Date of Award


Degree Type




First Advisor

Gogonea, Valentin

Subject Headings

Hydrogenase, Enzyme kinetics, Reactivation, H-cluster, DFT, ONIOM, Residues


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 subs

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