Biomimetic assembly and activation of [FeFe]-hydrogenases.

Hydrogenases are the most active molecular catalysts for hydrogen production and uptake, and could therefore facilitate the development of new types of fuel cell. In [FeFe]-hydrogenases, catalysis takes place at a unique di-iron centre (the [2Fe] subsite), which contains a bridging dithiolate ligand, three CO ligands and two CN(-) ligands. Through a complex multienzymatic biosynthetic process, this [2Fe] subsite is first assembled on a maturation enzyme, HydF, and then delivered to the apo-hydrogenase for activation. Synthetic chemistry has been used to prepare remarkably similar mimics of that subsite, but it has failed to reproduce the natural enzymatic activities thus far. Here we show that three synthetic mimics (containing different bridging dithiolate ligands) can be loaded onto bacterial Thermotoga maritima HydF and then transferred to apo-HydA1, one of the hydrogenases of Chlamydomonas reinhardtii algae. Full activation of HydA1 was achieved only when using the HydF hybrid protein containing the mimic with an azadithiolate bridge, confirming the presence of this ligand in the active site of native [FeFe]-hydrogenases. This is an example of controlled metalloenzyme activation using the combination of a specific protein scaffold and active-site synthetic analogues. This simple methodology provides both new mechanistic and structural insight into hydrogenase maturation and a unique tool for producing recombinant wild-type and variant [FeFe]-hydrogenases, with no requirement for the complete maturation machinery.

Spectroscopic investigations of a semi-synthetic [FeFe] hydrogenase with propane di-selenol as bridging ligand in the binuclear subsite: comparison to the wild type and propane di-thiol variants.

[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 Maturation with a [2Fe] complex in which the essential ADT amine moiety has been replaced by CH2 (PDT = propane-dithiolate) results in a low activity enzyme with structural and spectroscopic properties similar to those of the native enzyme, but with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.

New light on methylthiolation reactions.

A novel enzyme, named RimO for ribosomal modification (Anton et al., 2008) catalyzes the methylthiolation of aspartate 88 of the S12 ribosomal protein in Escherichia coli and shows a strong similarity with the iron-sulfur enzyme MiaB involved in the methylthiolation of tRNAs.

Ruthenium-cobalt dinuclear complexes as photocatalysts for CO2 reduction.

A series of Ru-Co dinuclear complexes have been synthesized and assayed as photocatalysts for the reduction of CO2 to CO in organic solvents. The Ru-Co complexes, with tris-diimine coordination at the Co ion, are the best supramolecular photocatalysts for CO2 reduction with a non-noble metal catalytic center reported so far.

Copper amine oxidase: a novel use for a tyrosine.

The first three-dimensional structure of copper amine oxidase demonstrates that one tyrosine residue is converted into 2,4,5-trihydroxyphenylalanine quinone (TPQ). TPQ binds to copper in the inactive form of the enzyme but not in the active form.

Glycyl radical enzymes: a conservative structural basis for radicals.

The first structures of glycyl radical enzymes, the anaerobic ribonucleotide reductase from bacteriophage T4 and pyruvate formate lyase from Escherichia coli, have been recently determined. This work provides new insights into the structure and chemistry of glycyl radical sites.

NMR studies of binding of 5-FdUDP and dCDP to ribonucleoside-diphosphate reductase from Escherichia coli.

5-Fluoro-2'-deoxyuridine-5'-diphosphate (5-FdUDP) has been synthesised using an original route, previously applied to the synthesis of natural nucleoside diphosphates. The interaction between 5-FdUDP and the enzyme ribonucleoside-diphosphate reductase (EC 1.17.4.1) has been studied with 19F-NMR. The product analogue is shown to be in fast exchange with substrate binding sites on protein subunit 1 (R1) of ribonucleoside-diphosphate (NDP) reductase. The number of binding sites is reduced to half when the complete holoenzyme R1R2 is formed. The temperature dependence of the line broadening of 5-FdUDP was studied using 19F-NMR, and of dCDP and dUDP using 1H-NMR. The temperature dependences are complex and a molecular model in which R1 is in a temperature dependent equilibrium between at least two conformations is suggested in order to explain the observed behaviour. Binding of a ligand to the substrate binding sites affects the conformational equilibrium in a ligand specific way. Formation of the holoenzyme R1R2 also affects the equilibrium.

Adenosylmethionine as a source of 5'-deoxyadenosyl radicals.

The combination of an iron-sulfur cluster and S-adenosylmethionine provides a novel mechanism for the initiation of radical catalysis in an unanticipated variety of metabolic processes. Molecular details of the cluster-mediated reductive cleavage of S-adenosylmethionine to methionine and, presumably, a 5'-deoxyadenosyl radical are the targets of recent studies.

Four crystal structures of the 60 kDa flavoprotein monomer of the sulfite reductase indicate a disordered flavodoxin-like module.

Escherichia coli NADPH-sulfite reductase (SiR) is a 780 kDa multimeric hemoflavoprotein composed of eight alpha-subunits (SiR-FP) and four beta-subunits (SiR-HP) that catalyses the six electron reduction of sulfite to sulfide. Each beta-subunit contains a Fe4S4 cluster and a siroheme, and each alpha-subunit binds one FAD and one FMN as prosthetic groups. The FAD gets electrons from NADPH, and the FMN transfers the electrons to the metal centers of the beta-subunit for sulfite reduction. We report here the 1.94 A X-ray structure of SiR-FP60, a recombinant monomeric fragment of SiR-FP that binds both FAD and FMN and retains the catalytic properties of the native protein. The structure can be divided into three domains. The carboxy-terminal part of the enzyme is composed of an antiparallel beta-barrel which binds the FAD, and a variant of the classical pyridine dinucleotide binding fold which binds NADPH. These two domains form the canonic FNR-like module, typical of the ferredoxin NADP+ reductase family. By analogy with the structure of the cytochrome P450 reductase, the third domain, composed of seven alpha-helices, is supposed to connect the FNR-like module to the N-terminal flavodoxine-like module. In four different crystal forms, the FMN-binding module is absent from electron density maps, although mass spectroscopy, amino acid sequencing and activity experiments carried out on dissolved crystals indicate that a functional module is present in the protein. Our results clearly indicate that the interaction between the FNR-like and the FMN-like modules displays lower affinity than in the case of cytochrome P450 reductase. The flexibility of the FMN-binding domain may be related, as observed in the case of cytochrome bc1, to a domain reorganisation in the course of electron transfer. Thus, a movement of the FMN-binding domain relative to the rest of the enzyme may be a requirement for its optimal positioning relative to both the FNR-like module and the beta-subunit.

A noble metal-free proton-exchange membrane fuel cell based on bio-inspired molecular catalysts.

Hydrogen is a promising energy vector for storing renewable energies: obtained from water-splitting, in electrolysers or photoelectrochemical cells, it can be turned back to electricity on demand in fuel cells (FCs). Proton exchange membrane (PEM) devices with low internal resistance, high compactness and stability are an attractive technology optimized over decades, affording fast start-up times and low operating temperatures. However, they rely on the powerful catalytic properties of noble metals such as platinum, while lower cost, more abundant materials would be needed for economic viability. Replacing these noble metals at both electrodes has long proven to be a difficult task, so far incompatible with PEM technologies. Here we take advantage of newly developed bio-inspired molecular H2 oxidation catalysts and noble metal-free O2-reducing materials, to fabricate a noble metal-free PEMFC, with an 0.74 V open circuit voltage and a 23 µW cm-2 output power under technologically relevant conditions. X-ray absorption spectroscopy measurements confirm that the catalysts are stable and retain their structure during turnover.

NADPH-sulfite reductase flavoprotein from Escherichia coli: contribution to the flavin content and subunit interaction.

The flavoprotein component (SiR-FP) of the sulfite reductase of E. coli is an octamer of the 66 kDa alpha subunit. It was shown to be cleaved in two peptide fragments. The 23 kDa fragment has been purified as a polymer of 8-10 subunits. It corresponds to the N-terminal part of the native protein and was shown to contain essentially FMN as cofactor. The 43 kDa fragment is monomeric. It contains exclusively FAD and remains able to catalyze efficiently NADPH-dependent reductions. One can conclude that each alpha-chain of SiR-FP is composed of two distinct domains, one binding FAD and the other FMN and that the FMN-binding domains cooperate for a head-to-head subunit interaction.

The lipoate synthase from Escherichia coli is an iron-sulfur protein.

Lipoate synthase catalyzes the last step of the biosynthesis of lipoic acid in microorganisms and plants. The protein isolated from an overexpressing Escherichia coli strain was purified from inclusion bodies. Spectroscopic (UV-visible and electron paramagnetic resonance) properties of the reconstituted protein demonstrate the presence of a (2Fe-2S) center per protein. As observed in biotin synthase, these clusters are converted to (4Fe-4S) centers during reduction under anaerobic conditions. The possible involvement of the cluster in the insertion of sulfur atoms into the octanoic acid backbone is discussed.

Pulse radiolysis studies on superoxide reductase from Treponema pallidum.

Superoxide reductases (SORs) are small metalloenzymes, which catalyze reduction of O2*- to H2O2. The reaction of the enzyme from Treponema pallidum with superoxide was studied by pulse radiolysis methods. The first step is an extremely fast bi-molecular reaction of the ferrous center with O2, with a rate constant of 6 x 10 (8) M(-1) s(-1). A first intermediate is formed which is converted to a second one with a slower rate constant of 4800 s(-1). This latter value is 10 times higher than the corresponding one previously reported in the case of SOR from Desulfoarculus baarsii. The reconstituted spectra for the two intermediates are consistent with formation of transient iron-peroxide species.

Resveratrol, a remarkable inhibitor of ribonucleotide reductase.

Resveratrol, a natural phytoalexin found in grapes, is well known for its presumed role in the prevention of heart disease, associated with red wine consumption. We show here that it is a remarkable inhibitor of ribonucleotide reductase and DNA synthesis in mammalian cells, which might have further applications as an antiproliferative or a cancer chemopreventive agent in humans.

The irreversible inactivation of ribonucleotide reductase from Escherichia coli by superoxide radicals.

The expression of superoxide dismutase in all aerobic living organisms supports the concept that superoxide radicals are toxic species. However, because of the limited chemical reactivity of superoxide, the mechanisms of this toxicity are still uncertain. Protein R2, the small component of ribonucleotide reductase, a key enzyme for DNA synthesis, is shown here to be irreversibly inactivated during incubation with an enzymatic generator of superoxide radicals, at neutral pH. During inactivation the essential tyrosyl radical of protein R2 is irreversibly destroyed. Full protection is afforded by superoxide dismutase. It is proposed that coupling between superoxide radicals and the radical protein R2 generates oxidized forms of tyrosine, tyrosine peroxide and 3,4-dihydroxyphenylalanine.

Iron: metabolism, toxicity and therapy.

This paper is an overview on iron metabolism, iron toxicity and therapeutics against this toxicity. The attention has been focused on: i) the solubilization of iron by living organisms; ii) the iron metabolism in human; iii) the toxicity of iron (Fe-catalyzed reduction of oxygen, Fenton reaction, decomposition of lipid peroxides); iv) the natural protective mechanisms; v) the relations between iron, free radicals and human diseases; and vi) the iron overload and its treatment.

Reduction of benzyl halides by liver microsomes. Formation of 478 NM-absorbing sigma-alkyl-ferric cytochrome P-450 complexes.

The benzyl halides benzyl bromide and 4-nitrobenzyl chloride are reduced anaerobically by NADPH and rat liver microsomes to yield toluene and 4-nitrotoluene, respectively. These reductions and cytochrome P-450-dependent since they are inhibited by CO and metyrapone, and are increased after pretreatment of rats by phenobarbital and 3-methylcholanthrene. During benzyl halide reduction, cytochrome P-450 complexes, which are very unstable to O2 and characterized by a Soret peak at 478 nm, are formed in steady-state concentrations. These concentrations are very dependent on pretreatment of rats and on the nature of the reducing agent (NADPH or dithionite) and the benzyl halide:4-methylbenzyl bromide and benzyl bromide lead to 478 nm absorbing complexes in the presence of NADPH whereas 4-nitrobenzyl chloride and benzyl chloride lead to such completes only in the presence of dithionite. Microsomal reductions of 4-nitrobenzyl chloride and benzyl bromide in D2O lead to partially deuterated 4-nitrotoluene and toluene. From these results, we propose a mechanism for anaerobic microsomal reduction of benzyl halides involving the intermediate formation of sigma-alkyl cytochrome P-450-Fe(III)-CH2Ar complexes which exhibit red-shifted Soret peaks around 478 nm. Toluenes, ArCH3, are formed either by protonation of the sigma-alkyl complexes or by hydrogen abstraction by the intermediate free radical ArCH2.

Dye-Sensitized Nanostructured Crystalline Mesoporous Tin-doped Indium Oxide Films with Tunable Thickness for Photoelectrochemical Applications.

A simple route towards nanostructured mesoporous Indium-Tin Oxide (templated nano-ITO) electrodes exhibiting both high conductivities and optimized bicontinuous pore-solid network is reported. The ITO films are first produced as an X-ray-amorphous, high surface area material, by adapting recently established template-directed sol-gel methods using Sn(IV) and In(III) salts. Carefully controlled temperature/atmosphere treatments convert the as-synthesized ITO films into nano-crystalline coatings with the cubic bixbyite structure. Specially, a multi-layered synthesis was successfully undertaken for tuning the film thickness. In order to evaluate the performances of templated nano-ITO as an electrode substrate for photoelectrochemical applications, photoelectrodes were prepared by covalent grafting of a redox-active dye, the complex [Ru(bpy)2(4,4'-(CH2PO3H2)2-bpy)]Cl21 (bpy=bipyridine). Surface coverage was shown to increase with the film thickness, from 0.7 × 10-9 mol.cm-2 (one layer, 45 nm) to 3.5 × 10-9 mol.cm-2 (ten layers, 470 nm), the latter value being ~ 100 times larger than that for commercially available planar ITO. In the presence of an electron mediator, photocurrents up to 50 µA.cm-2 have been measured under visible light irradiation, demonstrating the potential of this new templated nano-ITO preparation for the construction of efficient photoelectrochemical devices.

Cobalt stress in Escherichia coli and Salmonella enterica: molecular bases for toxicity and resistance.

Cobalt (Co) is present in trace amounts in the environment but it can be toxic when it accumulates in cells. The question of how Co produces its toxic effects and how living organisms protect themselves from, and resist to, such a stress remains to be clarified. Studies pertaining to these issues were recently carried out in Escherichia coli and Salmonella enterica. Iron-sulfur proteins were identified as primary targets of Co ions. Perturbation of iron homeostasis, oxidative stress and possible effects on sulfur assimilation were noticed. Cells were found to respond by up-regulating genes involved in the biosynthesis of Fe-S clusters as well as genes involved in Co efflux. These data are summarized in this review article to provide a preliminary general view of Co toxicity mechanisms in these two bacterial models.

Is the presence of oxygen on an exoplanet a reliable biosignature?

We revisit the validity of the presence of O(2) or O(3) in the atmosphere of a rocky planet as being a biosignature. Up to now, the false positive that has been identified applies to a planet during a hot greenhouse runaway, which is restricted to planets outside the habitable zone (HZ) of the star that are closer to the star. In this paper, we explore a new possibility based on abiotic photogeneration of O(2) at the surface of a planet that could occur inside the HZ. The search for such a process is an active field of laboratory investigation that has resulted from an ongoing interest in finding efficient systems with the capacity to harvest solar energy on Earth. Although such a process is energetically viable, we find it to be a very unlikely explanation for the observation of O(2) or O(3) in the atmosphere of a telluric exoplanet in the HZ. It requires an efficient photocatalyst to be present and abundant under natural planetary conditions, which appears unlikely according to our discussion of known mineral photochemical processes. In contrast, a biological system that synthesizes its constituents from abundant raw materials and energy has the inherent adaptation advantage to become widespread and dominant (Darwinist argument). Thus, O(2) appears to continue to be a good biosignature.