Pierre Dorlet

Effect of Ca2+/Sr2+ Substitution on the Electronic Structure of the Oxygen-Evolving Complex of Photosystem II: A Combined Multifrequency EPR, 55Mn-ENDOR, and DFT Study of the S2 State

The electronic structures of the native Mn(4)O(x)Ca cluster and the biosynthetically substituted Mn(4)O(x)Sr cluster of the oxygen evolving complex (OEC) of photosystem II (PSII) core complexes isolated from Thermosynechococcus elongatus, poised in the S(2) state, were studied by X- and Q-band CW-EPR and by pulsed Q-band (55)Mn-ENDOR spectroscopy. Both wild type and tyrosine D less mutants grown photoautotrophically in either CaCl(2) or SrCl(2) containing media were measured. The obtained CW-EPR spectra of the S(2) state displayed the characteristic, clearly noticeable differences in the hyperfine pattern of the multiline EPR signal [Boussac et al. J. Biol. Chem.2004, 279, 22809-22819]. In sharp contrast, the manganese ((55)Mn) ENDOR spectra of the Ca and Sr forms of the OEC were remarkably similar. Multifrequency simulations of the X- and Q-band CW-EPR and (55)Mn-pulsed ENDOR spectra using the Spin Hamiltonian formalism were performed to investigate this surprising result. It is shown that (i) all four manganese ions contribute to the (55)Mn-ENDOR spectra; (ii) only small changes are seen in the fitted isotropic hyperfine values for the Ca(2+) and Sr(2+) containing OEC, suggesting that there is no change in the overall spin distribution (electronic coupling scheme) upon Ca(2+)/Sr(2+) substitution; (iii) the changes in the CW-EPR hyperfine pattern can be explained by a small decrease in the anisotropy of at least two hyperfine tensors. It is proposed that modifications at the Ca(2+) site may modulate the fine structure tensor of the Mn(III) ion. DFT calculations support the above conclusions. Our data analysis also provides strong support for the notion that in the S(2) state the coordination of the Mn(III) ion is square-pyramidal (5-coordinate) or octahedral (6-coordinate) with tetragonal elongation. In addition, it is shown that only one of the currently published OEC models, the Siegbahn structure [Siegbahn, P. E. M. Acc. Chem. Res.2009, 42, 1871-1880, Pantazis, D. A. et al. Phys. Chem. Chem. Phys.2009, 11, 6788-6798], is consistent with all data presented here. These results provide important information for the structure of the OEC and the water-splitting mechanism. In particular, the 5-coordinate Mn(III) is a potential site for substrate water (H(2)O, OH(-)) binding. Its location within the cuboidal structural unit, as opposed to the external dangler position, may have important consequences for the mechanism of O-O bond formation.

Complete EPR Spectrum of the S3-State of the Oxygen-Evolving Photosystem II

Despite crystallographic structures now available and intensive work in the past decades, little is known about the higher redox states of the catalytic cycle of Photosystem II, the enzyme responsible for the presence of O(2) on Earth and at the beginning of the process that has produced both the biomass and the fossil fuels. In one of the highest oxidation states, the S(3)-state, only signals at g-values higher than 4 have been detected so far at the X-band. In this work, we report for the first time the complete X-band EPR spectrum for the S(3)-state of Photosystem II. Simulations show that, for a spin state S = 1, as was previously suggested for S(3), it is not possible to account for all the features observed. A satisfactory simulated spectrum was obtained for a spin state S = 3 with zero-field splitting parameters D = 0.175 cm(-1) and E/D = 0.275. The detection of the full EPR signal for S(3) opens the door for new investigations and a better understanding of the catalytic cycle of Photosystem II.

Manganese and tyrosyl radical function in photosynthetic oxygen evolution

Photosystem II catalyzes the photosynthetic oxidation of water to O2. The structural and functional basis for this remarkable process is emerging. The catalytic site contains a tetramanganese cluster, calcium, chloride and a redox-active tyrosine organized so as to promote electroneutral hydrogen atom abstraction from manganese-bound substrate water by the tyrosyl radical. Recent work is assessed within the framework of this model for the water oxidizing process.

Copper and Heme-Mediated Abeta Toxicity: Redox Chemistry, Abeta Oxidations and Anti-ROS Compounds

Oxidative stress mediated by reactive oxygen or nitrogen species (ROS/RNS) seems to be implicated in several diseases including neurodegenerative ones. In one of them, namely Alzheimers disease, there is a large body of evidence that the aggregation of the peptide amyloid-beta (Abeta) is implicated in the generation of the oxidative stress. Redox active metal ions play a key role in oxidative stress, either in the production of ROS/RNS by enzymes or loosely bound metals or in the protection against ROS, mostly as catalytic centers in enzymes. In Alzheimers disease, it is thought that metals (mostly Cu, Fe and heme) can bind to amyloid-beta and that such systems are involved in the generation of oxidative stress. In the present article, we review the role of ROS/RNS produced by redox active Cu ions and heme compounds in the context of the amyloid cascade. We focus on (i) the coordination chemistry of Cu and heme to Abeta; (ii) the role of the corresponding Abeta adducts in the (catalytic) production of ROS/RNS; (iii) the subsequent degradation of Abeta by these reactive species and (iv) the use of antioxidants, in particular metal sequestering compounds and direct antioxidants like polyphenols as a therapeutic strategies.

New Insights into the Coordination of Cu(II) by the Amyloid-B 16 Peptide from Fourier Transform IR Spectroscopy and Isotopic Labeling

Alzheimers disease is a neurodegenerative disorder in which the formation of amyloid-β (Aβ) aggregates plays a causative role. There is ample evidence that Cu(II) can bind to Aβ and modulate its aggregation. Moreover, Cu(II) bound to Aβ might be involved in the production of reactive oxygen species, a process supposed to be involved in the Alzheimers disease. The native Aβ40 contains a high affinity binding site for Cu(II), which is comprised in the N-terminal portion. Thus, Aβ16 (amino acid 1-16 of Aβ) has often been used as a model for Cu(II)-binding to monomeric Aβ. The Cu(II)-binding to Aβ is pH dependent and at pH 7.4, two different type of Cu(II) coordinations exist in equilibrium. These two forms are predominant at pH 6.5 and pH 9.0. In either form, a variety of studies show that the N-terminal Asp and the three His play a key role in the coordination, although the exact binding of these amino acids has not been addressed. Therefore, we studied the coordination modes of Cu(II) at pH 6.5 and 9.0 with the help of Fourier transform infrared (FTIR) spectroscopy. Combined with isotopic labeling of the amino acids involved in the coordination sphere, the data points toward the coordination of Cu(II) via the carboxylate of Asp1 at both pH values in a pseudobridging monovalent fashion. At low pH, His6 binds copper via Nτ, while His13 and His14 are bound via Nπ. At high pH, direct evidence is given on the coordination of Cu(II) via the Nτ atom of His6. Additionally, this study clearly shows the effect of Cu(II) binding on the protonation state of the His residues where a proton displacement takes places on the nitrogen atoms of the imidazole ring.

Heme binding properties of glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that also functions in transcriptional regulation, oxidative stress, vesicular trafficking, and apoptosis. Because GAPDH is required for the insertion of cellular heme into inducible nitric oxide synthase [Chakravarti, R., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 18004-18009], we extensively characterized the heme binding properties of GAPDH. Substoichiometric amounts of ferric heme bound to GAPDH (one heme per GAPDH tetramer) to form a low-spin complex with UV-visible maxima at 362, 418, and 537 nm and when reduced to ferrous gave maxima at 424, 527, and 559 nm. Ferric heme association and dissociation rate constants at 10 °C were as follows: k(on) = 17800 M(-1) s(-1), k(off1) = 7.0 × 10(-3) s(-1), and k(off2) = 3.3 × 10(-4) s(-1) (giving approximate affinities of 19-390 nM). Ferrous heme bound more poorly to GAPDH and dissociated with a k(off) of 4.2 × 10(-3) s(-1). Magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopic data on the ferric, ferrous, and ferrous-CO complexes of GAPDH showed that the heme is bis-ligated with His as the proximal ligand. The distal ligand in the ferric complex was not displaced by CN(-) or N(3)(-) but in the ferrous complex could be displaced by CO at a rate of 1.75 s(-1) (for >0.2 mM CO). Studies with heme analogues revealed selectivity toward the coordinating metal and porphyrin ring structure. The GAPDH-heme complex was isolated from bacteria induced to express rabbit GAPDH in the presence of δ-aminolevulinic acid. Our finding of heme binding to GAPDH expands the proteins potential roles. The strength, selectivity, reversibility, and redox sensitivity of heme binding to GAPDH are consistent with it performing heme sensing or heme chaperone-like functions in cells.

Evidence that D1-His332 in Photosystem II from Thermosynechococcus elongatus Interacts with the S3-State and not with the S2-State

Oxygen evolution by Photosystem II (PSII) is catalyzed by a Mn(4)Ca cluster. Thus far, from the crystallographic three-dimensional (3D) structures, seven amino acid residues have been identified as possible ligands of the Mn(4)Ca cluster. Among them, there is only one histidine, His332, which belongs to the D1 polypeptide. The relationships of the D1-His332 amino acid with kinetics and thermodynamic properties of the Mn(4)Ca cluster in the S(2)- and S(3)-states of the catalytic cycle were investigated in purified PSII from Thermosynechococcus elongatus. This was done by examining site-directed D1-His332Gln and D1-His332Ser mutants by a variety of spectroscopic techniques such as time-resolved UV-visible absorption change spectroscopy, cw- and pulse-EPR, thermoluminescence, and measurement of substrate water exchange. Both mutants grew photo-autotrophically and active PSII could be purified. On the basis of the parameters assessed in this work, the D1-His332(Gln, Ser) mutations had no effect in the S(2)-state. Electron spin-echo envelope modulation (ESEEM) spectroscopy also showed that possible interactions between the nuclear spin of the nitrogen(s) of D1-His332 with the electronic spin S = 1/2 of the Mn(4)Ca cluster in the S(2)-state were not detectable and that the D1-His332Ser mutation did not affect the detected hyperfine couplings. In contrast, the following changes were observed in the S(3)-state of the D1-His332 mutants: (1) The redox potential of the S(3)/S(2) couple was slightly increased by < or = 20 meV, (2) The S(3)-EPR spectrum was slightly modified, (3) The D1-His332Gln mutation resulted in a approximately 3 fold decrease of the slow (tightly bound) exchange rate and a approximately 2 fold increase of the fast exchange rate of the water substrate molecules. All these results suggest that the D1-His332 would be more involved in S(3) than in S(2). This could be one element of the conformational changes put forward in the S(2) to S(3) transition.

Remote His50 Acts as a Coordination Switch in the High-Affinity N-Terminal Centered Copper(II) Site of α-Synuclein

Parkinsons disease (PD) etiology is closely linked to the aggregation of α-synuclein (αS). Copper(II) ions can bind to αS and may impact its aggregation propensity. As a consequence, deciphering the exact mode of Cu(II) binding to αS is important in the PD context. Several previous reports have shown some discrepancies in the description of the main Cu(II) site in αS, which are resolved here by a new scenario. Three Cu(II) species can be encountered, depending on the pH and the Cu:αS ratio. At low pH, Cu(II) is bound to the N-terminal part of the protein by the N-terminal amine, the adjacent deprotonated amide group of the Asp2 residue, and the carboxylate group from the side chain of the same Asp2. At pH 7.4, the imidazole group of remote His50 occupies the fourth labile equatorial position of the previous site. At high Cu(II):αS ratio (>1), His50 leaves the coordination sphere of the first Cu site centered at the N-terminus, because a second weak affinity site centered on His50 is now filled with Cu(II). In this new scheme, the remote His plays the role of a molecular switch and it can be anticipated that the binding of the remote His to the Cu(II) ion can induce different folding of the αS protein, having various aggregation propensity.

The Proximal Hydrogen Bond Network Modulates Bacillus subtilis Nitric-oxide Synthase Electronic and Structural Properties

Bacterial nitric-oxide synthase (NOS)-like proteins are believed to be genuine NOSs. As for cytochromes P450 (CYPs), NOS-proximal ligand is a thiolate that exerts a push effect crucial for the process of dioxygen activation. Unlike CYPs, this catalytic electron donation seems controlled by a hydrogen bond (H-bond) interaction between the thiolate ligand and a vicinal tryptophan. Variations of the strength of this H-bond could provide a direct way to tune the stability along with the electronic and structural properties of NOS. We generated five different mutations of bsNOS Trp66, which can modulate this proximal H-bond. We investigated the effects of these mutations on different NOS complexes (FeIII, FeIICO, and FeIINO), using a combination of UV-visible absorption, EPR, FTIR, and resonance Raman spectroscopies. Our results indicate that (i) the proximal H-bond modulation can selectively decrease or increase the electron donating properties of the proximal thiolate, (ii) this modulation controls the σ-competition between distal and proximal ligands, (iii) this H-bond controls the stability of various NOS intermediates, and (iv) a fine tuning of the electron donation by the proximal ligand is required to allow at the same time oxygen activation and to prevent uncoupling reactions.

The proximal H-bond network modulates Bacillus subtilis NO-synthase electronic and structural properties

Bacterial nitric-oxide synthase (NOS)-like proteins are believed to be genuine NOSs. As for cytochromes P450 (CYPs), NOS-proximal ligand is a thiolate that exerts a push effect crucial for the process of dioxygen activation. Unlike CYPs, this catalytic electron donation seems controlled by a hydrogen bond (H-bond) interaction between the thiolate ligand and a vicinal tryptophan. Variations of the strength of this H-bond could provide a direct way to tune the stability along with the electronic and structural properties of NOS. We generated five different mutations of bsNOS Trp66, which can modulate this proximal H-bond. We investigated the effects of these mutations on different NOS complexes (FeIII, FeIICO, and FeIINO), using a combination of UV-visible absorption, EPR, FTIR, and resonance Raman spectroscopies. Our results indicate that (i) the proximal H-bond modulation can selectively decrease or increase the electron donating properties of the proximal thiolate, (ii) this modulation controls the σ-competition between distal and proximal ligands, (iii) this H-bond controls the stability of various NOS intermediates, and (iv) a fine tuning of the electron donation by the proximal ligand is required to allow at the same time oxygen activation and to prevent uncoupling reactions.