Studies on catalytic mechanism of [Fe]-hydrogenase from methanogenic archaea based on crystal structures

Hydrogenases are promising templates for designing new H2-based catalysts. [Fe]-hydrogenase reversibly catalyzes H2 cleavage and transfer of a hydride to the C14a of methenyl-tetrahydromethanopterin (methenyl-H4MPT+, a C1 carrier). Different from di-nuclear [NiFe]- and [FeFe]-hydrogenases, [Fe]-hydrogenase harbors the iron-guanylylpyridinol (FeGP) cofactor containing a single iron. The FeGP cofactor is consist of the iron site, the pyridinol ring and the guanosine monophosphate (GMP). [Fe]-hydrogenase is a dimer, in which each N-terminal domain contains one FeGP cofactor, and two C-terminal domains form the central domain. [Fe]-hydrogenase has open and closed forms. In the resting-open form, the iron site is 6-coordinated with two CO ligands, one acyl-C, one pyridinol-N, one Cys-S and a water ligand. In this work, a detailed catalytic mechanism of [Fe]-hydrogenase based on the 1.06-Å resolution structure of [Fe]-hydrogenase from Methanococcus aeolicus complexed with methenyl-H4MPT+ in a closed-active form was studied. In the closed-active form, the iron site is changed into 5-coordinated state due to removal of the water ligand by closure of the active cleft formed by the central and the N-terminal domains, generating an empty coordination site for H2-binding and the deprotonated 2-OH of the FeGP cofactor as the catalytic base. In this situation, the Fe site of the FeGP cofactor is near the C14a (hydride acceptor) of methenyl-H4MPT+ at 3.8 Å. The Quantum mechanics/molecular mechanics (QM/MM) computations based on the structure of the closed-active form indicated the nearly thermo-neutral H2-binding and smooth H2-cleavage and hydride transfer. The distorted imidazoline ring of methenyl-H4MPT+ might stabilize the active state for H2 binding/cleavage. Based on the results, a complete reaction cycle of [Fe]-hydrogenase was proposed. In addition, we found that [Fe]-hydrogenase catalyzes the reduction of O2 to H2O2 in the presence of reducing substrates (methylene-H4MPT or methenyl-H4MPT+/H2). The most possible reductant was the iron-hydride intermediate. This result provided the first evidence for the existence of the iron-hydride intermediate in the catalytic cycle. Subsequently, the crystal structure of [Fe]-hydrogenase from Methanothermobacter marburgensis indicated that this enzyme is in hexamer rather than dimer, in which an aspartate ligand (Asp189) of the loop from nearby monomer bound the Fe site of the FeGP cofactor. In this case, the enzyme can protect its active center against light and oxidative stresses. When substrates bound, this enzyme dissociates into active dimers. This finding also confirmed the catalytic function of the open coordination site of the Fe site