Iron-Sulfur Cluster States of the Hydrogenase Maturase HydF.
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ABSTRACT:
SUBMITTER: Shepard EM
PROVIDER: S-EPMC5940351 | biostudies-literature | 2017 Sep
REPOSITORIES: biostudies-literature
Publications
Iron-Sulfur Cluster States of the Hydrogenase Maturase HydF.
Shepard Eric M EM Byer Amanda S AS Broderick Joan B JB
Biochemistry 20170830 36
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Project description:[FeFe]-hydrogenases are nature's most prolific hydrogen catalysts, excelling at facilely interconverting H2 and protons. The catalytic core common to all [FeFe]-hydrogenases is a complex metallocofactor, referred to as the H-cluster, which is composed of a standard [4Fe-4S] cluster linked through a bridging thiolate to a 2Fe subcluster harboring dithiomethylamine, carbon monoxide, and cyanide ligands. This 2Fe subcluster is synthesized and inserted into [FeFe]-hydrogenase by three maturase enzymes denoted HydE, HydF, and HydG. HydE and HydG are radical S-adenosylmethionine enzymes and synthesize the nonprotein ligands of the H-cluster. HydF is a GTPase that functions as a scaffold or carrier for 2Fe subcluster production. Herein, we utilize UV-visible, circular dichroism, and electron paramagnetic resonance spectroscopic studies to establish the existence of redox active [4Fe-4S] and [2Fe-2S] clusters bound to HydF. We have used spectroelectrochemical titrations to assign iron-sulfur cluster midpoint potentials, have shown that HydF purifies with a reduced [2Fe-2S] cluster in the absence of exogenous reducing agents, and have tracked iron-sulfur cluster spectroscopic changes with quaternary structural perturbations. Our results provide an important foundation for understanding the maturation process by defining the iron-sulfur cluster content of HydF prior to its interaction with HydE and HydG. We speculate that the [2Fe-2S] cluster of HydF either acts as a placeholder for HydG-derived Fe(CO)2CN species or serves as a scaffold for 2Fe subcluster assembly.
| S-EPMC5501864 | biostudies-literature
Project description:[FeFe] hydrogenases have attracted extensive attention in the field of renewable energy research because of their remarkable efficiency for H2 gas production. H2 formation is catalyzed by a biologically unique hexanuclear iron cofactor denoted the H-cluster. The assembly of this cofactor requires a dedicated maturation machinery including HydF, a multidomain [4Fe4S] cluster protein with GTPase activity. HydF is responsible for harboring and delivering a precatalyst to the apo-hydrogenase, but the details of this process are not well understood. Here, we utilize gas-phase electrophoretic macromolecule analysis to show that a HydF dimer forms a transient interaction complex with the hydrogenase and that the formation of this complex depends on the cofactor content on HydF. Moreover, Fourier transform infrared, electron paramagnetic resonance, and UV-visible spectroscopy studies of mutants of HydF show that the isolated iron-sulfur cluster domain retains the capacity for binding the precatalyst in a reversible fashion and is capable of activating apo-hydrogenase in in vitro assays. These results demonstrate the central role of the iron-sulfur cluster domain of HydF in the final stages of H-cluster assembly, i.e. in binding and delivering the precatalyst.
| S-EPMC7450098 | biostudies-literature
Project description:Nature utilizes [FeFe]-hydrogenase enzymes to catalyze the interconversion between H2 and protons and electrons. Catalysis occurs at the H-cluster, a carbon monoxide-, cyanide-, and dithiomethylamine-coordinated 2Fe subcluster bridged via a cysteine to a [4Fe-4S] cluster. Biosynthesis of this unique metallocofactor is accomplished by three maturase enzymes denoted HydE, HydF, and HydG. HydE and HydG belong to the radical S-adenosylmethionine superfamily of enzymes and synthesize the nonprotein ligands of the H-cluster. These enzymes interact with HydF, a GTPase that acts as a scaffold or carrier protein during 2Fe subcluster assembly. Prior characterization of HydF demonstrated the protein exists in both dimeric and tetrameric states and coordinates both [4Fe-4S]2+/+ and [2Fe-2S]2+/+ clusters [Shepard, E. M., Byer, A. S., Betz, J. N., Peters, J. W., and Broderick, J. B. (2016) Biochemistry 55, 3514-3527]. Herein, electron paramagnetic resonance (EPR) is utilized to characterize the [2Fe-2S]+ and [4Fe-4S]+ clusters bound to HydF. Examination of spin relaxation times using pulsed EPR in HydF samples exhibiting both [4Fe-4S]+ and [2Fe-2S]+ cluster EPR signals supports a model in which the two cluster types either are bound to widely separated sites on HydF or are not simultaneously bound to a single HydF species. Gel filtration chromatographic analyses of HydF spectroscopic samples strongly suggest the [2Fe-2S]+ and [4Fe-4S]+ clusters are coordinated to the dimeric form of the protein. Lastly, we examined the 2Fe subcluster-loaded form of HydF and showed the dimeric state is responsible for [FeFe]-hydrogenase activation. Together, the results indicate a specific role for the HydF dimer in the H-cluster biosynthesis pathway.
| S-EPMC5490485 | biostudies-literature
Project description:[FeFe] hydrogenases carry out the redox interconversion of protons and molecular hydrogen (2H+ + 2e- ⇌ H2) at a complex Fe-S active site known as the H-cluster. The H-cluster consists of a [4Fe-4S] subcluster, denoted here as [4Fe]H, linked via a cysteine sulfur to an interesting organometallic [2Fe]H subcluster thought to be the subsite where the catalysis occurs. This [2Fe]H subcluster consists of two Fe atoms, linked with a bridging CO and a bridging SCH2NHCH2S azadithiolate (adt), with additional terminal CO and CN ligands bound to each Fe. Synthesizing such a complex organometallic unit is a fascinating problem in biochemistry, complicated by the toxic nature of both the CO and CN- species and the relative fragility of the azadithiolate bridge. It has been known for a number of years that this complex biosynthesis is carried out by a set of three essential Fe-S proteins, HydE, HydF, and HydG. HydF is a GTPase, while HydE and HydG are both members of the large family of radical S-adenosylmethionine (rSAM) enzymes. In this perspective we describe the history of research and discovery concerning these three Fe-S "maturase" proteins and describe recent evidence for a sequential biosynthetic pathway beginning with the synthesis of a mononuclear organometallic [Fe(ii)(CO)2CN(cysteine)] complex by the rSAM enzyme HydG and its subsequent activation by the second rSAM enzyme HydE to form a highly reactive Fe(i)(CO)2(CN)S species. In our model a pair of these Fe(i)(CO)2(CN)S units condense to form the [Fe(CO)2(CN)S]2 diamond core of the [2Fe]H cluster, requiring only the installation of the central CH2NHCH2 portion of the azadithiolate bridge, whose atoms are all sourced from the amino acid serine. This final step likely occurs with an interplay of HydE and HydF, the details of which yet remain to be elucidated.
| S-EPMC8162317 | biostudies-literature
Project description: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. Electron paramagnetic spectroscopy reveals that HydF binds both [4Fe-4S] and [2Fe-2S] clusters; however, when HydF is expressed in the presence of HydE and HydG (HydF(EG)), only the [4Fe-4S] cluster is observed by EPR. Insight into the fate of the [2Fe-2S] cluster harbored by HydF is provided by FTIR, which shows the presence of carbon monoxide and cyanide ligands in HydF(EG). 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.
| S-EPMC2890834 | biostudies-literature
Project description:Hydrogenases use complex metal cofactors to catalyze the reversible formation of hydrogen. In [FeFe]-hydrogenases, the H-cluster cofactor includes a diiron subcluster containing azadithiolate, three CO, and two CN(-) ligands. During the assembly of the H cluster, the radical S-adenosyl methionine (SAM) enzyme HydG lyses the substrate tyrosine to yield the diatomic ligands. These diatomic products form an enzyme-bound Fe(CO)x(CN)y synthon that serves as a precursor for eventual H-cluster assembly. To further elucidate the mechanism of this complex reaction, we report the crystal structure and EPR analysis of HydG. At one end of the HydG (βα)8 triosephosphate isomerase (TIM) barrel, a canonical [4Fe-4S] cluster binds SAM in close proximity to the proposed tyrosine binding site. At the opposite end of the active-site cavity, the structure reveals the auxiliary Fe-S cluster in two states: one monomer contains a [4Fe-5S] cluster, and the other monomer contains a [5Fe-5S] cluster consisting of a [4Fe-4S] cubane bridged by a μ2-sulfide ion to a mononuclear Fe(2+) center. This fifth iron is held in place by a single highly conserved protein-derived ligand: histidine 265. EPR analysis confirms the presence of the [5Fe-5S] cluster, which on incubation with cyanide, undergoes loss of the labile iron to yield a [4Fe-4S] cluster. We hypothesize that the labile iron of the [5Fe-5S] cluster is the site of Fe(CO)x(CN)y synthon formation and that the limited bonding between this iron and HydG may facilitate transfer of the intact synthon to its cognate acceptor for subsequent H-cluster assembly.
| S-EPMC4321296 | biostudies-literature
Project description:[Fe] hydrogenase (iron-sulfur-cluster-free hydrogenase) catalyzes the reversible reduction of methenyltetrahydromethanopterin (methenyl-H4MPT+) with H2 to methylene-H4MPT, a reaction involved in methanogenesis from H2 and CO2 in many methanogenic archaea. The enzyme harbors an iron-containing cofactor, in which a low-spin iron is complexed by a pyridone, two CO and a cysteine sulfur. [Fe] hydrogenase is thus similar to [NiFe] and [FeFe] hydrogenases, in which a low-spin iron carbonyl complex, albeit in a dinuclear metal center, is also involved in H2 activation. Like the [NiFe] and [FeFe] hydrogenases, [Fe] hydrogenase catalyzes an active exchange of H2 with protons of water; however, this activity is dependent on the presence of the hydride-accepting methenyl-H4MPT+. In its absence the exchange activity is only 0.01% of that in its presence. The residual activity has been attributed to the presence of traces of methenyl-H4MPT+ in the enzyme preparations, but it could also reflect a weak binding of H2 to the iron in the absence of methenyl-H4MPT+. To test this we reinvestigated the exchange activity with [Fe] hydrogenase reconstituted from apoprotein heterologously produced in Escherichia coli and highly purified iron-containing cofactor and found that in the absence of added methenyl-H4MPT+ the exchange activity was below the detection limit of the tritium method employed (0.1 nmol min(-1) mg(-1)). The finding reiterates that for H2 activation by [Fe] hydrogenase the presence of the hydride-accepting methenyl-H4MPT+ is essentially required. This differentiates [Fe] hydrogenase from [FeFe] and [NiFe] hydrogenases, which actively catalyze H2/H2O exchange in the absence of exogenous electron acceptors.
| S-EPMC2757585 | biostudies-other
Project description:[FeFe]-hydrogenases catalyze the reversible production of H2 in some bacteria and unicellular eukaryotes. These enzymes require ancillary proteins to assemble the unique active site H-cluster, a complex structure composed of a 2Fe center bridged to a [4Fe-4S] cubane. The first crystal structure of a key factor in the maturation process, HydF, has been determined at 3 Å resolution. The protein monomer present in the asymmetric unit of the crystal comprises three domains: a GTP-binding domain, a dimerization domain, and a metal cluster-binding domain, all characterized by similar folding motifs. Two monomers dimerize, giving rise to a stable dimer, held together mainly by the formation of a continuous β-sheet comprising eight β-strands from two monomers. Moreover, in the structure presented, two dimers aggregate to form a supramolecular organization that represents an inactivated form of the HydF maturase. The crystal structure of the latter furnishes several clues about the events necessary for cluster generation/transfer and provides an excellent model to begin elucidating the structure/function of HydF in [FeFe]-hydrogenase maturation.
| S-EPMC3243517 | biostudies-literature
Project description:Cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. This delivery process is regulated by availability of iron and oxygen, but it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we investigated the organization of CIA machinery under various conditions. We developed a targeted proteomics assay monitoring known CIA factors. Using this assay, we were able to detect NUBP1, CIAO3 and CIA substrates in immunoprecipitates of NUBP2, a component of the CIA scaffold complex. We also revealed that NUBP2 transiently associates with the CIA targeting complex (MMS19, CIAO1, CIAO2B), indicating the possible existence of CIA metabolons. We observed stronger interactions between CIAO3 and the CIA scaffold complex upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrated that CIAO3 mutant with defective Fe-S cluster binding failed to integrate into the metabolons. However, these mutants unexpectedly exhibit stronger association with CIA substrates regardless of their reduced association with the CIA targeting complex, implicating that CIAO3 and CIA substrates may present in complexes independent of the CIA targeting complex. Together, our data suggested that CIA components potentially form metabolons whose assembly are regulated by environmental cues and require Fe-S cluster incorporation in CIAO3. These findings provided additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization.
2021-11-16 | MSV000088394 | MassIVE
Project description:Hybrid methods, which combine and integrate several biochemical and biophysical techniques, have rapidly caught up in the last twenty years to provide a way to obtain a fuller description of proteins and molecular complexes with sizes and complexity otherwise not easily affordable. Here, we review the use of a robust hybrid methodology based on a mixture of NMR, SAXS, site directed mutagenesis and molecular docking which we have developed to determine the structure of weakly interacting molecular complexes. We applied this technique to gain insights into the structure of complexes formed amongst proteins involved in the molecular machine, which produces the essential iron-sulfur cluster prosthetic groups. Our results were validated both by X-ray structures and by other groups who adopted the same approach. We discuss the advantages and the limitations of our methodology and propose new avenues, which could improve it.
| S-EPMC5346568 | biostudies-literature