Enrico Salvadori

Water Oxidation at a Tetraruthenate Core Stabilized by Polyoxometalate Ligands: Experimental and Computational Evidence To Trace the Competent Intermediates

Converging UV-vis, EPR, rRaman, and DFT calculations highlight the evolution of [Ru(4)(H(2)O)(4)(mu-O)(4)(mu-OH)(2)(gamma-SiW(10)O(36))(2)](10-), 1, to high-valent intermediates. In analogy with the natural enzyme, five different oxidation states, generated from 1, have been found to power the catalytic cycle for water oxidation. A high electrophilic tetraruthenium(V)-hydroxo species is envisaged as the competent intermediate, undergoing nucleophilic attack by an external water molecule as a key step in the formation of a new O-O bond under catalytic conditions.

Biochemical and Kinetic Characterization of Radical S-Adenosyl-l-methionine Enzyme HydG

The radical S-adenosyl-L-methionine (AdoMet) enzyme HydG is one of three maturase enzymes involved in [FeFe]-hydrogenase H-cluster assembly. It catalyzes L-tyrosine cleavage to yield the H-cluster cyanide and carbon monoxide ligands as well as p-cresol. Clostridium acetobutylicum HydG contains the conserved CX3CX2C motif coordinating the AdoMet binding [4Fe-4S] cluster and a C-terminal CX2CX22C motif proposed to coordinate a second [4Fe-4S] cluster. To improve our understanding of the roles of each of these iron-sulfur clusters in catalysis, we have generated HydG variants lacking either the N- or C-terminal cluster and examined these using spectroscopic and kinetic methods. We have used iron analyses, UV-visible spectroscopy, and electron paramagnetic resonance (EPR) spectroscopy of an N-terminal C96/100/103A triple HydG mutant that cannot coordinate the radical AdoMet cluster to unambiguously show that the C-terminal cysteine motif coordinates an auxiliary [4Fe-4S] cluster. Spectroscopic comparison with a C-terminally truncated HydG (ΔCTD) harboring only the N-terminal cluster demonstrates that both clusters have similar UV-visible and EPR spectral properties, but that AdoMet binding and cleavage occur only at the N-terminal radical AdoMet cluster. To elucidate which steps in the catalytic cycle of HydG require the auxiliary [4Fe-4S] cluster, we compared the Michaelis-Menten constants for AdoMet and L-tyrosine for reconstituted wild-type, C386S, and ΔCTD HydG and demonstrate that these C-terminal modifications do not affect the affinity for AdoMet but that the affinity for L-tyrosine is drastically reduced compared to that of wild-type HydG. Further detailed kinetic characterization of these HydG mutants demonstrates that the C-terminal cluster and residues are not essential for L-tyrosine cleavage to p-cresol but are necessary for conversion of a tyrosine-derived intermediate to cyanide and CO.

The [4Fe-4S]-cluster coordination of [FeFe]-hydrogenase maturation protein HydF as revealed by EPR and HYSCORE spectroscopies.

[FeFe] hydrogenases are key enzymes for bio(photo)production of molecular hydrogen, and several efforts are underway to understand how their complex active site is assembled. This site contains a [4Fe-4S]-2Fe cluster and three conserved maturation proteins are required for its biosynthesis. Among them, HydF has a double task of scaffold, in which the dinuclear iron precursor is chemically modified by the two other maturases, and carrier to transfer this unit to a hydrogenase containing a preformed [4Fe-4S]-cluster. This dual role is associated with the capability of HydF to bind and dissociate an iron-sulfur center, due to the presence of the conserved FeS-cluster binding sequence CxHx(46-53)HCxxC. The recently solved three-dimensional structure of HydF from Thermotoga neapolitana described the domain containing the three cysteines which are supposed to bind the FeS cluster, and identified the position of two conserved histidines which could provide the fourth iron ligand. The functional role of two of these cysteines in the activation of [FeFe]-hydrogenases has been confirmed by site-specific mutagenesis. On the other hand, the contribution of the three cysteines to the FeS cluster coordination sphere is still to be demonstrated. Furthermore, the potential role of the two histidines in [FeFe]-hydrogenase maturation has never been addressed, and their involvement as fourth ligand for the cluster coordination is controversial. In this work we combined site-specific mutagenesis with EPR (electron paramagnetic resonance) and HYSCORE (hyperfine sublevel correlation spectroscopy) to assign a role to these conserved residues, in both cluster coordination and hydrogenase maturation/activation, in HydF proteins from different microorganisms.

Triplet–triplet energy transfer in the major intrinsic light-harvesting complex of Amphidinium carterae as revealed by ODMR and EPR spectroscopies

We present an optically detected magnetic resonance (ODMR) and electron paramagnetic resonance (EPR) spectroscopic study on the quenching of photo-induced chlorophyll triplet states by carotenoids, in the intrinsic light-harvesting complex (LHC) from the dinoflagellate Amphidinium carterae. Two carotenoid triplet states, differing in terms of optical and magnetic spectroscopic properties, have been identified and assigned to peridinins located in different protein environment. The results reveal a parallelism with the triplet-triplet energy transfer (TTET) process involving chlorophyll a and luteins observed in the LHC-II complex of higher plants. Starting from the hypothesis of a conserved alignment of the amino acid sequences at the cores of the LHC and LHC-II proteins, the spin-polarized time-resolved EPR spectra of the carotenoid triplet states of LHC have been calculated by a method which exploits the conservation of the spin momentum during the TTET process. The analysis of the spectra shows that the data are compatible with a structural model of the core of LHC which assigns the photo-protective function to two central carotenoids surrounded by the majority of Chl a molecules present in the protein, as found in LHC-II. However, the lack of structural data, and the uncertainty in the pigment composition of LHC, leaves open the possibility that this complex posses a different arrangement of the pigments with specific centers of Chl triplet quenching.

Archaeal TFEα/β is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39

Transcription initiation of archaeal RNA polymerase (RNAP) and eukaryotic RNAPII is assisted by conserved basal transcription factors. The eukaryotic transcription factor TFIIE consists of α and β subunits. Here we have identified and characterised the function of the TFIIEβ homologue in archaea that on the primary sequence level is related to the RNAPIII subunit hRPC39. Both archaeal TFEβ and hRPC39 harbour a cubane 4Fe-4S cluster, which is crucial for heterodimerization of TFEα/β and its engagement with the RNAP clamp. TFEα/β stabilises the preinitiation complex, enhances DNA melting, and stimulates abortive and productive transcription. These activities are strictly dependent on the β subunit and the promoter sequence. Our results suggest that archaeal TFEα/β is likely to represent the evolutionary ancestor of TFIIE-like factors in extant eukaryotes. DOI: http://dx.doi.org/10.7554/eLife.08378.001

Evidence for water-mediated triplet–triplet energy transfer in the photoprotective site of the peridinin–chlorophyll a–protein

Experimental and theoretical studies indicate that water molecules between redox partners can significantly affect their electron-transfer and possibly also the triplet-triplet energy transfer (TTET) properties when in the vicinity of chromophores. In the present work, the interaction of an intervening water molecule with the peridinin triplet state in the peridinin-chlorophyll a-protein (PCP) from Amphidinium carterae is studied by using orientation selective (2)H electron spin echo envelope modulation (ESEEM) spectroscopy, in conjunction with quantum mechanical calculations. This water molecule is located at the interface between the chlorophyll and peridinin pigments involved in the photoprotection mechanism (Chl601(602)-Per614(624), for nomenclature see reference [1]), based on TTET. The characteristic deuterium modulation pattern is observed in the electron spin-echo envelopes for the PCP complex exchanged against (2)H2O. Simulations of the time- and frequency-domain two-pulse and three-pulse ESEEM require two types of coupled (2)H. The more strongly coupled (2)H has an isotropic coupling constant (aiso) of -0.4MHz. This Fermi contact contribution for one of the two water protons and the precise geometry of the water molecule at the interface between the chlorophyll and peridinin pigments, resulting from the analysis, provide experimental evidence for direct involvement of this structured water molecule in the mechanism of TTET. The PCP antenna, characterised by a unity efficiency of the process, represents a model for future investigations on protein- and solvent-mediated TTET in the field of natural/artificial photosynthesis.

The electronic structure of the lutein triplet state in plant light-harvesting complex II

Carotenoid molecules are essential for the life of photosynthetic organisms in that they protect the cell from the photo-oxidative damage induced by light-stress conditions. One of the photo-protective mechanisms involves triplet-triplet energy transfer from the chlorophyll molecules to the carotenoids: a process that is strongly dependent on the electronic properties of the triplet states involved. Here, we obtain a clear description of the triplet state of lutein in LHCII from higher plants for the first time by density functional theory (DFT) calculations. DFT predictions have been validated by comparison with hyperfine couplings obtained with pulsed-ENDOR spectroscopy. Knowledge of the spin density distribution, the frontier orbitals and orbital excitations forms a basis for discussing the requirements for an efficient triplet-triplet energy transfer. The results obtained for the lutein in LHCII are compared with those of the highly-substituted carotenoid peridinin in PCP from Amphidinium carterae [Di Valentin et al., Biochim. Biophys. Acta, 2008, 1777, 295-307]. The presence of substituents in the peridinin molecule does not alter significantly the triplet state electronic structure compared to lutein. Despite the unusual spectroscopic behaviour of the peridinin excited singlet state, lutein and peridinin have similar triplet state properties. In both molecules the unpaired spins are delocalized uniformly over the whole π-conjugated system in an alternating even-odd pattern.

Cryo-EM Structure of a Relaxase Reveals the Molecular Basis of DNA Unwinding during Bacterial Conjugation

Summary Relaxases play essential roles in conjugation, the main process by which bacteria exchange genetic material, notably antibiotic resistance genes. They are bifunctional enzymes containing a trans-esterase activity, which is responsible for nicking the DNA strand to be transferred and for covalent attachment to the resulting 5′-phosphate end, and a helicase activity, which is responsible for unwinding the DNA while it is being transported to a recipient cell. Here we show that these two activities are carried out by two conformers that can both load simultaneously on the origin of transfer DNA. We solve the structure of one of these conformers by cryo electron microscopy to near-atomic resolution, elucidating the molecular basis of helicase function by relaxases and revealing insights into the mechanistic events taking place in the cell prior to substrate transport during conjugation.

Triplet-triplet energy transfer in Peridinin-Chlorophyll a-protein reconstituted with Chl a and Chl d as revealed by optically detected magnetic resonance and pulse EPR: comparison with the native PCP complex from Amphidinium carterae.

The triplet state of the carotenoid peridinin, populated by triplet-triplet energy transfer from photoexcited chlorophyll triplet state, in the reconstituted Peridinin-Chlorophyll a-protein, has been investigated by ODMR (Optically detected magnetic resonance), and pulse EPR spectroscopies. The properties of peridinins associated with the triplet state formation in complexes reconstituted with Chl a and Chl d have been compared to those of the main-form peridinin-chlorophyll protein (MFPCP) isolated from Amphidinium carterae. In the reconstituted samples no signals due to the presence of chlorophyll triplet states have been detected, during either steady state illumination or laser-pulse excitation. This demonstrates that reconstituted complexes conserve total quenching of chlorophyll triplet states, despite the biochemical treatment and reconstitution with the non-native Chl d pigment. Zero field splitting parameters of the peridinin triplet states are the same in the two reconstituted samples and slightly smaller than in native MFPCP. Analysis of the initial polarization of the photoinduced Electron-Spin-Echo detected spectra and their time evolution, shows that, in the reconstituted complexes, the triplet state is probably localized on the same peridinin as in native MFPCP although, when Chl d replaces Chl a, a local rearrangement of the pigments is likely to occur. Substitution of Chl d for Chl a identifies previously unassigned bands at approximately 620 and approximately 640 nm in the Triplet-minus-Singlet (T-S) spectrum of PCP detected at cryogenic temperature, as belonging to peridinin.

Conservation of spin polarization during triplet-triplet energy transfer in reconstituted peridinin-chlorophyll-protein complexes

Peridinin-chlorophyll-protein (PCP) complexes, where the N-terminal domain of native PCP from Amphidinium carterae has been reconstituted with different chlorophyll (Chl) species, have been investigated by time-resolved EPR in order to elucidate the details of the triplet-triplet energy transfer (TTET) mechanism. This spectroscopic approach exploits the concept of spin conservation during TTET, which leads to recognizable spin-polarization effects in the observed time-resolved EPR spectra. The spin polarization produced at the acceptor site (peridinin) depends on the initial polarization of the donor (chlorophyll) and on the relative geometric arrangement of the donor-acceptor spin axes. A variation of the donor triplet state properties in terms of population probabilities or triplet spin axis directions, as produced by replacement of chlorophyll a (Chl a) with non-native chlorophyll species (ZnChl a and BacterioChl a) in the reconstituted complexes, is unambiguously reflected in the polarization pattern of the carotenoid triplet state. For the first time, in the present investigation spin-polarization conservation has been shown to occur among natural cofactors in protein complexes during the TTET process. Proving the validity of the assumption of spin conservation adopted in the EPR spectral analysis, the results reinforce the hypothesis that in PCP proteins peridinin 614, according to X-ray nomenclature (Hofmann, E.; et al. Science 1996, 272, 1788-1791), is the carotenoid of election in the photoprotection mechanism based on TTET.