Catherine Berthomieu

Fourier transform infrared (FTIR) spectroscopy

Fourier transform infrared (FTIR) spectroscopy probes the vibrational properties of amino acids and cofactors, which are sensitive to minute structural changes. The lack of specificity of this technique, on the one hand, permits us to probe directly the vibrational properties of almost all the cofactors, amino acid side chains, and of water molecules. On the other hand, we can use reaction-induced FTIR difference spectroscopy to select vibrations corresponding to single chemical groups involved in a specific reaction. Various strategies are used to identify the IR signatures of each residue of interest in the resulting reaction-induced FTIR difference spectra. (Specific) Isotope labeling, site-directed mutagenesis, hydrogen/deuterium exchange are often used to identify the chemical groups. Studies on model compounds and the increasing use of theoretical chemistry for normal modes calculations allow us to interpret the IR frequencies in terms of specific structural characteristics of the chemical group or molecule of interest. This review presents basics of FTIR spectroscopy technique and provides specific important structural and functional information obtained from the analysis of the data from the photosystems, using this method.

Influence of uranium on bacterial communities: a comparison of natural uranium-rich soils with controls.

This study investigated the influence of uranium on the indigenous bacterial community structure in natural soils with high uranium content. Radioactive soil samples exhibiting 0.26% - 25.5% U in mass were analyzed and compared with nearby control soils containing trace uranium. EXAFS and XRD analyses of soils revealed the presence of U(VI) and uranium-phosphate mineral phases, identified as sabugalite and meta-autunite. A comparative analysis of bacterial community fingerprints using denaturing gradient gel electrophoresis (DGGE) revealed the presence of a complex population in both control and uranium-rich samples. However, bacterial communities inhabiting uraniferous soils exhibited specific fingerprints that were remarkably stable over time, in contrast to populations from nearby control samples. Representatives of Acidobacteria, Proteobacteria, and seven others phyla were detected in DGGE bands specific to uraniferous samples. In particular, sequences related to iron-reducing bacteria such as Geobacter and Geothrix were identified concomitantly with iron-oxidizing species such as Gallionella and Sideroxydans. All together, our results demonstrate that uranium exerts a permanent high pressure on soil bacterial communities and suggest the existence of a uranium redox cycle mediated by bacteria in the soil.

Modulating uranium binding affinity in engineered calmodulin EF-hand peptides: effect of phosphorylation

To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T9TKE12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from Kd = 25±6 nM to Kd = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (Kd = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the νas(P-O) and νs(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in νas(UO2)2+ vibration (from 923 cm−1 to 908 cm−1) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH.

Molecular Analysis by Vibrational Spectroscopy

Vibrational spectroscopy, which includes infrared and Raman spectroscopies, provides structural information of molecules by detecting molecular vibrations based on chemical bonds and interactions. These methods have been applied to the study of various cofactors in Photosystem II. In particular, light-induced Fourier transform infrared (FTIR) difference spectroscopy has proven to be a powerful method to reveal detailed structures of the binding sites of cofactors including protein moieties and water molecules. Information available by FTIR difference spectroscopy includes hydrogen bonding and protonation state of chemical groups, which play an essential role in proton transfer and also in controlling redox reactions, but are often not available by X-ray crystallography. The FTIR investigations cover all the redox cofactors of Photosystem II in both the main and peripheral electron-transfer pathways, i.e., the manganese-cluster, the redox-active tyrosines Yz and YD, the primary donor P680, the primary acceptor pheophytin, the quinone acceptors QA and QB, the non-heme iron, cytochrome b559, chlorophyll Z, and β-carotene. This article reviews how the structures and reactions of these cofactors have been studied using mainly FTIR spectroscopy with the assistance of Raman spectroscopy.

Crystal Structure of ChrR -- A Quinone Reductase with the Capacity to Reduce Chromate

The Escherichia coli ChrR enzyme is an obligatory two-electron quinone reductase that has many applications, such as in chromate bioremediation. Its crystal structure, solved at 2.2 Å resolution, shows that it belongs to the flavodoxin superfamily in which flavin mononucleotide (FMN) is firmly anchored to the protein. ChrR crystallized as a tetramer, and size exclusion chromatography showed that this is the oligomeric form that catalyzes chromate reduction. Within the tetramer, the dimers interact by a pair of two hydrogen bond networks, each involving Tyr128 and Glu146 of one dimer and Arg125 and Tyr85 of the other; the latter extends to one of the redox FMN cofactors. Changes in each of these amino acids enhanced chromate reductase activity of the enzyme, showing that this network is centrally involved in chromate reduction.

Assessing the Role of the Active-site Cysteine Ligand in the Superoxide Reductase from Desulfoarculus baarsii

Superoxide reductase is a novel class of non-heme iron proteins that catalyzes the one-electron reduction of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{.}}}\) \end{document} to H2O2, providing an antioxidant defense in some bacteria. Its active site consists of an unusual non-heme Fe2+ center in a [His4 Cys1] square pyramidal pentacoordination. In this class of enzyme, the cysteine axial ligand has been hypothesized to be an essential feature in the reactivity of the enzyme. Previous Fourier transform infrared spectroscopy studies on the enzyme from Desulfoarculus baarsii revealed that a protonated carboxylate group, proposed to be the side chain of Glu114, is in interaction with the cysteine ligand. In this work, using pulse radiolysis, Fourier transform infrared, and resonance Raman spectroscopies, we have investigated to what extent the presence of this Glu114 carboxylic lateral chain affects the strength of the S—Fe bond and the reaction of the iron active site with superoxide. The E114A mutant shows significantly modified pulse radiolysis kinetics for the protonation process of the first reaction intermediate. Resonance Raman spectroscopy demonstrates that the E114A mutation results in both a strengthening of the S—Fe bond and an increase in the extent of freeze-trapping of a Fe-peroxo species after treatment with H2O2 by a specific strengthening of the Fe—O bond. A fine tuning of the strength of the S—Fe bond by the presence of Glu114 appears to be an essential factor for both the strength of the Fe—O bond and the pKa value of the Fe3+-peroxo intermediate species to form the reaction product H2O2.

Detoxification of superoxide without production of H2O2: Antioxidant activity of superoxide reductase complexed with ferrocyanide

The superoxide radical O2·̅ is a toxic by-product of oxygen metabolism. Two O2·̅ detoxifying enzymes have been described so far, superoxide dismutase and superoxide reductase (SOR), both forming H2O2 as a reaction product. Recently, the SOR active site, a ferrous iron in a [Fe2+ (N-His)4 (S-Cys)] pentacoordination, was shown to have the ability to form a complex with the organometallic compound ferrocyanide. Here, we have investigated in detail the reactivity of the SOR–ferrocyanide complex with O2·̅ by pulse and γ-ray radiolysis, infrared, and UV-visible spectroscopies. The complex reacts very efficiently with O2·̅. However, the presence of the ferrocyanide adduct markedly modifies the reaction mechanism of SOR, with the formation of transient intermediates different from those observed for SOR alone. A one-electron redox chemistry appears to be carried out by the ferrocyanide moiety of the complex, whereas the SOR iron site remains in the reduced state. Surprisingly, the toxic H2O2 species is no longer the reaction product. Accordingly, in vivoexperiments showed that formation of the SOR–ferrocyanide complex increased the antioxidant capabilities of SOR expressed in an Escherichia coli sodA sodB recA mutant strain. Altogether, these data describe an unprecedented O2·̅ detoxification activity, catalyzed by the SOR–ferrocyanide complex, which does not conduct to the production of the toxic H2O2 species.

Coordination of proton and electron transfer from the redox-active tyrosine, YZ, of Photosystem II and examination of the electrostatic influence of oxidized tyrosine, YD˙(H+)

The redox active tyrosines, YZ and YD, of Photosystem II are oxidized by P680+ to the neutral radical. Such oxidation requires coupling of electron transfer to the transfer of the phenolic proton. Studies of the multiphasic kinetics of YZ oxidation in Mn-depleted PSII core complexes have shown that the relative amplitudes of the kinetic components are pH-dependent with one component showing a pH-dependent t1/2 in the microsecond to tens of microsecond range (pH 4–8). Sjodin and coworkers (M. Sjodin, S. Styring, B. Akemark, L. Sun and L. Hammarstrom, Philos. Trans. R. Soc. London, Ser. B, 2002, 357, 1471–1479) have suggested that the increase in rate of this latter component with pH reflects an increase in the driving force of the reaction by lowering the reduction potential of YZ˙/ YZ, consistent with concerted electron and proton transfer (CEP mechanism). A similar dependence of the rate of YZ oxidation on ΔG° is reported here through modification of the reduction potential of P680+/P680, that is, without modifying either the proton acceptor or the pathway for proton transfer. The results reported here support a CEP mechanism, though formation of the tyrosinate followed by electron transfer cannot be completely ruled out.The presence of oxidized tyrosine YD˙(H+) has been shown to accelerate the photoactivation of the oxygen evolving complex, possibly by an increase in the reduction potential of P680+/P680. The influence of YD˙(H+) on the P680+/P680 reduction potential is examined here by measuring the rate of YZ oxidation in Mn-depleted core complexes from the WT strain and from a YD-less strain of Synechocystis 6803. Also examined is the influence of YD˙(H+) on the P680+–P680 difference spectrum. These comparisons show that the electrostatic contribution of YD˙(H+) to the reduction potential of redox couple P680+/P680 is very small (≤10 mV), implying that the role of YD˙(H+) in photoactivation may have more to do with its providing an oxidizing equivalent during assembly of the manganese cluster.

Use of combined microscopic and spectroscopic techniques to reveal interactions between uranium and Microbacterium sp. A9, a strain isolated from the Chernobyl exclusion zone

Although uranium (U) is naturally found in the environment, soil remediation programs will become increasingly important in light of certain human activities. This work aimed to identify U(VI) detoxification mechanisms employed by a bacteria strain isolated from a Chernobyl soil sample, and to distinguish its active from passive mechanisms of interaction. The ability of the Microbacterium sp. A9 strain to remove U(VI) from aqueous solutions at 4 °C and 25 °C was evaluated, as well as its survival capacity upon U(VI) exposure. The subcellular localisation of U was determined by TEM/EDX microscopy, while functional groups involved in the interaction with U were further evaluated by FTIR; finally, the speciation of U was analysed by TRLFS. We have revealed, for the first time, an active mechanism promoting metal efflux from the cells, during the early steps following U(VI) exposure at 25 °C. The Microbacterium sp. A9 strain also stores U intracellularly, as needle-like structures that have been identified as an autunite group mineral. Taken together, our results demonstrate that this strain exhibits a high U(VI) tolerance based on multiple detoxification mechanisms. These findings support the potential role of the genus Microbacterium in the remediation of aqueous environments contaminated with U(VI) under aerobic conditions.

Molecular origin of the pH dependence of tyrosine D oxidation kinetics and radical stability in photosystem II.

A role for redox-active tyrosines has been demonstrated in many important biological processes, including water oxidation carried out by photosystem II (PSII) of oxygenic photosynthesis. The rates of tyrosine oxidation and reduction and the Tyr/Tyr reduction potential are undoubtedly controlled by the immediate environment of the tyrosine, with the coupling of electron and proton transfer, a critical component of the kinetic and redox behavior. It has been demonstrated by Faller et al. that the rate of oxidation of tyrosine D (Tyr(D)) at room temperature and the extent of Tyr(D) oxidation at cryogenic temperatures, following flash excitation, dramatically increase as a function of pH with a pK(a) of approximately 7.6 [Faller et al. 2001 Proc. Natl. Acad. Sci. USA 98, 14368-14373; Faller et al. 2001 Biochemistry 41, 12914-12920]. In this work, we investigated, using FTIR difference spectroscopy, the mechanistic reasons behind this large pH dependence. These studies were carried out on Mn-depleted PSII core complexes isolated from Synechocystis sp. PCC 6803, WT unlabeled and labeled with (13)C(6)-, or (13)C(1)(4)-labeled tyrosine, as well as on the D2-Gln164Glu mutant. The main conclusions of this work are that the pH-induced changes involve the reduced Tyr(D) state and not the oxidized Tyr(D)() state and that Tyr(D) does not exist in the tyrosinate form between pH 6 and 10. We can also exclude a change in the protonation state of D2-His189 as being responsible for the large pH dependence of Tyr(D) oxidation. Indeed, our data are consistent with D2-His189 being neutral both in the Tyr(D) and Tyr(D)() states in the whole pH6-10 range. We show that the interactions between reduced Tyr(D) and D2-His189 are modulated by the pH. At pH greater than 7.5, the nu(CO) mode frequency of Tyr(D) indicates that Tyr(D) is involved in a strong hydrogen bond, as a hydrogen bond donor only, in a fraction of the PSII centers. At pH below 7.5, the hydrogen-bonding interaction formed by Tyr(D) is weaker and Tyr(D) could be also involved as a hydrogen bond acceptor, according to calculations performed by Takahashi and Noguchi [J. Phys. Chem. B 2007 111, 13833-13844]. The involvement of Tyr(D) in this strong hydrogen-bonding interaction correlates with the ability to oxidize Tyr(D) at cryogenic temperatures and rapidly at room temperature. A strong hydrogen-bonding interaction is also observed at pH 6 in the D2-Gln164Glu mutant, showing that the residue at position D2-164 regulates the properties of Tyr(D.) The IR data point to the role of a protonatable group(s) (with a pK(a) of approximately 7) other than D2-His189 and Tyr(D), in modifying the characteristics of the Tyr(D) hydrogen-bonding interactions, and hence its oxidation properties. It remains to be determined whether the strong hydrogen-bonding interaction involves D2-His189 and if Tyr(D) oxidation involves the same proton transfer route at low and at high pH.