HscA and HscB Stimulate [2Fe-2S] Cluster Transfer from IscU to Apoferredoxin in an ATP-Dependent Reaction

Chandramouli, Kala; Johnson, Michael K.

doi:10.1021/bi061237w

Cited by 152 publications

(199 citation statements)

References 39 publications

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“…The presence of NTPase enzymes in the assembly of metal clusters appears to be an emerging theme in metallobiochemistry, although definitive roles have not yet been established in many cases (28)(29)(30)(31)(32)(33)(34). HydF contains an N-terminal GTPase domain comprised of the Walker A P-loop and Walker B Mg 2þ binding motifs (8), as well as a C-terminal domain containing the conserved amino acids CXH X44-53 HCXXC comprising the putative ligands for an iron-sulfur cluster (7).…”

Section: Discussionmentioning

confidence: 99%

Synthesis of the 2Fe subcluster of the [FeFe]-hydrogenase H cluster on the HydF scaffold

Shepard

McGlynn

Bueling

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

126

214

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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...

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Section: Discussionmentioning

confidence: 99%

Synthesis of the 2Fe subcluster of the [FeFe]-hydrogenase H cluster on the HydF scaffold

Shepard

McGlynn

Bueling

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

126

214

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“…The apo form of ferredoxin (aFd), has classically served as a [2Fe-2S] cluster acceptor protein in different biochemical studies (Wu et al, 2002;Wu and Cowan, 2003;Chandramouli and Johnson, 2006). We therefore used At-NEET as a potential [2Fe-2S] cluster donor and aFd as a potential [2Fe-2S] cluster acceptor ( Figure 7) to test the hypothesis that NEET could act as an Fe-S transfer protein.…”

Section: Knockdown and Rnai Mutants Of At-neet Show Late Bolting And mentioning

confidence: 99%

Characterization of Arabidopsis NEET Reveals an Ancient Role for NEET Proteins in Iron Metabolism

et al. 2012

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The NEET family is a newly discovered group of proteins involved in a diverse array of biological processes, including autophagy, apoptosis, aging, diabetes, and reactive oxygen homeostasis. They form a novel structure, the NEET fold, in which two protomers intertwine to form a two-domain motif, a cap, and a unique redox-active labile 2Fe-2S cluster binding domain. To accelerate the functional study of NEET proteins, as well as to examine whether they have an evolutionarily conserved role, we identified and characterized a plant NEET protein. Here, we show that the Arabidopsis thaliana At5g51720 protein (At-NEET) displays biochemical, structural, and biophysical characteristics of a NEET protein. Phenotypic characterization of At-NEET revealed a key role for this protein in plant development, senescence, reactive oxygen homeostasis, and Fe metabolism. A role in Fe metabolism was further supported by biochemical and cell biology studies of At-NEET in plant and mammalian cells, as well as mutational analysis of its cluster binding domain. Our findings support the hypothesis that NEET proteins have an ancient role in cells associated with Fe metabolism.

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“…This motif is missing in SufU-type and NifU-type scaffolds, which are not usually encoded with HscA or HscB, suggesting that SufU and NifU either do not require molecular chaperones or utilize a completely separate chaperone system (46). Recent work has shown that A. vinelandii HscA and HscB can enhance transfer of [2Fe-2S] clusters from IscU to apoproteins in an ATP-dependent manner (20). Deletion of hscA or hscB does diminish the activity of succinate dehydrogenase (SDH or complex II) in E. coli (121).…”

Section: Molecluar Chaperones For Fe-s Cluster Assemblymentioning

confidence: 99%

Fe-S Cluster Assembly Pathways in Bacteria

Ayala-Castro

Saini

Outten

2008

Microbiol Mol Biol Rev

273

258

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SUMMARY Iron-sulfur (Fe-S) clusters are required for critical biochemical pathways, including respiration, photosynthesis, and nitrogen fixation. Assembly of these iron cofactors is a carefully controlled process in cells to avoid toxicity from free iron and sulfide. Multiple Fe-S cluster assembly pathways are present in bacteria to carry out basal cluster assembly, stress-responsive cluster assembly, and enzyme-specific cluster assembly. Although biochemical and genetic characterization is providing a partial picture of in vivo Fe-S cluster assembly, a number of mechanistic questions remain unanswered. Furthermore, new factors involved in Fe-S cluster assembly and repair have recently been identified and are expanding the complexity of current models. Here we attempt to summarize recent advances and to highlight new avenues of research in the field of Fe-S cluster assembly.

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HscA and HscB Stimulate [2Fe-2S] Cluster Transfer from IscU to Apoferredoxin in an ATP-Dependent Reaction

Cited by 152 publications

References 39 publications

Synthesis of the 2Fe subcluster of the [FeFe]-hydrogenase H cluster on the HydF scaffold

Synthesis of the 2Fe subcluster of the [FeFe]-hydrogenase H cluster on the HydF scaffold

Characterization of Arabidopsis NEET Reveals an Ancient Role for NEET Proteins in Iron Metabolism

Fe-S Cluster Assembly Pathways in Bacteria

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