A [4Fe-4S]-Fe(CO)(CN)-L-cysteine intermediate is the first organometallic precursor in [FeFe] hydrogenase H-cluster bioassembly.
ABSTRACT: Biosynthesis of the [FeFe] hydrogenase active site (the 'H-cluster') requires the interplay of multiple proteins and small molecules. Among them, the radical S-adenosylmethionine enzyme HydG, a tyrosine lyase, has been proposed to generate a complex that contains an Fe(CO)2(CN) moiety that is eventually incorporated into the H-cluster. Here we describe the characterization of an intermediate in the HydG reaction: a [4Fe-4S][(Cys)Fe(CO)(CN)] species, 'Complex A', in which a CO, a CN- and a cysteine (Cys) molecule bind to the unique 'dangler' Fe site of the auxiliary [5Fe-4S] cluster of HydG. The identification of this intermediate-the first organometallic precursor to the H-cluster-validates the previously hypothesized HydG reaction cycle and provides a basis for elucidating the biosynthetic origin of other moieties of the H-cluster.
Project description:Hydrogenases catalyze the redox interconversion of protons and H2, an important reaction for a number of metabolic processes and for solar fuel production. In FeFe hydrogenases, catalysis occurs at the H cluster, a metallocofactor comprising a [4Fe-4S]H subcluster coupled to a [2Fe]H subcluster bound by CO, CN(-), and azadithiolate ligands. The [2Fe]H subcluster is assembled by the maturases HydE, HydF, and HydG. HydG is a member of the radical S-adenosyl-L-methionine family of enzymes that transforms Fe and L-tyrosine into an [Fe(CO)2(CN)] synthon that is incorporated into the H cluster. Although it is thought that the site of synthon formation in HydG is the "dangler" Fe of a [5Fe] cluster, many mechanistic aspects of this chemistry remain unresolved including the full ligand set of the synthon, how the dangler Fe initially binds to HydG, and how the synthon is released at the end of the reaction. To address these questions, we herein show that L-cysteine (Cys) binds the auxiliary [4Fe-4S] cluster of HydG and further chelates the dangler Fe. We also demonstrate that a [4Fe-4S]aux[CN] species is generated during HydG catalysis, a process that entails the loss of Cys and the [Fe(CO)2(CN)] fragment; on this basis, we suggest that Cys likely completes the coordination sphere of the synthon. Thus, through spectroscopic analysis of HydG before and after the synthon is formed, we conclude that Cys serves as the ligand platform on which the synthon is built and plays a role in both Fe(2+) binding and synthon release.
Project description:The radical SAM enzyme HydG generates CO- and CN--containing Fe complexes that are involved in the bioassembly of the [FeFe] hydrogenase active cofactor, the H-cluster. HydG contains a unique 5Fe-4S cluster in which the fifth "dangler" Fe and the coordinating cysteine molecule have both been shown to be essential for its function. Here, we demonstrate that this dangler Fe can be replaced with Ni2+ or Co2+ and that the cysteine can be replaced with selenocysteine. The resulting HydG variants were characterized by electron paramagnetic resonance and X-ray absorption spectroscopy, as well as subjected to a Tyr cleavage assay. Both Ni2+ and Co2+ are shown to be exchange-coupled to the 4Fe-4S cluster, and selenocysteine substitution does not alter the electronic structure significantly. XAS data provide details of the coordination environments near the Ni, Co, and Se atoms and support a close interaction of the dangler metal with the FeS cluster via an asymmetric SeCys bridge. Finally, while we were unable to observe the formation of novel organometallic species for the Ni2+ and Co2+ variants, the selenocysteine variant retains the activity of wild type HydG in forming [Fe(CO)x(CN)y] species. Our results provide more insights into the unique auxiliary cluster in HydG and expand the scope of artificially generated Fe-S clusters with heteroatoms.
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.
Project description:Three maturase enzymes-HydE, HydF, and HydG-synthesize and insert the organometallic component of the [FeFe]-hydrogenase active site (the H-cluster). HydG generates the first organometallic intermediates in this process, ultimately producing an [Fe(CO)2(CN)] complex. A limitation in understanding the mechanism by which this complex forms has been uncertainty regarding the precise metallocluster composition of HydG that comprises active enzyme. We herein show that the HydG auxiliary cluster must bind both l-cysteine and a dangler Fe in order to generate the [Fe(CO)2(CN)] product. These findings support a mechanistic framework in which a [(Cys)Fe(CO)2(CN)](-) species is a key intermediate in H-cluster maturation.
Project description:Hydrogenase enzymes catalyze the rapid and reversible interconversion of H2 with protons and electrons. The active site of the [FeFe] hydrogenase is the H cluster, which consists of a [4Fe-4S]H subcluster linked to an organometallic [2Fe]H subcluster. Understanding the biosynthesis and catalytic mechanism of this structurally unusual active site will aid in the development of synthetic and biological hydrogenase catalysts for applications in solar fuel generation. The [2Fe]H subcluster is synthesized and inserted by three maturase enzymes-HydE, HydF, and HydG-in a complex process that involves inorganic, organometallic, and organic radical chemistry. HydG is a member of the radical S-adenosyl-l-methionine (SAM) family of enzymes and is thought to play a prominent role in [2Fe]H subcluster biosynthesis by converting inorganic Fe(2+), l-cysteine (Cys), and l-tyrosine (Tyr) into an organometallic [(Cys)Fe(CO)2(CN)](-) intermediate that is eventually incorporated into the [2Fe]H subcluster. In this Forum Article, the mechanism of [2Fe]H subcluster biosynthesis is discussed with a focus on how this key [(Cys)Fe(CO)2(CN)](-) species is formed. Particular attention is given to the initial metallocluster composition of HydG, the modes of substrate binding (Fe(2+), Cys, Tyr, and SAM), the mechanism of SAM-mediated Tyr cleavage to CO and CN(-), and the identification of the final organometallic products of the reaction.
Project description:The H-cluster of [FeFe]-hydrogenase consists of a [4Fe-4S]H-subcluster linked by a cysteinyl bridge to a unique organometallic [2Fe]H-subcluster assigned as the site of interconversion between protons and molecular hydrogen. This [2Fe]H-subcluster is assembled by a set of Fe-S maturase enzymes HydG, HydE and HydF. Here we show that the HydG product [FeII(Cys)(CO)2(CN)] synthon is the substrate of the radical SAM enzyme HydE, with the generated 5'-deoxyadenosyl radical attacking the cysteine S to form a C5'-S bond concomitant with reduction of the central low-spin Fe(II) to the Fe(I) oxidation state. This leads to the cleavage of the cysteine C3-S bond, producing a mononuclear [FeI(CO)2(CN)S] species that serves as the precursor to the dinuclear Fe(I)Fe(I) center of the [2Fe]H-subcluster. This work unveils the role played by HydE in the enzymatic assembly of the H-cluster and expands the scope of radical SAM enzyme chemistry.
Project description:Three iron-sulfur proteins--HydE, HydF, and HydG--play a key role in the synthesis of the [2Fe](H) component of the catalytic H-cluster of FeFe hydrogenase. The radical S-adenosyl-L-methionine enzyme HydG lyses free tyrosine to produce p-cresol and the CO and CN(-) ligands of the [2Fe](H) cluster. Here, we applied stopped-flow Fourier transform infrared and electron-nuclear double resonance spectroscopies to probe the formation of HydG-bound Fe-containing species bearing CO and CN(-) ligands with spectroscopic signatures that evolve on the 1- to 1000-second time scale. Through study of the (13)C, (15)N, and (57)Fe isotopologs of these intermediates and products, we identify the final HydG-bound species as an organometallic Fe(CO)2(CN) synthon that is ultimately transferred to apohydrogenase to form the [2Fe](H) component of the H-cluster.
Project description:The synthesis and assembly of the active site [FeFe] unit of [FeFe]-hydrogenases require at least three maturases. The radical S-adenosyl-l-methionine HydG, the best characterized of these proteins, is responsible for the synthesis of the hydrogenase CO and CN(-) ligands from tyrosine-derived dehydroglycine (DHG). We speculated that CN(-) and the CO precursor (-):CO2H may be generated through an elimination reaction. We tested this hypothesis with both wild type and HydG variants defective in second iron-sulfur cluster coordination by measuring the in vitro production of CO, CN(-), and (-):CO2H-derived formate. We indeed observed formate production under these conditions. We conclude that HydG is a multifunctional enzyme that produces DHG, CN(-), and CO at three well-differentiated catalytic sites. We also speculate that homocysteine, cysteine, or a related ligand could be involved in Fe(CO)x(CN)y transfer to the HydF carrier/scaffold.
Project description:The preparation and spectroscopic characterization of a CO-inhibited [FeFe] hydrogenase with a selectively (57)Fe-labeled binuclear subsite is described. The precursor [(57)Fe2(adt)(CN)2(CO)4](2-) was synthesized from the (57)Fe metal, S8, CO, (NEt4)CN, NH4Cl, and CH2O. (Et4N)2[(57)Fe2(adt)(CN)2(CO)4] was then used for the maturation of the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii, to yield the enzyme selectively labeled at the [2Fe]H subcluster. Complementary (57)Fe enrichment of the [4Fe-4S]H cluster was realized by reconstitution with (57)FeCl3 and Na2S. The Hox-CO state of [2(57)Fe]H and [4(57)Fe-4S]H HydA1 was characterized by Mössbauer, HYSCORE, ENDOR, and nuclear resonance vibrational spectroscopy.
Project description:S-Adenosyl methionine (SAM) is employed as a [4Fe-4S]-bound cofactor in the superfamily of radical SAM (rSAM) enzymes, in which one-electron reduction of the [4Fe-4S]-SAM moiety leads to homolytic cleavage of the S-adenosyl methionine to generate the 5'-deoxyadenosyl radical (5'dAdo•), a potent H-atom abstractor. HydG, a member of this rSAM family, uses the 5'dAdo• radical to lyse its substrate, tyrosine, producing CO and CN that bind to a unique Fe site of a second HydG Fe-S cluster, ultimately producing a mononuclear organometallic Fe-l-cysteine-(CO)2CN complex as an intermediate in the bioassembly of the catalytic H-cluster of [Fe-Fe] hydrogenase. Here we report the use of non-native tyrosine substrate analogues to further probe the initial radical chemistry of HydG. One such non-native substrate is 4-hydroxy phenyl propanoic acid (HPPA) which lacks the amino group of tyrosine, replacing the C?H-NH2 with a CH2 at the C2 position. Electron paramagnetic resonance (EPR) studies show the generation of a strong and relatively stable radical in the HydG reaction with natural abundance and 13C2-HPPA, with appreciable spin density localized at C2. These results led us to try parallel experiments with the more oxidized non-native substrate coumaric acid, which has a C2=C3 alkene substitution relative to HPPA's single bond. Interestingly, the HydG reaction with the cis-p-coumaric acid isomer led to the trapping of a new radical EPR signal, and EPR studies using cis-p-coumaric acid along with isotopically labeled SAM reveal that we have for the first time trapped and characterized the 5'dAdo• radical in an actual rSAM enzyme reaction, here by using this specific non-native substrate cis-p-coumaric acid. Density functional theory energetics calculations show that the cis-p-coumaric acid has approximately the same C-H bond dissociation free energy as 5'dAdo•, providing a possible explanation for our ability to trap an appreciable fraction of 5'dAdo• in this specific rSAM reaction. The radical's EPR line shape and its changes with SAM isotopic substitution are nearly identical to those of a 5'dAdo• radical recently generated by cryophotolysis of a prereduced [4Fe-4S]-SAM center in another rSAM enzyme, pyruvate formate-lyase activating enzyme, further supporting our assignment that we have indeed trapped and characterized the 5'dAdo• radical in a radical SAM enzymatic reaction by appropriate tuning of the relative radical free energies via the judicious selection of a non-native substrate.