Distinct Valence States of the [4Fe4S] Cluster Revealed in the Hydrogenase CrHydA1.

Iron-sulfur clusters play a crucial role in electron transfer for many essential enzymes, including [FeFe]-hydrogenases. This study focuses on the [4Fe4S] cluster ([4Fe]H) of the minimal [FeFe]-hydrogenase from Chlamydomonas reinhardtii (CrHydA1) and employs advanced spectroscopy, site-directed mutagenesis, molecular dynamics simulations, and QM/MM calculations. We provide insights into the complex electronic structure of [4Fe]H and its role in the catalytic reaction of CrHydA1, serving as paradigm for understanding [FeFe]-hydrogenases.

Structural determinants of oxygen resistance and Zn-mediated stability of the [FeFe]-hydrogenase from .

[FeFe]-hydrogenases catalyze the reversible two-electron reduction of two protons to molecular hydrogen. Although these enzymes are among the most efficient H-converting biocatalysts in nature, their catalytic cofactor (termed H-cluster) is irreversibly destroyed upon contact with dioxygen. The [FeFe]-hydrogenase CbA5H from has a unique mechanism to protect the H-cluster from oxygen-induced degradation.

Mesoporous Electrodes Enhance the Electrocatalytic Performance of [FeFe]-Hydrogenase.

The metalloenzyme [FeFe]-hydrogenase is of interest to future biotechnologies targeting the production of "green" hydrogen (H). We recently developed a simple two-step functionalized procedure to immobilize the [FeFe]-hydrogenase from Clostridium pasteurianum ("CpI") on mesoporous indium tin oxide (ITO) electrodes to achieve elevated H production with high operational stability and current densities of 8 mA cm. Here, we use a combination of atomic force microscopy (AFM), scanning electron microscopy (SEM) and electrochemical quartz crystal microbalance (EQCM) to understand how mesoporous ITO stabilizes and activates CpI for electroenzymatic H production.

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.