The organometallic H cluster at the active site of [FeFe]-hydrogenase consists of a 2Fe subcluster coordinated by cyanide, carbon monoxide, and a nonprotein dithiolate bridged to a [4Fe-4S] cluster via a cysteinate ligand. Biosynthesis of this cluster requires three accessory proteins, two of which (HydE and HydG) are radical S-adenosylmethionine enzymes. The third, HydF, is a GTPase. We present here spectroscopic and kinetic studies of HydF that afford fundamental new insights into the mechanism of H-cluster assembly. The thorough kinetic characterization of the GTPase activity of HydF shows that activity can be gated by monovalent cations and further suggests that GTPase activity is associated with synthesis of the 2Fe subcluster precursor on HydF, rather than with transfer of the assembled precursor to hydrogenase. Interestingly, we show that whereas the GTPase activity is independent of the presence of the FeS clusters on HydF, GTP perturbs the EPR spectra of the clusters, suggesting communication between the GTP-and cluster-binding sites. Together, the results indicate that the 2Fe subcluster of the H cluster is synthesized on HydF from a [2Fe-2S] cluster framework in a process requiring HydE, HydG, and GTP.T he reversible reduction of protons, a reaction central to bioenergy and fuel cell applications, is a conceptually simple but chemically challenging reaction. In biology, these reactions occur at unique organometallic metal centers that contain biochemically unusual nonprotein ligands such as carbon monoxide and cyanide. In the case of the [FeFe]-hydrogenase, the site of catalysis is a metal cluster, termed the H cluster, consisting of a [4Fe-4S] cubane bridged by a cysteine thiolate to a 2Fe unit coordinated by carbon monoxide, cyanide, and a bridging dithiolate ligand (Fig. 1) (1-6). The [FeFe]-hydrogenase is of particular interest for bioenergy applications because of its high catalytic rates of proton reduction; however, a limiting factor in its practical utilization is the lack of understanding of the biosynthesis of the organometallic active site cluster. Assembly of a catalytically competent H cluster requires the actions of three hydrogenase-specific accessory proteins, two of which (HydE and HydG) are radical S-adenosylmethionine (SAM) enzymes and the third of which (HydF) is a GTPase (7, 8). These accessory proteins are directed at synthesis of the 2Fe subcluster of the H cluster, which is subsequently transferred to the hydrogenase structural protein (HydA) containing a preformed [4Fe-4S] cluster (9, 10) to produce the active hydrogenase. The detailed stepwise mechanism of H-cluster assembly, as well as the specific roles of and interactions between the three accessory proteins in this assembly process, remains largely unknown. Herein we provide evidence that the 2Fe subcluster of the H cluster is synthesized on HydF from a [2Fe-2S] precursor by the activities of HydE and HydG and that GTP hydrolysis likely plays a role in the assembly of this precursor on HydF.Radical SAM enzymes are charact...

In an effort to determine the specific protein component(s) responsible for in vitro activation of the [FeFe] hydrogenase (HydA), the individual maturation proteins HydE, HydF, and HydG from Clostridium acetobutylicum were purified from heterologous expressions in Escherichia coli. Our results demonstrate that HydF isolated from a strain expressing all three maturation proteins is sufficient to confer hydrogenase activity to purified inactive heterologously expressed HydA (expressed in the absence of HydE, HydF, and HydG). These results represent the first in vitro maturation of [FeFe] hydrogenase with purified proteins, and suggest that HydF functions as a scaffold upon which an H-cluster intermediate is synthesized.

A simple, yet powerful wave function manipulation method was introduced utilizing a generalized ionic fragment approach that allows for systematic mapping of the wave function space for multispin systems with antiferromagnetic coupling. The use of this method was demonstrated for developing ground state electronic wave function for [2Fe-2S] and [Mo-3Fe-4S] clusters. Using well-defined ionic wave functions for ferrous and ferric irons, sulfide, and thiolate fragments, the accuracy of various density functionals and basis sets including effective core potentials were evaluated on a [4Fe-4S] cluster by comparing the calculated geometric and electronic structures with crystallographic data and experimental atomic spin densities from X-ray absorption spectroscopy, respectively. We found that the most reasonable agreement for both geometry and atomic spin densities is obtained by a hybrid functional with 5% HF exchange and 95% density functional exchange supplemented with Perdew's 1986 correlation functional. The basis set seems to saturate only at the triple-level with polarization and diffuse functions. Reasonably preoptimized structures can be obtained by employing computationally less expensive effective core potentials, such as the Stuttgart-Dresden potential with a triple-valence basis set. The extension of the described calibration methodology to other biologically important and more complex iron-sulfur clusters, such as hydrogenase H-cluster and nitrogenase FeMo-co will follow.

This review identifies how the classical/non‐classical renin‐angiotensin system (RAS) and exercise influence muscle wasting. The classical RAS axis enhances muscle loss through the interaction with NADPH oxidase (NOX), ubiquitin proteasome system (UPS), protein synthesis and fibrosis pathways. The mainstream hypothesis identifies reactive oxygen species (ROS) as the key pathway in muscle, this review recognizes alternative pathways that lead to an increase in muscle wasting through the classical RAS axis. In addition, pathways in which the non‐classical RAS axis and exercise inhibit the classical RAS axis are also explored. The non‐classical RAS axis and exercise have a significant negative impact on ROS production and protein synthesis. The non‐classical RAS axis has been identified in this review to directly affect protein synthesis pathways not by altering the pre‐existing intracellular ROS level, further supporting the idea that muscle wasting caused by the classical RAS system is not entirely due to ROS production. Exercise has been identified to modify the RAS axes making it a therapeutic option.