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 [2Fe_H], 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 [2Fe_H] precursor, termed [2Fe_P], to the [FeFe]-hydrogenase. The binding site of [2Fe_P] in HydF has not been elucidated, however, the [4Fe-4S]-cluster of HydF was considered as a possible binding partner of [2Fe_P]. 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 [2Fe_P]. Surprisingly, our results suggest that binding or transfer of [2Fe_P] does not involve the proposed binding mechanism or the presence of a [4Fe-4S]-cluster at all.     

The Arabidopsis Mitochondrial Glutaredoxin GRXS15 Provides [2Fe-2S] Clusters for ISCA-Mediated [4Fe-4S] Cluster Maturation

Iron-sulfur (Fe-S) proteins are crucial for many cellular functions, particularly those involving electron transfer and metabolic reactions. An essential monothiol glutaredoxin GRXS15 plays a key role in the maturation of plant mitochondrial Fe-S proteins. However, its specific molecular function is not clear, and may be different from that of the better characterized yeast and human orthologs, based on known properties. Hence, we report here a detailed characterization of the interactions between Arabidopsis thaliana GRXS15 and ISCA proteins using both in vivo and in vitro approaches. Yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that GRXS15 interacts with each of the three plant mitochondrial ISCA1a/1b/2 proteins. UV-visible absorption/CD and resonance Raman spectroscopy demonstrated that coexpression of ISCA1a and ISCA2 resulted in samples with one [2Fe-2S]²⁺ cluster per ISCA1a/2 heterodimer, but cluster reconstitution using as-purified [2Fe-2S]-ISCA1a/2 resulted in a [4Fe-4S]²⁺ cluster-bound ISCA1a/2 heterodimer. Cluster transfer reactions monitored by UV-visible absorption and CD spectroscopy demonstrated that [2Fe-2S]-GRXS15 mediates [2Fe-2S]²⁺ cluster assembly on mitochondrial ferredoxin and [4Fe-4S]²⁺ cluster assembly on the ISCA1a/2 heterodimer in the presence of excess glutathione. This suggests that ISCA1a/2 is an assembler of [4Fe-4S]²⁺ clusters, via two-electron reductive coupling of two [2Fe-2S]²⁺ clusters. Overall, the results provide new insights into the roles of GRXS15 and ISCA1a/2 in effecting [2Fe-2S]²⁺ to [4Fe-4S]²⁺ cluster conversions for the maturation of client [4Fe-4S] cluster-containing proteins in plants.     

On the role of conserved proline residues in the structure and function of Clostridium pasteurianum 2[4Fe-4S] ferredoxin

The widespread occurrence of Pro residues adjacent to Cys ligandsin the sequences of [4Fe-4S] electron transfer proteins hasnot yet found a functional basis. The two such Pro of Clostridiumpasteurianum 2[4Fe-4S] ferredoxin have been probed by site-directedmutagenesis. Any one of them, but not both simultaneously, canbe substituted without impairing the proper folding of the protein.The reduction potentials of the ferredoxin variants fall ina narrow range of <20 mV above the potential of the nativeprotein. The biological activities with C.pasteurianum hydrogenaseand pyruvate-ferredoxin oxidoreductase do not change significantly,except when Lys replaces Pro. In these cases, the data suggestthat the two clusters of 2[4Fe-4S] ferredoxin may not alwaysbe equivalent in the interaction with the redox partners. Destabilizationof the structure has been observed as the consequence of theProl9 or Pro48 substitutions. Using 2-D NMR, this effect hasbeen associated with perturbations of both the hydrogen bondnetwork and one amino acid side chain around the [4Fe-4S] clusters.Thus, the conserved Pro found in the binding motif of [4Fe-4S]clusters in proteins strongly stabilizes the active site butdoes not play an essential role in the mechanism of electrontransfer.     

Mechanism and Application of the Catalytic Reaction of [NiFe] Hydrogenase: Recent Developments

Hydrogenases (H₂ase) catalyze the oxidation of dihydrogen and the reduction of protons with remarkable efficiency, thereby attracting considerable attention in the energy field due to their biotechnological potential. For this simple reaction, [NiFe] H₂ase has developed a sophisticated but intricate mechanism with the heterolytic cleavage of dihydrogen, where its Ni−Fe active site exhibits various redox states. Recently, new spectroscopic and crystal structure studies of [NiFe] H₂ases have been reported, providing significant insights into the catalytic reaction mechanism, hydrophobic gas-access tunnel, proton-transfer pathway, and electron-transfer pathway of [NiFe] H₂ases. In addition, [NiFe] H₂ases have been shown to play an important role in biofuel cell and solar dihydrogen production. This concept provides an overview of the biocatalytic reaction mechanism and biochemical application of [NiFe] H₂ases based on the new findings.     

Hydrogen Sulfide Oxidation by Sulfide Quinone Oxidoreductase

Hydrogen sulfide (H₂S) is an environmental toxin and a heritage of ancient microbial metabolism that has stimulated new interest following its discovery as a neuromodulator. While many physiological responses have been attributed to low H₂S levels, higher levels inhibit complex IV in the electron transport chain. To prevent respiratory poisoning, a dedicated set of enzymes that make up the mitochondrial sulfide oxidation pathway exists to clear H₂S. The committed step in this pathway is catalyzed by sulfide quinone oxidoreductase (SQOR), which couples sulfide oxidation to coenzyme Q₁₀ reduction in the electron transport chain. The SQOR reaction prevents H₂S accumulation and generates highly reactive persulfide species as products; these can be further oxidized or can modify cysteine residues in proteins by persulfidation. Here, we review the kinetic and structural characteristics of human SQOR, and how its unconventional redox cofactor configuration and substrate promiscuity lead to sulfide clearance and potentially expand the signaling potential of H₂S. This dual role of SQOR makes it a promising target for H₂S-based therapeutics.     

[FeII(MeCN)4]2+(ClO4−)2 and [FeIIICl33] as Mimics for the Catalytic Centers of Peroxidase,Catalase and Cvtochrome P-450

Addition of [Fe^II(MeCN)²₄⁺(ClO⁻₄)₂ to solutions of hydrogen peroxide in dry acetonitrile (MeCN) catalyzes a rapid disproportionation of H₂O₂ via the initial formation of an adduct, [Fe^II(HOOH)↔Fe(O)(OH₂)]²⁺, which oxidizes a second H₂O₂ to dioxygen. This intermediate also cleanly oxidizes substituted hydrazines, alcohols, aldehydes, and thioethers by a two-electron process. The products for these H₂O₂ oxidations are consistent with those that result from catalase- and some peroxidase-catalyzed processes. In the same aprotic medium (MeCN) anhydrous Fe^IIICl₃ catalyzes the demethylation of N,N-dimethylaniline, the epoxidation of olefins, and the oxidative cleavage of 1-phenyl-1,2-ethanediol (and other 1,2-diols) by hydrogen peroxide. A mechanism is proposed in which an initial Lewis acid-base interaction of Fe^IIICl₃ with H₂O₂ generates a highly electrophilic Fe^III-oxene species as the reactive intermediate. For each class of substrate the products closely parallel those that result from their enzymatic oxidation by cytochrome P-450. Because of (a) the close congruence of products, (b) the catalytic nature of the Fe^IIICl₃/H₂O₂ reaction mimic, and (c) the similarity of the dipolar aprotic solvent (acetonitrile) to the proteinaceous lipid matrix of the biomembrane, the form of the reactive intermediate may be the same in each case. This is in contrast to the prevailing view that cytochrome P-450 acts as a redox catalyst to generate an Fe(V)-oxo species or an Fe(IV)-oxo cation radical as the reactive intermediate.     

New Insight into Dearomatization and Decarbonylation of Antitubercular 4H-Benzo[e][1,3]thiazinones: Stable 5H- and 7H-Benzo[e][1,3]thiazines

8-Nitro-4H-benzo[e][1,3]thiazinones (BTZs) are potent in vitro antimycobacterial agents. New chemical transformations, viz. dearomatization and decarbonylation, of two BTZs and their influence on the compounds’ antimycobacterial properties are described. Reactions of 8-nitro-2-(piperidin-1-yl)-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one and the clinical drug candidate BTZ043 with the Grignard reagent CH₃MgBr afford the corresponding dearomatized stable 4,5-dimethyl-5H- and 4,7-dimethyl-7H-benzo[e][1,3]thiazines. These methine compounds are structurally characterized by X-ray crystallography for the first time. Reduction of the BTZ carbonyl group, leading to the corresponding markedly non-planar 4H-benzo[e][1,3]thiazine systems, is achieved using the reducing agent (CH₃)₂S ⋅ BH₃. Double methylation with dearomatization and decarbonylation renders the two BTZs studied inactive against Mycobacterium tuberculosis and Mycobacterium smegmatis, as proven by in vitro growth inhibition assays.     

Characterization of the acid properties of [Al]-, [Ga]- and [Fe]-HZSM-5 by low-temperature FTIR spectroscopy of adsorbed dihydrogen and ethylbenzene disproportionation

[Al]-, [Ga]- and [Fe]-HZSM-5 having closely similar Brønsted acid site densities were prepared. The low-temperature adsorption of H₂ and D₂ was studied by FTIR spectroscopy and the frequency shifts Δν_OH and Δν_HH (Δν_DD) were measured and compared with corresponding frequency shifts observed for CO and N₂ probes. A linear correlation between |Δν_OH|^1/2 which is proportional to the heat of formation &;/Delta;H_B of the H-bonded complex O-H···B (B representing the H-bond acceptor), and the proton affinities of the three probe molecules was found for [Al]-HZSM-5. The ΔνOH-values measured for CO and H2 (D2) on the three isomorphously substituted zeolites suggested the following acid strength ranking: [Al]-HZSM-5 > [Ga]-HZSM-5 ≈ [Fe]-HZSM-5. This sequence is clearly reflected in the relative activities of the materials for the acid-catalyzed disproportionation of ethylbenzene.     

Two potential pyridine-borane oligomer and polymer building blocks. Structural characterisation of [NC5H4.C5H4N.B10H12.NC5H4.C5H4N] and [Me2S.B10H12.NC4H4N.B10H12.SMe2] by conventional and synchrotron X-ray methods

Two five-unit linear compounds, [NC₅H₄.C₅H₄N.B₁₀H₁₂.NC₅H₄.C₅H₄N] and [Me₂S.B₁₀H₁₂.NC₄H₄N.B₁₀H₁₂.SMe₂], have been isolated from the reactions of [6,9-(SMe₂)₂-arachno-B₁₀H₁₂] with 4,4′-bipyridyl and pyrazine (1,4-diazabenzene), respectively, and are characterised by single-crystal X-ray diffraction analysis.     

2-[1-(2,6-Dibenzhydryl-4-chlorophenylimino)ethyl]-6-[1-(arylimino)ethyl]pyridyliron(II) dichlorides: Synthesis,characterization and ethylene polymerization behavior

A series of 2-[1-(2,6-dibenzhydryl-4-chlorophenylimino)ethyl]-6-[1-(arylimino)ethyl]pyridine ligands (L1–L5) as well as the ligand 2,6-bis[1-(2,6-dibenzhydryl-4-chloro-phenylimino)ethyl]pyridine (L6) were synthesized and reacted with FeCl₂·4H₂O to afford the iron(II) dichloride complexes [LFeCl₂] (Fe1–Fe6). All new compounds were fully characterized by elemental and spectroscopic analysis, and the molecular structures of the complexes Fe1, Fe2 and Fe4 were determined by single-crystal X-ray diffraction, which revealed a pseudo-square-pyramidal geometry at iron. Upon activation with either MAO or MMAO, all iron pre-catalysts exhibited very high activity in ethylene polymerization with good thermal stability. To the best of our knowledge, the current system showed the highest activity amongst iron bis(imino)pyridine pre-catalysts reported to-date. The polymerization parameters were explored to determine the optimum conditions for catalytic activity, which were typically found to be 2500 eq. Al to Fe at 60 °C in the presence of MMAO, and 80 °C in the presence of MAO. The resultant polyethylene possessed a narrow molecular polydispersity index (PDI) consistent with the formation of single-site active species.     

Synthesis of New Ferrocenylenesilylene Polymers by Direct Hydrosilylation/Polymerization of [FC–SiRH], FC?=?(η5-C5H4)Fe(η5-C5H4), Using Organometallic Alkenes and Alkynes

Synthesis of new ferrocenylenesilylene polymers was effected by direct hydrosilylation/polymerization of 1,1′-methylsilyl-ferrocenophane [FC–SiMeH] (FC = (η⁵-C₅H₄)Fe(η⁵-C₅H₄) with acetylenes and organometallic olefins using a Pt⁰ catalyst. The reaction of [FC–SiMeH] with HC₂Ph, HC₂SiMe₃, CH₂=CHSiMe₂Fp, Fp = (η⁵-C₅H₅)Fe(CO)₂ and CH₂=CHCH₂SiMe₂Fp in the presence of a platinum catalyst resulted in high yields of the corresponding hydrosilylated polymers. In the case of the acetylenes only β products were obtained, both E and Z isomers, whereas for the olefins both α and β-isomers were noted. Only in the case of the more bulky allylsilicon material was any unreacted SiH functionality retained in the polymer, an effect also noted when using [FCSiPhH] as the starting ferrocenophane. Cyclic voltammetric studies on these polymers revealed metal–metal interaction with two redox processes associated with the ferrocenylene Fe center along with an irreversible oxidation at higher potential for the Fp Fe atom. Ian: A pleasure to contribute; keep up the good work-aptp, cheers, Keith.     

[Fe]-Hydrogenase,Cofactor Biosynthesis and Engineering

[Fe]-hydrogenase catalyzes the heterolytic cleavage of H₂ and reversible hydride transfer to methenyl-tetrahydromethanopterin. The iron-guanylylpyridinol (FeGP) cofactor is the prosthetic group of this enzyme, in which mononuclear Fe(II) is ligated with a pyridinol and two CO ligands. The pyridinol ligand fixes the iron by an acyl carbon and a pyridinol nitrogen. Biosynthetic proteins for this cofactor are encoded in the hmd co-occurring (hcg) genes. The function of HcgB, HcgC, HcgD, HcgE, and HcgF was studied by using structure-to-function analysis, which is based on the crystal structure of the proteins and subsequent enzyme assays. Recently, we reported the catalytic properties of HcgA and HcgG, novel radical S-adenosyl methionine enzymes, by using an in vitro biosynthesis assay. Here, we review the properties of [Fe]-hydrogenase and the FeGP cofactor, and the biosynthesis of the FeGP cofactor. Finally, we discuss the expected engineering of [Fe]-hydrogenase and the FeGP cofactor.     

Recovery of [BMIM]FeCl4 from homogeneous mixture using a simple chemical method

[BMIM]FeCl₄ (1-butyl-3-methylimidazolium tetrachloroferrate) was successfully separated from a homogeneous mixture of [BMIM]FeCl₄ and H₂O via a simple two-step method of phase-division by adding inorganic salt plus chemical extraction, or alternatively, ultracentrifugation, or ultrastrong magnetic field. NaCl showed excellent and effective phase-dividing performance combined with chemical extraction method from the homogeneous mixture of [BMIM]FeCl₄ and H₂O lower to 1 v%.     

[(C_2H_5)_4N]_2｛Fe_4S_4[S_2CN(C_2H_5)_2]_4｝的晶体和分子结构

本文报导含类立方烷型簇核Ｆｅ４ｓ４的金硫配位化合物［（Ｃ２Ｈ５）４Ｎ］２｛Ｆｅ４Ｓ４［Ｓ２ＣＮ（Ｃ２Ｈ５）２］４｝的合成、晶体和分子结构测定的结果。     

Redox Signaling through DNA

Biological electron transfer reactions between metal cofactors are critical to many essential processes within the cell. Duplex DNA is, moreover, capable of mediating the transport of charge through its π-stacked nitrogenous bases. Increasingly, [4Fe4S] clusters, generally redox-active cofactors, have been found to be associated with enzymes involved in DNA processing. DNA-binding enzymes containing [4Fe4S] clusters can thus utilize DNA charge transport (DNA CT) for redox signaling to coordinate reactions over long molecular distances. In particular, DNA CT signaling may represent the first step in the search for DNA lesions by proteins containing [4Fe4S] clusters that are involved in DNA repair. Here we describe research carried out to examine the chemical characteristics and biological consequences of DNA CT. We are finding that DNA CT among metalloproteins represents powerful chemistry for redox signaling at long range within the cell.     

Two types of tetrasubstitution in [H4Ru4(CO)12]: spectroscopic characterization and crystal structure determination of [H4Ru4(CO)8(PMe2Ph)4] and [H2Ru4(CO)9(PMe2Ph)4]

Two tetrasubstituted tetranuclear ruthenium clusters containing dimethylphenylphosphine [H₄Ru₄(CO)₈(PMe₂Ph)₄] and [H₂Ru₄(CO)₉(PMe₂Ph)₄] were prepared and characterized spectroscopically as well as by X-ray crystallography. Both compounds show a structure based on the usual 60-electron tetrahedral framework with each ruthenium atom being bonded to one phosphine ligand. In the case of the nonacarbonyl derivative, one of the ruthenium atoms has one terminal carbonyl group and is also bonded to two semibridging carbonyl groups; two other metal–metal bonds are bridged by the hydride groups. In the case of the octacarbonyl compound all the ruthenium atoms are bonded to two terminal carbonyls and to two bridging hydrides. NMR spectroscopical information of [H₄Ru₄(CO)₈(PMe₂Ph)₄] suggests the presence of two isomers in solution which we propose to have D_2d and C_s symmetries. The observation of the NMR properties gives some observations about conditions that favour coupling between substituents in different ruthenium atoms.     

Effect of replacing a conserved proline residue on the function and stability of bovine adrenodoxin

A proline residue in the C-terminal part of the polypeptide chain is highly conserved among many [2Fe-2S] ferredoxins. To investigate the requirement for proline at this position, we constructed steric (4- 108W), charged (4-108K), polar (4-108S) and non-polar (4-108A) truncated mutants of adrenodoxin and studied them for biological function and stability. Although the variants were expressed in Escherichia coli with a significantly lower yield compared with wild- type adrenodoxin, successful incorporation of the iron-sulfur cluster suggested their proper folding. Similar absorption, CD and EPR spectra indicated that the cluster environment was not affected by the mutations. No evidence for an essential role of Pro108 in determining the redox potential of adrenodoxin or its interactions with the redox partners was found. However, replacement of this residue results in a dramatic decrease in the overall protein stability. The differences in the Gibbs energy of unfolding at 37 degrees C, delta[delta(d)G(37 degrees C)], are -5.0, -7.8, -10.1 and -10.7 kJ/mol for 4-108A, 4-108S, 4-108W and 4-108K mutants, respectively, compared with 4-108P as a control. We conclude that the principle function of Pro108 is to stabilize adrenodoxin threefold: (i) through limitation of the conformation of the polypeptide chain in this region, (ii) through a hydrogen bond to Arg14 and (iii) favorable hydrophobic contacts.      

A catalytic method for synthesis of 6-aryl-1H-pyrazolo[3,4-d]pyrimidin-4[5H]-ones by heteropolyacids: H14[NaP5W29MoO110] and H3PMo12O40

Derivatives of 6-aryl-1H-pyrazolo[3,4-d]pyrimidin-4[5H]-ones 4, are synthesized from reaction of 5-amino-1-phenyl-1H-pyrazolo-4-carboxamide 1 with aromatic aldehyde in the presence of heteropolyacids, H₃PMo₁₂O₄₀, H₃-PMo, and H₁₄[NaP₅W₂₉MoO₁₁₀], H₁₄P₅-Mo with high yields. The catalytic activity of H₃-PMo is found to be lower than H₁₄P₅-Mo.     

CT interactions of the cuboidal cluster Fe4S4. Photoredox decomposition of (C5H5)4Fe4S4

The cluster complex (C₅H₅)₄Fe₄S₄ formally contains Fe(III) and sulfide as constituents. In agreement with this assignment, the long-wavelength absorptions of the cluster are attributed to S^2 − to Fe(III) LMCT transitions. In solutions of acetonitrile, LMCT excitation of the cluster complex leads to a photoredox decomposition yielding Fe(II) and elemental sulfur. Upon a one-electron oxidation of (C₅H₅)₄Fe₄S₄, the cation [(C₅H₅)₄Fe₄S₄]⁺ is obtained, which is much more stable to water, air and light than the neutral parent cluster. Upon addition of [M(CN)₆]^4 − with MRu and Os to aqueous solutions of this cation, dark blue and violet colors, respectively, immediately develop, which are attributed to outer-sphere CT absorptions of the ion pairs [(C₅H₅)₄Fe₄S₄]⁺/[M(CN)₆]^4 −.     

Functionalizing nanocrystalline metal oxide electrodes with robust synthetic redox proteins

De novo designed synthetic redox proteins (maquettes) are structurally simpler, working counterparts of natural redox proteins. The robustness and adaptability of the maquette protein scaffold are ideal for functionalizing electrodes. A positive amino acid patch has been designed into a maquette surface for strong electrostatic anchoring to the negatively charged surfaces of nanocrystalline, mesoporous TiO(2) and SnO(2) films. Such mesoporous metal oxide electrodes offer a major advantage over conventional planar gold electrodes by facilitating formation of high optical density, spectroelectrochemically active thin films with protein loading orders of magnitude greater (up to 8 nmol cm(-2)) than that achieved with gold electrodes. The films are stable for weeks, essentially all immobilized-protein display rapid, reversible electrochemistry. Furthermore, carbon monoxide ligand binding to the reduced heme group of the protein is maintained, can be sensed optically and reversed electrochemically. Pulsed UV excitation of the metal oxide results in microsecond or faster photoreduction of an immobilized cytochrome and millisecond reoxidation. Upon substitution of the heme-group Fe by Zn, the light-activated maquette injects electrons from the singlet excited state of the Zn protoporphyrin IX into the metal oxide conduction band. The kinetics of cytochrome/metal oxide interfacial electron transfer obtained from the electrochemical and photochemical data obtained are discussed in terms of the free energies of the observed reactions and the electronic coupling between the protein heme group and the metal oxide surface.