe4-S4]2+p. [Fep-Fed] is coordinated by two cyanides (CN-), two terminal carbonyls (COt), and a bridging carbonyl (COb)*. An Fe atom from [Fe4-S4]2+p connects Fep through a cysteinyl sulfur (of Cys382). Density functional theory calculations on the native and ruthenium-modified H-cluster (gas phase) have been performed using the B3LYP functional with 6-31+G** and 6-311+G** bases sets. We have ascertained that there is a thermodynamically favorable pathway for the reactivation of the OH- inhibited H-cluster, which proceeds by an initial protonation of Fed-OH- complex. The proposed reaction pathway has all of its intermediate reactions proceed exergonically. The aqueous enzyme phase investigation uses the hybrid quantum mechanics/molecular mechanics (QM/MM) method to study reactivation pathways for the exogenously inhibited enzyme matrix. ONIOM calculations performed on the enzyme agree with experimental results, i.e., the hydrogenase H-cluster is inhibited by oxygen metabolites. To investigate potential inhibitory residues that prevent H2O from leaving the catalytic site, and reactivate the hydrogenase H-cluster, an enzyme spherical region of radius 8 A (from the distal iron, Fed, of [Fe-Fe]-hydrogenase H-cluster) was screened. In the screening process, polar residues were removed, one at a time, and frequency calculations provided the change in Gibbs energy of water dissociation (due to their deletion). When residue deletion resulted in significant Gibbs energy decrease, further residue substitutions have been carried out. Following each sub

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

In this 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 (Dogaru et al., Int J Quantum Chem 2008, 108; Motiu et al., Int J Quantum Chem 2007, 107, 1248). 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 (Liu et al., J Am Chem Soc 2002, 124, 5175), that is, 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\u27 energy for the dissociation of water (due to their deletion). When residue deletion resulted in significant Gibbs\u27 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\u27 energy for the elimination of 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, that is, 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. Nevertheless, enzyme reactivation can be achieved by means of appropriate amino acid substitutions

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

In this 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 (Dogaru et al., Int J Quantum Chem 2008, 108; Motiu et al., Int J Quantum Chem 2007, 107, 1248). 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 (Liu et al., J Am Chem Soc 2002, 124, 5175), that is, 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\u27 energy for the dissociation of water (due to their deletion). When residue deletion resulted in significant Gibbs\u27 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\u27 energy for the elimination of 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, that is, 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. Nevertheless, enzyme reactivation can be achieved by means of appropriate amino acid substitutions

Residue Mutations in [Fe-Fe]-Hydrogenase Impedes O 2 Binding: A QM/MM Investigation

[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 H(2) 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 Fe(p) (II)-Fe(d) (II)-O-H(-), or Fe(p) (II)-Fe(d) (II)-O-O-H, thus O(2) has to be prevented from binding to Fe(d) 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 O(2) binding to distal iron (Fe(d)) of Desulfovibrio desulfuricans hydrogenase (DdH). Individual residue deletions were carried out in the 8 A apoenzyme layer radial outward from Fe(d) to determine what residue substitutions should be made to weaken O(2) binding. Residue deletions and substitutions were performed for three di-iron subcluster oxidation states, Fe(p) (II)-Fe(d) (II), Fe(p) (II)-Fe(d) (I), and Fe(p) (I)-Fe(d) (I) of [Fe-Fe]-hydrogenase. Two deletions (DeltaThr(152) and DeltaSer(202)) were found most effective in weakening O(2) binding to Fe(d) in Fe(p) (II)-Fe(d) (I) hydrogenase (DeltaG(QM/MM) = +5.4 kcal/mol). An increase in Gibbs\u27 energy (+2.2 kcal/mol and +4.4 kcal/mol) has also been found for Fe(p) (II)-Fe(d) (II), and Fe(p) (I)-Fe(d) (I) hydrogenase respectively. pi-backdonation considerations for frontier molecular orbital and geometrical analysis corroborate the Gibbs\u27s energy results

Inactivation of [Fe-Fe]-Hydrogenase by O2. Thermodynamics and Frontier Molecular Orbitals Analyses

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 isalso 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 nonspontaneous 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

A Theoretical Study for the Reactivation of O2 Inhibited [Fe-Fe]-Hydrogenase

The current investigation presents a reactivation pathway of the exogenously inhibited H-cluster (viz., by O2, or OH-, which metabolizes to H2O), for both vacuum and aqueous enzyme phase. The H-cluster is the catalytic site of [Fe-Fe]-hydrogenase, with the latter extracted from Desulfovibrio desulfuricans (Dd) bacteria. It consists of proximal iron, Fep, and distal Fed subunit, [Fep-Fed], which is bridged by di(thiomethyl)amine (DTMA) ligand, and a proximal cubane subunit, [Fe4-S4]2+p. [Fep-Fed] is coordinated by two cyanides (CN-), two terminal carbonyls (COt), and a bridging carbonyl (COb)*. An Fe atom from [Fe4-S4]2+p connects Fep through a cysteinyl sulfur (of Cys382). Density functional theory calculations on the native and ruthenium-modified H-cluster (gas phase) have been performed using the B3LYP functional with 6-31+G** and 6-311+G** bases sets. We have ascertained that there is a thermodynamically favorable pathway for the reactivation of the OH- inhibited H-cluster, which proceeds by an initial protonation of Fed-OH- complex. The proposed reaction pathway has all of its intermediate reactions proceed exergonically. The aqueous enzyme phase investigation uses the hybrid quantum mechanics/molecular mechanics (QM/MM) method to study reactivation pathways for the exogenously inhibited enzyme matrix. ONIOM calculations performed on the enzyme agree with experimental results, i.e., the hydrogenase H-cluster is inhibited by oxygen metabolites. To investigate potential inhibitory residues that prevent H2O from leaving the catalytic site, and reactivate the hydrogenase H-cluster, an enzyme spherical region of radius 8 A (from the distal iron, Fed, of [Fe-Fe]-hydrogenase H-cluster) was screened. In the screening process, polar residues were removed, one at a time, and frequency calculations provided the change in Gibbs energy of water dissociation (due to their deletion). When residue deletion resulted in significant Gibbs energy decrease, further residue substitutions have been carried out. Following each sub

Inactivation of [Fe-Fe]-Hydrogenase by O2. Thermodynamics and Frontier Molecular Orbitals Analyses

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 isalso 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 nonspontaneous 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