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A Theoretical Study for the Reactivation of O2 Inhibited [Fe-Fe]-hydrogenase
| Title: | A Theoretical Study for the Reactivation of O2 Inhibited [Fe-Fe]-hydrogenase |
| Author: | Motiu, Stefan |
| Description: | 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 substitution, geometry optimization, and frequency calculations have been performed to assess the change in the Gibbs energy of H2O elimination. Favorable thermodynamic results have been obtained for both single residue removal (cdGcdGlu374 = -1.6 kcal/mol)**, single substitution (cdGGlu374His = - 3.1 kcal/mol), and combined residue substitutions (cdGArg111Glu; 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 by one, the endergonic step was made to proceed more spontaneously. Thus, the most promising residue deletions which enhance H2O elimination are cdArg111, cdThr145, cdSer177, cdGlu240, cdGlu374, and cdTyr375. Hence, both single and combined residue substituted [Fe-Fe]-hydrogenase show increased spontaneity for H2O removal. The thermodynamics and electronic structure analyses show that COb 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. But enzyme reactivation can be achieved by suitable residue substitutions. *The di-iron atoms are named proximal and distal, Fep-Fed (for Fep is closest to the proximal cubane, while Fed is distal from the cubane). **Where cd in front of G, Glu374, etc. means capital delta. |
| Permanent Link: |
http://rave.ohiolink.edu/etdc/view?acc_num=csu1234364653
http://hdl.handle.net/2374.OX/104967 |
| Date: | 2008 |
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