Jean-Marie Mouesca

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.

Two Fe-S clusters catalyze sulfur insertion by radical-SAM methylthiotransferases

How living organisms create carbon-sulfur bonds during the biosynthesis of critical sulfur-containing compounds is still poorly understood. The methylthiotransferases MiaB and RimO catalyze sulfur insertion into tRNAs and ribosomal protein S12, respectively. Both belong to a subgroup of radical-S-adenosylmethionine (radical-SAM) enzymes that bear two [4Fe-4S] clusters. One cluster binds S-adenosylmethionine and generates an Ado• radical via a well-established mechanism. However, the precise role of the second cluster is unclear. For some sulfur-inserting radical-SAM enzymes, this cluster has been proposed to act as a sacrificial source of sulfur for the reaction. In this paper, we report parallel enzymological, spectroscopic and crystallographic investigations of RimO and MiaB, which provide what is to our knowledge the first evidence that these enzymes are true catalysts and support a new sulfation mechanism involving activation of an exogenous sulfur cosubstrate at an exchangeable coordination site on the second cluster, which remains intact during the reaction.

Carbon Monoxide Dehydrogenase Reaction Mechanism: A Likely Case of Abnormal CO2 Insertion to a Ni−H− Bond

Ni-containing carbon monoxide dehydrogenases (CODH), present in many anaerobic microorganisms, catalyze the reversible oxidation of CO to CO(2) at the so-called C-cluster. This atypical active site is composed of a [NiFe(3)S(4)] cluster and a single unusual iron ion called ferrous component II or Fe(u) that is bridged to the cluster via one sulfide ion. After additional refinement of recently published high-resolution structures of COOH(x)-, OH(x)-, and CN-bound CODH from Carboxydothermus hydrogenoformans (Jeoung and Dobbek Science 2007, 318, 1461-1464; J. Am. Chem. Soc. 2009, 131, 9922-9923), we have used computational methods on the predominant resulting structures to investigate the spectroscopically well-characterized catalytic intermediates, C(red1) and the two-electron more-reduced C(red2). Several models were geometry-optimized for both states using hybrid quantum mechanical/molecular mechanical potentials. The comparison of calculated Mössbauer parameters of these active site models with experimental data allows us to propose that the C(red1) state has a Fe(u)-Ni(2+) bridging hydroxide ligand and the C(red2) state has a hydride terminally bound to Ni(2+). Using our combined structural and theoretical data, we put forward a revised version of an earlier proposal for the catalytic cycle of Ni-containing CODH (Volbeda and Fontecilla-Camps Dalton Trans. 2005, 21, 3443-3450) that agrees with available spectroscopic and structural data. This mechanism involves an abnormal CO(2) insertion into the Ni(2+)-H(-) bond.

The Structural Plasticity of the Proximal [4Fe3S] Cluster is Responsible for the O2 Tolerance of Membrane‐Bound [NiFe] Hydrogenases

Hydrogen metabolism is restricted to three classes of enzymes, [NiFe], [FeFe], and [Fe] hydrogenases. As H2 oxidation catalysts, [NiFe] hydrogenases are competitive with the more expensive Pt. Consequently, there has been a significant effort to understand the H2 uptake/evolution mechanism for its application in hydrogen-based technologies. These enzymes are sensitive to oxygen; an exception is a subclass of periplasmic membrane-bound [NiFe] hydrogenases represented by Ralstonia eutropha (ReMBH), Escherichia coli hydrogenase 1 (EcHyd-1), and Aquifex aeolicus hydrogenase 1 (AaHase-1). This O2-tolerant subclass has already been used in potentially relevant technological applications. [NiFe] designates the active site, which is located in the large subunit and consists of a bimetallic NiFe center coordinated by four cysteine residues and CO/CN ligands to Fe. Upon oxidation, two EPR-active redox states are detected in standard (i.e., O2-sensitive) hydrogenases, called Ni-A and Ni-B. The Ni-B species, characterized by a Ni-Fe center with a bridging hydroxide ion, is a “ready” state, as it reactivates rapidly under reducing conditions. Conversely, the Ni-A state, a Ni-Fe center with most probably a bridging (hydro)peroxo, is “unready” and requires prolonged reactivation, which may not be achievable in vivo. EPR and FTIR studies on ReMBH and AaHase-1 have shown that, with the exception of the Ni-A species, most redox states observed in standard hydrogenases are also observed in these O2-tolerant enzymes. In standard hydrogenases, electron transfer is ensured by proximal and distal (relative to the active site) [4Fe4S] clusters and a medial [3Fe4S] cluster. Unexpectedly, an EPR study on ReMBH recorded a putative additional paramagnetic species at a fixed redox potential of + 290 mV, coupled to both the active site and the medial cluster. This observation suggested the presence of a modification near or at the proximal cluster, as further confirmed by an X-ray absorption spectroscopy study. Furthermore, assuming a proximal [4Fe4S] cluster, redox potentials for + 2/ + 1 and + 3/ + 2 transitions were determined at 60/ + 160 mV and + 98/ + 232 mV for ReMBH and AaHase-1, respectively. The narrow potential difference of less than 220 mV between these two redox couples has been recently also observed in EcHyd-1. Notably, the nonphysiological superreduction to the + 1 state of high-potential iron–sulfur protein (HiPIP) [4Fe4S] cluster occurs at a potential that is about 1000 mV more negative than that of the + 3/ + 2 redox couple. The possible + 3/ + 2 superoxidation of the proximal cluster in O2-tolerant [NiFe] hydrogenases was thus proposed to be a redox switch against oxidative stress. One specificity of these enzymes is the presence of two supernumerary conserved cysteine residues (Cys19 and Cys120 in EcHyd-1) close to the proximal cluster. Site-directed mutagenesis studies indicated that these residues are essential for the unusual redox property of this cluster and the associated O2 tolerance. Recently, the crystal structures of oxidized and H2reduced forms of Hydrogenovibrio marinus (Hm) MBH and of the reduced form of ReMBH showed that the highly symmetrical four-cysteine-coordinated [4Fe4S] cluster in standard hydrogenases is substituted by a [4Fe3S] cluster. Remarkably, the absent sulfide is replaced by both the bridging Cys19 thiolate and the terminally bound Cys120, which coordinates a Fe atom also bound to another cysteine thiolate. This results in an unprecedented asymmetric FeS cluster, which is further deformed upon superoxidation as the Fe that is coordinated by Cys19 and Cys20 moves away from the remaining [3Fe3S] moiety and binds the deprotonated amide nitrogen atom of Cys20. We reported the crystal structures of the as-isolated and H2-reduced EcHyd-1, and proposed that a glutamate residue conserved in O2-tolerant hydrogenases (Glu76 in EcHyd-1) plays the role of the base that deprotonates the amide moiety and allows the formation of the Fe N bond. Herein, we call the three redox states of the reduced [4Fe3S], oxidized [4Fe3S], and superoxidized proximal [4Fe3S] clusters PC1, PC2, and PC3, respectively (Table 1). Previously, we reported quantum calculations that defined an electronic/spin state for the PC3 species and fitted available structural and spectroscopic data. In the present study, we examined the remarkable plasticity of the [4Fe3S] cluster in the PC2/PC3 transition with special attention to the role of Glu76. We found that the interplay between the high asymmetry of the cluster and the resulting Fe valence/spin localization in the three redox states is the key feature that explains the facile superoxidation. [*] Dr. J. C. Fontecilla-Camps, Dr. P. Amara Metalloproteins Unit, Institut de Biologie Structurale J.P. Ebel Commissariat l’ nergie Atomique, Centre National de la Recherche Scientifique, Universit Joseph Fourier 41 rue Jules Horowitz, 38027 Grenoble (France) E-mail: juan.fontecilla@ibs.fr patricia.amara@ibs.fr

Tuning of Ferromagnetic Spin Interactions in Polymeric Aromatic Amines via Modification of Their π-Conjugated System

Polyarylamine containing meta-para-para-aniline units in the main chain and meta-para-aniline units in the pendant chains was synthesized. The polymer can be oxidized to radical cations in chemical or electrochemical ways. The presence of meta-phenylenes in the polymer chemical structure allows for the ferromagnetic coupling of electronic spins, which leads to the formation of high spin states. Detailed pulsed-EPR study indicates that the S = 2 spin state was reached for the best oxidation level. Quantitative magnetization measurements reveal that the doped polymer contains mainly S = 2 spin states and a fraction of S = 3/2 spin states. The efficiency of the oxidation was determined to be 74%. To the best of our knowledge, this polymer is the first example of a linear doped polyarylamine combining such high spin states with high doping efficiency.

An artificial photosynthetic system for photoaccumulation of two electrons on a fused dipyridophenazine (dppz)–pyridoquinolinone ligand

The π-extended ligand of a ruthenium complex stores two photo-generated electrons, mimicking a key step in photosynthesis.

High-Spin Polymers: Ferromagnetic Coupling of S = 1 Hexaazacyclophane Units up to a Pure S = 2 Polycyclophane

Triarylamines oxidized to radical cations can be used as stable spins sources for the design of high-spin compounds. Here, we present the synthesis of the polyarylamine-containing hexaazacyclophanes linked via meta-terphenyl bridges. Spins, created after oxidation of the polymer, can be coupled magnetically in cyclophane moieties via meta-phenyl and along the polymer chain via meta-terphenyl units. The formation of a quintet spin state was evidenced by pulsed-EPR nutation spectroscopy. Two exchange coupling constants via both couplers were determined experimentally and corresponded to J/k = 89 K in the cyclophane moiety and j/k = 17 K via meta-terphenyl. Most importantly, in this polymer, four spins can be ferromagnetically ordered via both couplers, which leads to the high spin state.

Highly Efficient Tuning of Ferromagnetic Spin Interactions in High-Spin Arylamine Structures by Incorporation of Spin Bearing Carbazole Units

Arylamine moieties oxidized to radical cations are considered promising spin bearing units in high-spin-type compounds. Here, we report the first use of carbazole-3,6-diamine units as efficient, rigid spin containing units. The use of rigid spin bearing units enhances significantly spin exchange interactions. The design using density functional theory calculations shows the progressive increase of the exchange coupling constant dependent on the considered model molecules. Two of the most representative molecules containing flexible (dimer 1) and rigid spin coupling unit (dimer 2) were synthesized. Electrochemical and pulsed-electron paramagnetic resonance nutation studies showed that both dimers can be oxidized to yield a majority of dicationic diradicals exhibiting S = 1 ground states. The high values of the dimer 2 exchange coupling constant obtained both computationally ( J/ kB = 145 K; HHeis. = - JS1 S2) and experimentally ( J/ kB = 90-100 K) indicate the beneficial role of the carbazole moiety incorporated into spin bearing units.

Towards enhancing spin states in doped arylamine compounds through extended planarity of the spin coupling moieties

Arylamine moieties oxidized to radical cations are promising spin bearers for organic high-spin compounds. However, successive oxidations of next-neighbour sites as well as the resulting ferromagnetic exchange interactions depend strongly on the charge and spin distributions over the molecule. We report here that the use of rigid segments composed of m-phenylene spin couplers linked to arylamine spin bearers in a co-planar way facilitates successive oxidations, enhances spin exchange interactions and doubles the observed spin state (S = 2) in a polymer (PQA) when compared to the chemically equivalent (but locally free rotating) PA2 polymer (S = 1). Such a quintet spin state is observed for the first time for a linear polyarylamine with an m-phenylene coupler. DFT calculations of the dimer QA reproduce the experimental J value and indicate that oxidation of these co-planar compounds leads indeed to well localized radical cations, a fact of crucial importance for the preparation of arylamine-type high-spin materials.