Spectroscopic Investigations of [FeFe] Hydrogenase Maturated with [(57)Fe2(adt)(CN)2(CO)4](2-).
ABSTRACT: 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:[FeFe] Hydrogenases catalyze the reversible conversion of H2 into electrons and protons. Their catalytic site, the H-cluster, contains a generic [4Fe-4S]H cluster coupled to a [2Fe]H subsite [Fe2(ADT)(CO)3(CN)2]2-, ADT?=?µ(SCH2)2NH. Heterologously expressed [FeFe] hydrogenases (apo-hydrogenase) lack the [2Fe]H unit, but this can be incorporated through artificial maturation with a synthetic precursor [Fe2(ADT)(CO)4(CN)2]2-. Maturation with a [2Fe] complex in which the essential ADT amine moiety has been replaced by CH2 (PDT?=?propane-dithiolate) results in a low activity enzyme with structural and spectroscopic properties similar to those of the native enzyme, but with simplified redox behavior. Here, we study the effect of sulfur-to-selenium (S-to-Se) substitution in the bridging PDT ligand incorporated in the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii using magnetic resonance (EPR, NMR), FTIR and spectroelectrochemistry. The resulting HydA1-PDSe enzyme shows the same redox behavior as the parent HydA1-PDT. In addition, a state is observed in which extraneous CO is bound to the open coordination site of the [2Fe]H unit. This state was previously observed only in the native enzyme HydA1-ADT and not in HydA1-PDT. The spectroscopic features and redox behavior of HydA1-PDSe, resulting from maturation with [Fe2(PDSe)(CO)4(CN)2]2-, are discussed in terms of spin and charge density shifts and provide interesting insight into the electronic structure of the H-cluster. We also studied the effect of S-to-Se substitution in the [4Fe-4S] subcluster. The reduced form of HydA1 containing only the [4Fe-4Se]H cluster shows a characteristic S?=?7/2 spin state which converts back into the S?=?1/2 spin state upon maturation with a [2Fe]-PDT/ADT complex.
Project description:The [FeFe] hydrogenase from Clostridium pasteurianum (CpI) harbors four Fe-S clusters that facilitate the transfer of an electron to the H-cluster, a ligand-coordinated six-iron prosthetic group that catalyzes the redox interconversion of protons and H(2). Here, we have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the iron centers in CpI, and we compare our data to that for a [4Fe-4S] ferredoxin as well as a model complex resembling the [2Fe](H) catalytic domain of the H-cluster. To enrich the hydrogenase with (57)Fe nuclei, we used cell-free methods to post-translationally mature the enzyme. Specifically, inactive CpI apoprotein with (56)Fe-labeled Fe-S clusters was activated in vitro using (57)Fe-enriched maturation proteins. This approach enabled us to selectively label the [2Fe](H) subcluster with (57)Fe, which NRVS confirms by detecting (57)Fe-CO and (57)Fe-CN normal modes from the H-cluster nonprotein ligands. The NRVS and iron quantification results also suggest that the hydrogenase contains a second (57)Fe-S cluster. Electron paramagnetic resonance (EPR) spectroscopy indicates that this (57)Fe-enriched metal center is not the [4Fe-4S](H) subcluster of the H-cluster. This finding demonstrates that the CpI hydrogenase retained an (56)Fe-enriched [4Fe-4S](H) cluster during in vitro maturation, providing unambiguous evidence of stepwise assembly of the H-cluster. In addition, this work represents the first NRVS characterization of [FeFe] hydrogenases.
Project description:[FeFe]-hydrogenase enzymes employ a unique organometallic cofactor for efficient and reversible hydrogen conversion. This so-called H-cluster consists of a [4Fe-4S] cubane cysteine linked to a diiron complex coordinated by carbon monoxide and cyanide ligands and an azadithiolate ligand (adt?=?NH(CH2S)2)·[FeFe]-hydrogenase apo-protein binding only the [4Fe-4S] sub-complex can be fully activated in vitro by the addition of a synthetic diiron site precursor complex ([2Fe]adt). Elucidation of the mechanism of cofactor assembly will aid in the design of improved hydrogen processing synthetic catalysts. We combined electron paramagnetic resonance, Fourier-transform infrared, and X-ray absorption spectroscopy to characterize intermediates of H-cluster assembly as initiated by mixing of the apo-protein (HydA1) from the green alga Chlamydomonas reinhardtii with [2Fe]adt. The three methods consistently show rapid formation of a complete H-cluster in the oxidized, CO-inhibited state (Hox-CO) already within seconds after the mixing. Moreover, FTIR spectroscopy support a model in which Hox-CO formation is preceded by a short-lived Hred'-CO-like intermediate. Accumulation of Hox-CO was followed by CO release resulting in the slower conversion to the catalytically active state (Hox) as well as formation of reduced states of the H-cluster.
Project description:Active site mimics of [FeFe]-hydrogenase are shown to be bidirectional catalysts, producing H2 upon treatment with protons and reducing equivalents. This reactivity complements the previously reported oxidation of H2 by these same catalysts in the presence of oxidants. The complex Fe2(adt(Bn))(CO)3(dppv)(PFc*(Et2) ) ((0); adt(Bn) = (SCH2)2NBn, dppv = cis-1,2-bis(diphenylphosphino)ethylene, PFc*(Et2) = Et2PCH2C5Me4FeCp*) reacts with excess [H(OEt2)2]BAr(F)4 (BAr(F)4(-) = B(C6H3-3,5-(CF3)2)4(-)) to give ?0.5 equiv of H2 and [Fe2(adt(Bn)H)(CO)3(dppv)(PFc*(Et2) )](2+) ([1H](2+)). The species [1H](2+) consists of a ferrocenium ligand, an N-protonated amine, and an Fe(I)Fe(I) core. In the presence of additional reducing equivalents in the form of decamethylferrocene (Fc*), hydrogen evolution is catalytic, albeit slow. The related catalyst Fe2(adt(Bn))(CO)3(dppv)(PMe3) (3) behaves similarly in the presence of Fc*, except that in the absence of excess reducing agent it converts to the catalytically inactive ?-hydride derivative [?-H3](+). Replacement of the adt in (0) with propanedithiolate (pdt) results in a catalytically inactive complex. In the course of synthesizing [FeFe]-hydrogenase mimics, new routes to ferrocenylphosphine ligands and nonamethylferrocene were developed.
Project description:The active site of the [FeFe] hydrogenase (HydA1), the H-cluster, is a 6-Fe cofactor that contains CO and CN- ligands. It undergoes several different oxidation and protonation state changes in its catalytic cycle to metabolize H2. Among them, the well-known Hox state and the recently identified Hhyd state are thought to be directly involved in H2 activation and evolution, and they are both EPR active with net spin S = 1/2. Herein, we report the pulse electronic paramagnetic spectroscopic investigation of these two catalytic states in Chlamydomonas reinhardtii HydA1 ( CrHydA1). Using an in vitro biosynthetic maturation approach, we site-specifically installed 13C into the CO or CN- ligands and 57Fe into the [2Fe]H subcluster of the H-cluster in order to measure the hyperfine couplings to these magnetic nuclei. For Hox, we measured 13C hyperfine couplings (13CO aiso of 25.5, 5.8, and 4.5 MHz) corresponding to all three CO ligands in the H-cluster. We also observed two 57Fe hyperfine couplings (57Fe aiso of ?17 and 5.7 MHz) arising from the two Fe atoms in the [2Fe]H subcluster. For Hhyd, we only observed two distinct 13CO hyperfine interactions (13CO aiso of 0.16 and 0.08 MHz) and only one for 13CN- (13CN aiso of 0.16 MHz); the couplings to the 13CO/13CN- on the distal Fe of [2Fe]H may be too small to detect. We also observed a very small (<0.3 MHz) 57Fe HFI from the labeled [2Fe]H subcluster and four 57Fe HFI from the labeled [4Fe-4S]H subcluster (57Fe aiso of 7.2, 16.6, 28.2, and 35.3 MHz). These hyperfine coupling constants are consistent with the previously proposed electronic structure of the H-cluster at both Hox and Hhyd states and provide a basis for more detailed analysis.
Project description: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:The [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii has been studied using 1H NMR spectroscopy identifying the paramagnetically shifted 1H resonances associated with both the [4Fe-4S]H and the [2Fe]H subclusters of the active site "H-cluster". The signal pattern of the unmaturated HydA1 containing only [4Fe-4S]H is reminiscent of bacterial-type ferredoxins. The spectra of maturated HydA1, with a complete H-cluster in the active Hox and the CO-inhibited Hox-CO state, reveal additional upfield and downfield shifted 1H resonances originating from the four methylene protons of the azadithiolate ligand in the [2Fe]H subsite. The two axial protons are affected by positive spin density, while the two equatorial protons experience negative spin density. These protons can be used as important probes sensing the effects of ligand-binding to the catalytic site of the H-cluster.
Project description:The active site of [FeFe] hydrogenase features a binuclear iron cofactor Fe2ADT(CO)3(CN)2, where ADT represents the bridging ligand aza-propane-dithiolate. The terminal diatomic ligands all coordinate in a basal configuration, and one CO bridges the two irons leaving an open coordination site at which the hydrogen species and the competitive inhibitor CO bind. Externally supplied CO is expected to coordinate in an apical configuration. However, an alternative configuration has been proposed in which, due to ligand rotation, the CN- bound to the distal Fe becomes apical. Using selective 13C isotope labeling of the CN- and COext ligands in combination with pulsed 13C electron-nuclear-nuclear triple resonance spectroscopy, spin polarization effects are revealed that, according to density functional theory calculations, are consistent with only the "unrotated" apical COext configuration.
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.