Oxygen sensitivity of [FeFe]-hydrogenase: a comparative study of active site mimics inside outside the enzyme.

[FeFe]-hydrogenase is nature's most efficient proton reducing and H-oxidizing enzyme. However, biotechnological applications are hampered by the O sensitivity of this metalloenzyme, and the mechanism of aerobic deactivation is not well understood. Here, we explore the oxygen sensitivity of four mimics of the organometallic active site cofactor of [FeFe]-hydrogenase, [Fe(adt)(CO)(CN)] and [Fe(pdt)(CO)(CN)] ( = 1, 2) as well as the corresponding cofactor variants of the enzyme by means of infrared, Mössbauer, and NMR spectroscopy.

A Conserved Binding Pocket in HydF is Essential for Biological Assembly and Coordination of the Diiron Site of [FeFe]-Hydrogenases.

The active site cofactor of [FeFe]-hydrogenases consists of a cubane [4Fe-4S]-cluster and a unique [2Fe-2S]-cluster, harboring unusual CO- and CN-ligands. The biosynthesis of the [2Fe-2S]-cluster requires three dedicated maturation enzymes called HydG, HydE and HydF. HydG and HydE are both involved in synthesizing a [2Fe-2S]-precursor, still lacking parts of the azadithiolate (adt) moiety that bridge the two iron atoms.

Insights into the Molecular Mechanism of Formaldehyde Inhibition of [FeFe]-Hydrogenases.

[FeFe]-hydrogenases are efficient H converting biocatalysts that are inhibited by formaldehyde (HCHO). The molecular mechanism of this inhibition has so far not been experimentally solved. Here, we obtained high-resolution crystal structures of the HCHO-treated [FeFe]-hydrogenase CpI from , showing HCHO reacts with the secondary amine base of the catalytic cofactor and the cysteine C299 of the proton transfer pathway which both are very important for catalytic turnover.

A Dynamic Water Channel Affects O Stability in [FeFe]-Hydrogenases.

[FeFe]-hydrogenases are capable of reducing protons at a high rate. However, molecular oxygen (O ) induces the degradation of their catalytic cofactor, the H-cluster, which consists of a cubane [4Fe4S] subcluster (4Fe ) and a unique diiron moiety (2Fe ). Previous attempts to prevent O -induced damage have focused on enhancing the protein's sieving effect for O by blocking the hydrophobic gas channels that connect the protein surface and the 2Fe .

The [4Fe-4S]-Cluster of HydF is not Required for the Binding and Transfer of the Diiron Site of [FeFe]-Hydrogenases.

The active site of [FeFe]-hydrogenases contains a cubane [4Fe-4S]-cluster and a unique diiron cluster with biologically unusual CO and CN ligands. The biogenesis of this diiron site, termed [2Fe ], requires the maturation proteins HydE, HydF and HydG. During the maturation process HydF serves as a scaffold protein for the final assembly steps and the subsequent transfer of the [2Fe ] precursor, termed [2Fe ], to the [FeFe]-hydrogenase.

Increasing the O Resistance of the [FeFe]-Hydrogenase CbA5H through Enhanced Protein Flexibility.

The high turnover rates of [FeFe]-hydrogenases under mild conditions and at low overpotentials provide a natural blueprint for the design of hydrogen catalysts. However, the unique active site (H-cluster) degrades upon contact with oxygen. The [FeFe]-hydrogenase from (CbA5H) is characterized by the flexibility of its protein structure, which allows a conserved cysteine to coordinate to the active site under oxidative conditions.

[FeFe]-Hydrogenases: maturation and reactivity of enzymatic systems and overview of biomimetic models.

While hydrogen plays an ever-increasing role in modern society, nature has utilized hydrogen since a very long time as an energy carrier and storage molecule. Among the enzymatic systems that metabolise hydrogen, [FeFe]-hydrogenases are one of the most powerful systems to perform this conversion. In this light, we will herein present an overview on developments in [FeFe]-hydrogenase research with a strong focus on synthetic mimics and their application within the native enzymatic environment.

Shedding Light on Proton and Electron Dynamics in [FeFe] Hydrogenases.

[FeFe] hydrogenases are highly efficient catalysts for reversible dihydrogen evolution. H turnover involves different catalytic intermediates including a recently characterized hydride state of the active site (H-cluster). Applying cryogenic infrared and electron paramagnetic resonance spectroscopy to an [FeFe] model hydrogenase from (HydA1), we have discovered two new hydride intermediates and spectroscopic evidence for a bridging CO ligand in two reduced H-cluster states.