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' 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.