Diiron Site Research Articles

A Conserved Binding Pocket in HydF is Essential for Biological Assembly and Coordination of the Diiron Site of [FeFe]-Hydrogenases.

The active site cofactor of [FeFe]-hydrogenases consists of a cubane [4Fe-4S]-cluster and a unique [2Fe-2S]-cluster, harboring unusual CO- and CN--ligands. The biosynthesis of the [2Fe-2S]-cluster requires three dedicated maturation enzymes called HydG, HydE and HydF. HydG and HydE are both involved in synthesizing a [2Fe-2S]-precursor, still lacking parts of the azadithiolate (adt) moiety that bridge the two iron atoms. This [2Fe-2S]-precursor is then finalized within the scaffold protein HydF, which binds and transfers the [2Fe-2S]-precursor to the hydrogenase. However, its exact binding mode within HydF is still elusive. Herein, we identified the binding location of the [2Fe-2S]-precursor by altering size and charge of a highly conserved protein pocket via site directed mutagenesis (SDM). Moreover, we identified two serine residues that are essential for binding and assembling the [2Fe-2S]-precursor. By combining SDM and molecular docking simulations, we provide a new model on how the [2Fe-2S]-cluster is bound to HydF and demonstrate the important role of one highly conserved aspartate residue, presumably during the bioassembly of the adt moiety.

The Ferroxidase Centre of Escherichia coli Bacterioferritin Plays a Key Role in the Reductive Mobilisation of the Mineral Iron Core

AbstractFerritins are multimeric cage‐forming proteins that play a crucial role in cellular iron homeostasis. All H‐chain‐type ferritins harbour a diiron site, the ferroxidase centre, at the centre of a 4 α‐helical bundle, but bacterioferritins are unique in also binding 12 hemes per 24 meric assembly. The ferroxidase centre is known to be required for the rapid oxidation of Fe2+ during deposition of an immobilised ferric mineral core within the protein's hollow interior. In contrast, the heme of bacterioferritin is required for the efficient reduction of the mineral core during iron release, but has little effect on the rate of either oxidation or mineralisation of iron. Thus, the current view is that these two cofactors function in iron uptake and release, respectively, with no functional overlap. However, rapid electron transfer between the heme and ferroxidase centre of bacterioferritin from Escherichia coli was recently demonstrated, suggesting that the two cofactors may be functionally connected. Here we report absorbance and (magnetic) circular dichroism spectroscopies, together with in vitro assays of iron‐release kinetics, which demonstrate that the ferroxidase centre plays an important role in the reductive mobilisation of the bacterioferritin mineral core, which is dependent on the heme‐ferroxidase centre electron transfer pathway.

The Ferroxidase Centre of Escherichia coli Bacterioferritin Plays a Key Role in the Reductive Mobilisation of the Mineral Iron Core.

Ferritins are multimeric cage-forming proteins that play a crucial role in cellular iron homeostasis. All H-chain-type ferritins harbour a diiron site, the ferroxidase centre, at the centre of a 4 α-helical bundle, but bacterioferritins are unique in also binding 12 hemes per 24 meric assembly. The ferroxidase centre is known to be required for the rapid oxidation of Fe2+ during deposition of an immobilised ferric mineral core within the protein's hollow interior. In contrast, the heme of bacterioferritin is required for the efficient reduction of the mineral core during iron release, but has little effect on the rate of either oxidation or mineralisation of iron. Thus, the current view is that these two cofactors function in iron uptake and release, respectively, with no functional overlap. However, rapid electron transfer between the heme and ferroxidase centre of bacterioferritin from Escherichia coli was recently demonstrated, suggesting that the two cofactors may be functionally connected. Here we report absorbance and (magnetic) circular dichroism spectroscopies, together with in vitro assays of iron-release kinetics, which demonstrate that the ferroxidase centre plays an important role in the reductive mobilisation of the bacterioferritin mineral core, which is dependent on the heme-ferroxidase centre electron transfer pathway.

Use of the Asymmetrical Chelating N-Donor 2-Imino-Pyridine as a Redox [Fe4S4] Cubane Surrogate at a Di-Iron Site Related to [FeFe]-Hydrogenases

Two complexes, related to the active site of [FeFe]-hydrogenases, [Fe2(CO)4(κ2-pma)(µ-bdt)] (1) and [Fe2(CO)4(κ2-pma)(µ-pdt)] (2) (bdt = benzene-1,2-dithiolate, pdt = propane-1,2-dithiolate) featuring the diaza chelate ligand trans-N-(2-pyridylmethylene)aniline (pma) were prepared, in order to study the influence of such a redox ligand, potentially non-innocent, on their redox behaviours. Both complexes were synthesized by photolysis in moderate yields, and they were characterized by IR, 1H and 13C{1H} NMR spectroscopies, elemental analyses and X-ray diffraction. Their electrochemical study by cyclic voltammetry, in the presence and in the absence of protons, revealed different behaviours depending on the aliphatic or aromatic nature of the dithiolate bridge. Density functional theory (DFT) calculations showed the role of the pma ligand as an electron reservoir, allowing the rationalization of the proton reduction process of complex 1.

Nitrite Formation at a Diiron Dinitrosyl Complex.

Pathogenic bacteria employ iron-containing enzymes to detoxify nitric oxide (NO•) produced by mammals as part of their immune response. Two classes of diiron proteins, flavodiiron nitric oxide reductases (FNORs) and the hemerythrin-like proteins from mycobacteria (HLPs), are upregulated in bacteria in response to an increased local NO• concentration. While FNORs reduce NO• to nitrous oxide (N2O), the HLPs have been found to either reduce nitrite to NO• (YtfE), or oxidize NO• to nitrite (Mka-HLP). Various structural and functional models of the diiron site in FNORs have been developed over the years. However, the NO• oxidation reactivity of Mka-HLP has yet to be replicated with a synthetic complex. Compared to the FNORs, the coordination environment of the diiron site in Mka-HLP contains one less carboxylate ligand and, therefore, is expected to be more electron-poor. Herein, we synthesized a new diiron complex that models the electron-poor coordination environment of the Mka-HLP diiron site. The diferrous precursor FeIIFeII reacts with NO• to form a diiron dinitrosyl species ({FeNO}72), which is in equilibrium with a mononitrosyl diiron species (FeII{FeNO}7) in solution. Both complexes can be isolated and fully characterized. However, only oxidation of {FeNO}72 produced nitrite in high yield (71%). Our study provides the first model that reproduces the NO• oxidase reactivity of Mka-HLP and suggests intermediacy of an {FeNO}6/{FeNO}7 species.

The [4Fe-4S]-Cluster of HydF is not Required for the Binding and Transfer of the Diiron Site of [FeFe]-Hydrogenases.

The active site of [FeFe]-hydrogenases contains a cubane [4Fe-4S]-cluster and a unique diiron cluster with biologically unusual CO and CN- ligands. The biogenesis of this diiron site, termed [2FeH ], requires the maturation proteins HydE, HydF and HydG. During the maturation process HydF serves as a scaffold protein for the final assembly steps and the subsequent transfer of the [2FeH ] precursor, termed [2FeP ], to the [FeFe]-hydrogenase. The binding site of [2FeP ] in HydF has not been elucidated, however, the [4Fe-4S]-cluster of HydF was considered as a possible binding partner of [2FeP ]. By targeting individual amino acids in HydF from Thermosipho melanesiensis using site directed mutagenesis, we examined the postulated binding mechanism as well as the importance and putative involvement of the [4Fe-4S]-cluster for binding and transferring [2FeP ]. Surprisingly, our results suggest that binding or transfer of [2FeP ] does not involve the proposed binding mechanism or the presence of a [4Fe-4S]-cluster at all.

Structural investigation of 2'-azido-2'-deoxyribonucleoside-5'-diphosphate inhibition of class Ia Escherichia coli ribonucleotide reductase.

Bacterial class Ia ribonucleotide reductases (RNRs) are attractive antibiotic targets due to their essential role in the biosynthesis of deoxyribonucleotides from the corresponding ribonucleotides. The catalytically active state of class Ia RNRs consists of a heterodimer of two homodimers, α;2 and β;2, in which the α;2 subunit contains substrate-binding and allosteric sites and the β;2 subunit contains two di-iron sites responsible for the initial steps of a 32-Å radical transfer pathway that enables conversion of the ribonucleotide substrate. Recent structural work in the Drennan lab has elucidated the active state structure of this enzyme, but required a doubly-mutated variant of β;2, E52Q/F3Y122-β;2, to decrease the rate of subunit dissociation. We describe here the use of 2′-azido-2′-deoxyribonucleoside-5′-diphosphate, a nucleotide analog, to trap the active state structure of the wild type Escherichia coli class Ia RNR and subsequent structural efforts to elucidate the conformational trajectory of the dissociation. This work provides a basis for understanding the complex regulatory mechanisms of RNR, which may result in novel antibiotic targets.

Electrode Integration of Synthetic Hydrogenase as Bioinspired and Noble Metal-Free Cathodes for Hydrogen Evolution

Diiron complexes mimicking the H-cluster of [FeFe]-hydrogenases have been extensively studied as (electro-)catalysts for proton reduction under homogeneous conditions. The incorporation of such complexes as “active sites” within macromolecular scaffolds such as organic polymers is receiving increasing attention as this strategy allows controlling the environment, that is, the outer coordination sphere, around the molecular catalytic center, to tune its performance as well as its stability. Here, we report on the synthesis and characterization of a library of metallo-copolymers featuring a bioinspired diiron active site and internal proton relays based on a previous report [Brezinski et al. Angew. Chem. Int. Ed. 2018, 57, 11898–11902]. The polymers are further functionalized with various amounts of pyrene groups for efficient noncovalent anchoring onto multi-walled carbon nanotubes (MWNTs), enabling the preparation of molecularly engineered electrode materials. The addition of pyrene anchors resulted in improved activity and stability, with a pyrene loading of about ∼8% corresponding to an optimized balance between polymer hydrophilicity and surface affinity. The best material displayed an average turnover frequency (TOFH2) of 4.3 ± 0.6 s–1 and a conservative turnover number for H2 production (TONH2) of 3.1 ± 0.4 × 105 after 20 h of continuous bulk electrolysis in aqueous conditions at 0.39 V overpotential. Interestingly, comparing such activities with an analogous diiron site deprived from polymeric scaffold revealed that latter could only show TONH2 of ∼4 ± 2 × 103 and TOFH2 of 0.06 ± 0.02 s–1 in 20 h under the same conditions. Post operando analysis of the modified electrodes suggests that electrode inactivation occurs via leaching of the diiron core from MWNT. In addition, a life cycle assessment was carried out to evaluate the performance of the engineered electrode materials not only from a technical perspective but also from an environmental point of view.

Insights into Triazolylidene Ligands Behaviour at a Di-Iron Site Related to [FeFe]-Hydrogenases.

The behaviour of triazolylidene ligands coordinated at a {Fe2(CO)5(µ-dithiolate)} core related to the active site of [FeFe]-hydrogenases have been considered to determine whether such carbenes may act as redox electron-reservoirs, with innocent or non-innocent properties. A novel complex featuring a mesoionic carbene (MIC) [Fe2(CO)5(Pmpt)(µ-pdt)] (1; Pmpt = 1-phenyl-3-methyl-4-phenyl-1,2,3-triazol-5-ylidene; pdt = propanedithiolate) was synthesized and characterized by IR, 1H, 13C{1H} NMR spectroscopies, elemental analyses, X-ray diffraction, and cyclic voltammetry. Comparison with the spectroscopic characteristics of its analogue [Fe2(CO)5(Pmbt)(µ-pdt)] (2; Pmbt = 1-phenyl-3-methyl-4-butyl-1,2,3-triazol-5-ylidene) showed the effect of the replacement of a n-butyl by a phenyl group in the 1,2,3-triazole heterocycle. A DFT study was performed to rationalize the electronic behaviour of 1, 2 upon the transfer of two electrons and showed that such carbenes do not behave as redox ligands. With highly perfluorinated carbenes, electronic communication between the di-iron site and the triazole cycle is still limited, suggesting low redox properties of MIC ligands used in this study. Finally, although the catalytic performances of 2 towards proton reduction are weak, the protonation process after a two-electron reduction of 2 was examined by DFT and revealed that the protonation process is favoured by S-protonation but the stabilized diprotonated intermediate featuring a {Fe-H⋯H-S} interaction does not facilitate the release of H2 and may explain low efficiency towards HER (Hydrogen Evolution Reaction).

Direct photo-oxidation of methane to methanol over a mono-iron hydroxyl site.

Natural gas, consisting mainly of methane (CH4), has a relatively low energy density at ambient conditions (~36 kJ l-1). Partial oxidation of CH4 to methanol (CH3OH) lifts the energy density to ~17 MJ l-1 and drives the production of numerous chemicals. In nature, this is achieved by methane monooxygenase with di-iron sites, which is extremely challenging to mimic in artificial systems due to the high dissociation energy of the C-H bond in CH4 (439 kJ mol-1) and facile over-oxidation of CH3OH to CO and CO2. Here we report the direct photo-oxidation of CH4 over mono-iron hydroxyl sites immobilized within a metal-organic framework, PMOF-RuFe(OH). Under ambient and flow conditions in the presence of H2O and O2, CH4 is converted to CH3OH with 100% selectivity and a time yield of 8.81 ± 0.34 mmol gcat-1 h-1 (versus 5.05 mmol gcat-1 h-1 for methane monooxygenase). By using operando spectroscopic and modelling techniques, we find that confined mono-iron hydroxyl sites bind CH4 by forming an [Fe-OH···CH4] intermediate, thus lowering the barrier for C-H bond activation. The confinement of mono-iron hydroxyl sites in a porous matrix demonstrates a strategy for C-H bond activation in CH4 to drive the direct photosynthesis of CH3OH.

Trapping an Oxidized and Protonated Intermediate of the [FeFe]-Hydrogenase Cofactor under Mildly Reducing Conditions

The H-cluster is the catalytic cofactor of [FeFe]-hydrogenase, a metalloenzyme that catalyzes the formation of dihydrogen (H2). The catalytic diiron site of the H-cluster carries two cyanide and three carbon monoxide ligands, making it an excellent target for IR spectroscopy. In previous work, we identified an oxidized and protonated H-cluster species, whose IR signature differs from that of the oxidized resting state (Hox) by a small but distinct shift to higher frequencies. This "blue shift" was explained by a protonation at the [4Fe-4S] subcomplex of the H-cluster. The novel species, denoted HoxH, was preferentially accumulated at low pH and in the presence of the exogenous reductant sodium dithionite (NaDT). When HoxH was reacted with H2, the hydride state (Hhyd) was formed, a key intermediate of [FeFe]-hydrogenase turnover. A recent publication revisited our protocol for the accumulation of HoxH in wild-type [FeFe]-hydrogenase, concluding that inhibition by NaDT decay products rather than cofactor protonation causes the spectroscopic "blue shift". Here, we demonstrate that HoxH formation does not require the presence of NaDT (or its decay products), but accumulates also with the milder reductants tris(2-carboxyethyl)phosphine, dithiothreitol, or ascorbic acid, in particular at low pH. Our data consistently suggest that HoxH is accumulated when deprotonation of the H-cluster is impaired, thereby preventing the regain of the oxidized resting state Hox in the catalytic cycle.

Metal Substrate Translocation and Transport Mechanism of the Sinorhizobium melilotiP1B‐5‐type ATPase Nia Revealed by In‐vitro Transport Assays in Proteoliposomes

P1B‐type ATPases are a class of transmembrane transporter proteins responsible for metal substrate translocation, and export across cellular membranes. These ion pumps are classified into seven sub‐families (P1B‐1‐P1B‐7‐type) based on conserved amino acid motifs in their transmembrane helices that appear to control each sub‐family’s substrate selectivity. The P1B‐5‐type sub‐family is widely found in nitrogen fixing bacteria, such as Sinorhizobium meliloti,which form a unique symbiotic relationship with legumes. This symbiotic relationship is highly dependent on the nutrition trafficking between the two species, including a tight control of transition metal homeostasis. The P1B‐5‐type ATPase Nia from S. Meliloti is recognized as a putative iron and nickel transporter, and besides possessing the typical P‐type ATPase domains and topology, it features of a C‐terminal hemerythrin‐like domain containing a di‐iron binding site involved in oxygen sensing, as proposed by in‐vivo studies. However, the substrate transport across lipid bi‐layers, the overall mechanism for cargo translocation and the transport kinetic parameters remain elusive due to lack of molecular tools to study putative substrate transport across membranes in real‐time.In this study, we coupled metal‐stimulated ATPase activity assays with an experimental platform based on multiple fluorescence sensor probes, to study substrate transport mechanism and translocation kinetics in real‐time with Nia reconstituted in a native‐like environment. We expressed and purified Nia in detergent micelles and subsequently incorporated it in artificial small unilamellar lipid bi‐layer vesicles (proteoliposomes) which allow to study the substrate specificity and transport mechanism in real‐time. The proteoliposomes were used as an in‐vitrotool to determine the kinetic parameters for metal transport by encapsulating fluorescent detector probes featuring turn‐on florescence signal upon substrate ion binding and translocation.The study revealed that Fe2+, Ni2+ and Co2+are actively translocated by Nia and the kinetic parameter analysis demonstrated that Fe2+ is the preferential Nia substrate. In parallel, by encapsulating in the proteoliposome lumen pH and membrane sensitive fluorescent probes we are also revealing the chemistry underlying the mechanism of transport and potential ions involved as counter‐transported cargos in P1B‐5‐type ATPase catalytic cycle, with the aim of revealing unique molecular features for this subclass of transmembrane metal pumps.

The Di-Iron Protein YtfE Is a Nitric Oxide-Generating Nitrite Reductase Involved in the Management of Nitrosative Stress.

Previously characterized nitrite reductases fall into three classes: siroheme-containing enzymes (NirBD), cytochrome c hemoproteins (NrfA and NirS), and copper-containing enzymes (NirK). We show here that the di-iron protein YtfE represents a physiologically relevant new class of nitrite reductases. Several functions have been previously proposed for YtfE, including donating iron for the repair of iron–sulfur clusters that have been damaged by nitrosative stress, releasing nitric oxide (NO) from nitrosylated iron, and reducing NO to nitrous oxide (N2O). Here, in vivo reporter assays confirmed that Escherichia coli YtfE increased cytoplasmic NO production from nitrite. Spectroscopic and mass spectrometric investigations revealed that the di-iron site of YtfE exists in a mixture of forms, including nitrosylated and nitrite-bound, when isolated from nitrite-supplemented, but not nitrate-supplemented, cultures. Addition of nitrite to di-ferrous YtfE resulted in nitrosylated YtfE and the release of NO. Kinetics of nitrite reduction were dependent on the nature of the reductant; the lowest Km, measured for the di-ferrous form, was ∼90 μM, well within the intracellular nitrite concentration range. The vicinal di-cysteine motif, located in the N-terminal domain of YtfE, was shown to function in the delivery of electrons to the di-iron center. Notably, YtfE exhibited very low NO reductase activity and was only able to act as an iron donor for reconstitution of apo-ferredoxin under conditions that damaged its di-iron center. Thus, YtfE is a high-affinity, low-capacity nitrite reductase that we propose functions to relieve nitrosative stress by acting in combination with the co-regulated NO-consuming enzymes Hmp and Hcp.

Key carboxylate residues for iron transit through the prokaryotic ferritin SynFtn.

Ferritins are proteins forming 24meric rhombic dodecahedral cages that play a key role in iron storage and detoxification in all cell types. Their function requires the transport of Fe2+ from the exterior of the protein to buried di-iron catalytic sites, known as ferroxidase centres, where Fe2+ is oxidized to form Fe3+-oxo precursors of the ferritin mineral core. The route of iron transit through animal ferritins is well understood: the Fe2+ substrate enters the protein via channels at the threefold axes and conserved carboxylates on the inner surface of the protein cage have been shown to contribute to transient binding sites that guide Fe2+ to the ferroxidase centres. The routes of iron transit through prokaryotic ferritins are less well studied but for some, at least, there is evidence that channels at the twofold axes are the major route for Fe2+ uptake. SynFtn, isolated from the cyanobacterium Synechococcus CC9311, is an atypical prokaryotic ferritin that was recently shown to take up Fe2+ via its threefold channels. However, the transfer site carboxylate residues conserved in animal ferritins are absent, meaning that the route taken from the site of iron entry into SynFtn to the catalytic centre is yet to be defined. Here, we report the use of a combination of site-directed mutagenesis, absorbance-monitored activity assays and protein crystallography to probe the effect of substitution of two residues potentially involved in this pathway. Both Glu141 and Asp65 play a role in guiding the Fe2+ substrate to the ferroxidase centre. In the absence of Asp65, routes for Fe2+ to, and Fe3+ exit from, the ferroxidase centre are affected resulting in inefficient formation of the mineral core. These observations further define the iron transit route in what may be the first characterized example of a new class of ferritins peculiar to cyanobacteria.

Site-selective protonation of the one-electron reduced cofactor in [FeFe]-hydrogenase.

Hydrogenases are bidirectional redox enzymes that catalyze hydrogen turnover in archaea, bacteria, and algae. While all types of hydrogenase show H2 oxidation activity, [FeFe]-hydrogenases are excellent H2 evolution catalysts as well. Their active site cofactor comprises a [4Fe-4S] cluster covalently linked to a diiron site equipped with carbon monoxide and cyanide ligands. The active site niche is connected with the solvent by two distinct proton transfer pathways. To analyze the catalytic mechanism of [FeFe]-hydrogenase, we employ operando infrared spectroscopy and infrared spectro-electrochemistry. Titrating the pH under H2 oxidation or H2 evolution conditions reveals the influence of site-selective protonation on the equilibrium of reduced cofactor states. Governed by pKa differences across the active site niche and proton transfer pathways, we find that individual electrons are stabilized either at the [4Fe-4S] cluster (alkaline pH values) or at the diiron site (acidic pH values). This observation is discussed in the context of the complex interdependence of hydrogen turnover and bulk pH.

Front Cover: Synthesis, Characterization and Electrochemical Reductive Properties of Complexes [Fe2(CO)4(κ2‐PPh2NR2)(μ‐dithiolato)] Related to the H‐Cluster of [FeFe]‐H2ases (Eur. J. Inorg. Chem. 3/2021)

The Front Cover shows the fate of protons at a diiron site inspired by the H-cluster of [FeFe]-H2ases. Different conformations favor the capture of protons and their transfer to the diiron core, thus generating a suitable proton channel. Finding the right combinations of ligands for the design of efficient diiron electrocatalysts for proton reduction is like a 3D combination puzzle involving concerted electronic and steric effects through the choice of ligands around the dinuclear site. More information can be found in the Full Paper by C. Elleouet, P. Schollhammer and co-workers.

Temperature Dependence of Structural Dynamics at the Catalytic Cofactor of [FeFe]-hydrogenase.

[FeFe]-hydrogenases are nature's blueprint for efficient hydrogen turnover. Understanding their enzymatic mechanism may improve technological H2 fuel generation. The active-site cofactor (H-cluster) consists of a [4Fe-4S] cluster ([4Fe]H), cysteine-linked to a diiron site ([2Fe]H) carrying an azadithiolate (adt) group, terminal cyanide and carbon monoxide ligands, and a bridging carbon monoxide (μCO) in the oxidized protein (Hox). Recently, the debate on the structure of reduced H-cluster states was intensified by the assignment of new species under cryogenic conditions. We investigated temperature effects (4-280 K) in infrared (IR) and X-ray absorption spectroscopy (XAS) data of [FeFe]-hydrogenases using fit analyses and quantum-chemical calculations. IR data from our laboratory and literature sources were evaluated. At ambient temperatures, reduced H-cluster states with a bridging hydride (μH-, in Hred and Hsred) or with an additional proton at [4Fe]H (Hred') or at the distal iron of [2Fe]H (Hhyd) prevail. At cryogenic temperatures, these species are largely replaced by states that hold a μCO, lack [4Fe]H protonation, and bind an additional proton at the adt nitrogen (HredH+ and HsredH+). XAS revealed the atomic coordinate dispersion (i.e., the Debye-Waller parameter, 2σ2) of the iron-ligand bonds and Fe-Fe distances in the oxidized and reduced H-cluster. 2σ2 showed a temperature dependence typical for the so-called protein-glass transition, with small changes below ∼200 K and a pronounced increase above this "breakpoint". This behavior is attributed to the freezing-out of larger-scale anharmonic motions of amino acid side chains and water species. We propose that protonation at [4Fe]H as well as ligand rearrangement and μH- binding at [2Fe]H are impaired because of restricted molecular mobility at cryogenic temperatures so that protonation can be biased toward adt. We conclude that a H-cluster with a μCO, selective [4Fe]H or [2Fe]H protonation, and catalytic proton transfer via adt facilitates efficient H2 conversion in [FeFe]-hydrogenase.

Synthesis, Characterization and Electrochemical Reductive Properties of Complexes [Fe2(CO)4(κ2‐PPh2NR2)(µ‐dithiolato)] Related to the H‐Cluster of [FeFe]‐H2ases

A series of dithiolato carbonyl diiron complexes with cyclic azadiphosphine, related to the active site of [FeFe]‐hydrogenases, has been prepared. Several combinations of diphosphines and dithiolato bridges have been examined in order to obtain information on the influence of electronic and steric effects of the group linked to the nitrogen atom of the phosphine (phenyl or benzyl) and the bridge‐head of the dithiolate (CH2, CEt2, CBn2). Products were spectroscopically and structurally characterized. Protonation experiments show that depending on the nature of the amine in the diphosphine, protonation processes are different. With NBn group, the process is arrested at N‐protonated species, while with NPh, the proton is transferred to the diiron site giving bridging‐hydrido species. The effect of the bridge‐head group of the dithiolate is very slight on the electronic properties of the compounds. Depending on the steric crowding of the bridge, different isomers related to the dibasal or basal‐apical position of the diphosphine, are observed in solution or in solid state. Electrochemical study was performed to examine the activity of these compounds in the presence of acid.

FeFe]-hydrogenase maturation: H-cluster assembly intermediates tracked by electron paramagnetic resonance, infrared, and X-ray absorption spectroscopy

[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.Graphic abstract

The Catalytic Role of a Conserved Tyrosine in Nitric Oxide-Reducing Non-heme Diiron Enzymes

Recent studies have identified several key intermediates of the nitric oxide reductase (NOR) cycle of flavodiiron proteins (FDPs). These intermediates include, sequentially, a μ-hydroxo diferrous species, a mono-NO intermediate, a di-NO intermediate (FDPdiNO), and a bis-μ-hydroxo diferric species. This paper focuses on the reaction path connecting the last two intermediates on which the nitrous oxide product is released. On the basis of density functional theory calculations, a reaction sequence is proposed in which the enzyme passes through an intermediate with a bridging hyponitrite ligand, which accepts a proton, initiating a rate-determining ligand rotation from an N/N- to an N/O-coordinated conformation. This rotation is facilitated by a second-sphere tyrosine residue, which provides a transient hydrogen bond to one of the nitrogen atoms of the substrate near the transition state. The role of the tyrosine residue in the NOR activity has been tested by steady-state kinetics and rapid freeze-quench (RFQ) studies of the Y197F variant of the FDP from T. maritima in which the hydrogen bonding interaction is absent. The Y197F variant displayed little or no steady-state NOR activity in support of the importance of Y197. The RFQ samples, monitored by Mössbauer spectroscopy, showed that Y197F follows the same reaction path as a wild-type FDP up to and including the formation of FDPdiNO but diverges subsequently with the variant forming an inactive mono-NO species. The RFQ results demonstrate that Y197 enables the postFDPdiNO section of the reaction cycle in the wild-type FDP to proceed to N2O. The proposed refinement of the reaction mechanism provides an explanation for the lack of NOR activity of the variant Y197F of T. maritima and of other di-NO binding diiron enzymes and model compounds with active site structures like those of FDPs. The reported reductions of NO to N2O catalyzed by synthetic diiron complexes proceed only with the support of radiation, additional electrons, or an electron-rich ligand environment. However, higher potential diiron sites like those of FDPs require hydrogen bonding to second-sphere residues to turn over NO to N2O.