Dataset Information

Residue Mutations in [Fe-Fe]-hydrogenase Impedes O(2) Binding: A QM/MM Investigation.

ABSTRACT: [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' 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's energy results.

SUBMITTER: Dogaru D

PROVIDER: S-EPMC2872501 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Residue Mutations in [Fe-Fe]-hydrogenase Impedes O(2) Binding: A QM/MM Investigation.

Dogaru Daniela D Motiu Stefan S Gogonea Valentin V

International journal of quantum chemistry 20091001 9

[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) h ...[more]

PMID: 20485511

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Project description: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.

Project description:A combined quantum mechanical/molecular mechanical (QM/MM) potential function is used in a thermodynamic integration approach to calculate the pK(a) of residue 66 in two mutants (V66E, V66D) of Staphylococal nuclease relative to solution. Despite the similarity in chemical nature and experimentally measured pK(a) of the two buried titritable residues, the behaviors of the two mutants and the computed pK(a) values vary greatly in the simulations. For Glu66, the side chain is consistently observed to spontaneously flip out from the protein interior during titration, and the overall protein structure remains stable throughout the simulations. The computed pK(a) shifts using conventional sampling techniques with multiple nanoseconds per lambda window (Set A and B) are generally close to the experimental value, therefore indicating that large-scale conformational rearrangements are not as important for V66E as suggested by the recent study of Warshel and co-worker. For Asp66, by contrast, flipping of the shorter side chain is not sufficient for getting adequate solvent stabilization of the ionized state. As a result, more complex behaviors such as partial unfolding of a nearby beta-sheet region is observed, and the computed pK(a) shift is substantially higher than the experimental value unless Asp66 is biased to adopt the similar configurations as Glu66 in the V66E simulations. Collectively, these studies suggest that the lack of electronic polarization is not expected to be the dominant source of error in microscopic pK(a) shift calculations, while the need of enhanced sampling is more compelling for predicting the pK(a) of buried residues. Furthermore, the comparison between V66E and V66D also highlights that the microscopic interpretation of similar apparent pK(a) values and effective "dielectric constants" of proteins can vary greatly in terms of the residues that make key contributions and the scale of structural/hydration response to titration, the latter of which is difficult to predict a priori. Perturbative analyses of interactions that contribute to the titration free energy point to mutants that can be used to verify the microscopic mechanisms of titration in V66E/D SNase proteins.

Dataset Information

Residue Mutations in [Fe-Fe]-hydrogenase Impedes O(2) Binding: A QM/MM Investigation.

Publications

Residue Mutations in [Fe-Fe]-hydrogenase Impedes O(2) Binding: A QM/MM Investigation.

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