Biomedical subjects

Christophe Léger

Publications and source records attributed to Christophe Léger.

12 recordsLinked to original sources

PubMed2006

A needle in a haystack: the active site of the membrane-bound complex cytochrome c nitrite reductase.

Cytochrome c nitrite reductase is a multicenter enzyme that uses a five-coordinated heme to perform the six-electron reduction of nitrite to ammonium. In the sulfate reducing bacterium Desulfovibrio desulfuricans ATCC 27774, the enzyme is purified as a NrfA2NrfH complex that houses 14 hemes. The number of closely-spaced hemes in this enzyme and the magnetic interactions between them make it very difficult to study the active site by using traditional spectroscopic approaches such as EPR or UV-Vis. Here, we use both catalytic and non-catalytic protein film voltammetry to simply and unambiguously determine the reduction potential of the catalytic heme over a wide range of pH and we demonstrate that proton transfer is coupled to electron transfer at the active site.

Bacterial Proteins↗

PubMed2006

Regional analysis of the left ventricle of the heart.

This paper presents a method to analyze the local wall motion of the left ventricle of the heart. Data are sets of points (obtained from various medical imaging modalities) corresponding to surfaces of the left ventricle, which evolve as a function of time. After re-sampling, the surfaces are segmented in order to create regions of equivalent volume. Then, the local cardiac parameters are estimated: evolution of the regional volumes as a function of time, ejection fraction, end-diastolic and end-systolic volumes, end-diastolic and end-systolic instants. The method has been validated using deformable surfaces synthesized from an ellipsoidal model. It has also been tested in vivo on a set of 59 patients using a specially developed software product, which satisfies severe constraints of robustness, real-time, interactivity and ergonomics. The results obtained are similar to those provided by a reference nuclear medicine examination, but the proposed method is faster and gives a more precise localization of the cardiac wall motion anomalies.

Algorithms↗

PubMed2006

Changing the ligation of the distal [4Fe4S] cluster in NiFe hydrogenase impairs inter- and intramolecular electron transfers.

In NiFe hydrogenases, electrons are transferred from the active site to the redox partner via a chain of three Iron-Sulfur clusters, and the surface-exposed [4Fe4S] cluster has an unusual His(Cys)3 ligation. When this Histidine (H184 in Desulfovibrio fructosovorans) is changed into a cysteine or a glycine, a distal cubane is still assembled but the oxidative activity of the mutants is only 1.5 and 3% of that of the WT, respectively. We compared the activities of the WT and engineered enzymes for H2 oxidation, H+ reduction and H/D exchange, under various conditions: (i) either with the enzyme directly adsorbed onto an electrode or using soluble redox partners, and (ii) in the presence of exogenous ligands whose binding to the exposed Fe of H184G was expected to modulate the properties of the distal cluster. Protein film voltammetry proved particularly useful to unravel the effects of the mutations on inter and intramolecular electron transfer (ET). We demonstrate that changing the coordination of the distal cluster has no effect on cluster assembly, protein stability, active-site chemistry and proton transfer; however, it slows down the first-order rates of ET to and from the cluster. All-sulfur coordination is actually detrimental to ET, and intramolecular (uphill) ET is rate determining in the glycine variant. This demonstrates that although [4Fe4S] clusters are robust chemical constructs, the direct protein ligands play an essential role in imparting their ability to transfer electrons.

Desulfovibrio↗

PubMed2006

Electron flow in multicenter enzymes: theory, applications, and consequences on the natural design of redox chains.

In protein film voltammetry, a redox enzyme is directly connected to an electrode; in the presence of substrate and when the driving force provided by the electrode is appropriate, a current flow reveals the steady-state turnover. We show that, in the case of a multicenter enzyme, this signal reports on the energetics and kinetics of electron transfer (ET) along the redox chain that wires the active site to the electrode, and this provides a new strategy for studying intramolecular ET. We propose a model which takes into account all the enzyme's redox microstates, and we prove it useful to interpret data for various enzymes. Several general ideas emerge from this analysis. Considering the reversibility of ET is a requirement: the usual picture, where ET is depicted as a series of irreversible steps, is oversimplified and lacks the important features that we emphasize. We give justification to the concept of apparent reduction potential on the time scale of turnover and we explain how the value of this potential relates to the thermodynamic and kinetic properties of the system. When intramolecular ET does not limit turnover, the redox chain merely mediates the driving force provided by the electrode or the soluble redox partner, whereas when intramolecular ET is slow, the enzyme behaves as if its active active site had apparent redox properties which depend on the reduction potentials of the relays. This suggests an alternative to the idea that redox chains are optimized in terms of speed: evolutionary pressure may have resulted in slowing down intramolecular ET in order to tune the enzyme's "operating potential".

Animals↗

PubMed2004

Inhibition and aerobic inactivation kinetics of Desulfovibrio fructosovorans NiFe hydrogenase studied by protein film voltammetry.

We have used protein film voltammetry to study the NiFe hydrogenase from Desulfovibrio fructosovorans. We show how measurements of transient activity following the addition in the electrochemical cell of H(2), CO, or O(2) allow simple and virtually instantaneous determinations of the Michaelis constant, inhibition constant, or rate of inactivation, respectively, thus opening new opportunities to study the active site of NiFe hydrogenases. The binding and release of CO occur within a fraction of a second, and we determine and discuss how its affinity for the active site changes as the driving force for the H(+)/H(2) reaction is continuously varied. Inactivation by O(2) is a slow, bimolecular process (with pH-independent rate constant approximately 3 x 10(4) s(-1) M(-1) at 40 degrees C, under one atm of H(2)) that leads to a mixture of fully oxidized states, and unlike the case of CO inhibition, the active site is not fully protected by H(2). This experimental approach could be used to study the reaction of other multicentered metalloenzymes with their gaseous substrates or inhibitors.

Aerobiosis↗

PubMed2004

In Rhodobacter sphaeroides respiratory nitrate reductase, the kinetics of substrate binding favors intramolecular electron transfer.

The respiratory nitrate reductase (NapAB) from Rb. sphaeroides is a periplasmic molybdenum-containing enzyme which belongs to the DMSO reductase family. We report a study of NapAB by protein film voltammetry (PFV), and we present the first quantitative interpretation of the complex redox-state dependence of activity that has also been observed with other related enzymes. The model we use to fit the data assumes that binding of substrate partly limits turnover and is faster and weaker when the Mo ion is in the V oxidation state than when it is fully reduced. We explain how the presence in the catalytic cycle of such slow chemical steps coupled to electron transfer to the active site decreases the driving force required to reduce the MoV ion and makes exergonic the last intramolecular electron-transfer step (between the proximal cubane and the Mo cofactor). Importantly, comparison is made with all Mo enzymes for which PFV data are available, and we emphasize general features of the energetics of the catalytic cycles in enzymes of the DMSO reductase family.

Electrochemistry↗

PubMed2004

Three-dimensional analysis of left ventricle regional wall motion by using gated blood pool tomography.

BACKGROUND: We have elaborated a new software called ACS designed for left ventricle 3-dimensional reconstruction and quantitative analysis of regional wall motion using gated blood-pool tomography (GBPS). This paper presents the principle and the validation of this software. METHODS AND RESULTS: Blood pool tomography was reconstructed by filtered back-projection. Voxels corresponding to ventricular volumes were extracted using 3-D thresholding and 3-D elliptic mask. The auriculoventricular plane was located by phase analysis. Then, the reconstruction of the 3-D volume was initiated. First, a pre-processing based on a bi-dimensional Fourier transform re-sampled the initial points representing the wall of the left ventricle. Left ventricular contraction could be visualized in three dimensions. The points corresponding to the wall of the left ventricle were reorganized in order to obtain a regular sampling that allowed connection to the points during the cardiac cycle. Elementary volumes were defined by three adjacent wall points and the centre of the whole volume. The variation of elementary volumes during the cardiac cycle was related to the wall motion. Elementary volumes were regrouped to create regions of equivalent volume. Finally, regional and global ejection fractions could be calculated in 17 different regions. The method has been validated using a mathematical deformable ellipsoidal model. It had also been tested in vivo on a set of 59 patients in comparison to equilibrium radionuclide angiography for left ventricular ejection fraction and to echocardiography for regional wall motion evaluation. CONCLUSIONS: ACS is a new software that allows reconstruction and visualization of the left ventricle in three dimensions, and it quantified 3-dimensional regional wall motion analysis.

Algorithms↗

PubMed2004

Time-varying, 3-D echocardiography using a fast-rotating probe.

A fast continuous rotating ultrasound scan-head transducer was used to perform three-dimensional (3-D) echocardiography with 2-D images acquired during a single cardiac cycle. The 3-D images were reconstructed by interpolating 2-D data acquired with the probe. Two experiments were carried out to validate the image reconstructions. A dynamic cardiac phantom was used as a known reference to compare the minimal and maximal volumes estimated manually on the reconstructed 3-D images. The left ventricle (LV) volume of 30 healthy volunteers also were estimated using a semiautomatic ellipse approach and compared to measurements obtained with standard 2-D examination. Results showed a good agreement between 3-D and reference measurements.

Algorithms↗

PubMed2003

Enzyme electrokinetics: using protein film voltammetry to investigate redox enzymes and their mechanisms.

Protein film voltammetry is a relatively new approach to studying redox enzymes, the concept being that a sample of a redox protein is configured as a film on an electrode and probed by a variety of electrochemical techniques. The enzyme molecules are bound at the electrode surface in such a way that there is fast electron transfer and complete retention of the chemistry of the active site that is observed in more conventional experiments. Modulations of the electrode potential or catalytic turnover result in the movement of electrons to, from, and within the enzyme; this is detected as a current that varies in characteristic ways with time and potential. Henceforth, the potential dimension is introduced into enzyme kinetics. The presence of additional intrinsic redox centers for providing fast intramolecular electron transfer between a buried active site and the protein surface is an important factor. Centers which carry out cooperative two-electron transfer, most obviously flavins, produce a particularly sharp signal that allows them to be observed, even as transient states, when spectroscopic methods are not useful. High catalytic activity produces a large amplification of the current, and useful information can be obtained even if the coverage on the electrode is low. Certain enzymes display optimum activity at a particular potential, and this can be both mechanistically informative and physiologically relevant. This paper outlines the principles of protein film voltammetry by discussing some recent results from this laboratory.

Binding Sites↗

PubMed2003

Architecture of succinate dehydrogenase and reactive oxygen species generation.

The structure of Escherichia coli succinate dehydrogenase (SQR), analogous to the mitochondrial respiratory complex II, has been determined, revealing the electron transport pathway from the electron donor, succinate, to the terminal electron acceptor, ubiquinone. It was found that the SQR redox centers are arranged in a manner that aids the prevention of reactive oxygen species (ROS) formation at the flavin adenine dinucleotide. This is likely to be the main reason SQR is expressed during aerobic respiration rather than the related enzyme fumarate reductase, which produces high levels of ROS. Furthermore, symptoms of genetic disorders associated with mitochondrial SQR mutations may be a result of ROS formation resulting from impaired electron transport in the enzyme.

Aerobiosis↗

PubMed2002

Enzyme electrokinetics: hydrogen evolution and oxidation by Allochromatium vinosum [NiFe]-hydrogenase.

The mechanism of catalytic hydrogen evolution and oxidation by Allochromatium vinosum [NiFe]-hydrogenase has been studied by protein film voltammetry (PFV) with the enzyme adsorbed at a pyrolytic graphite edge electrode. By analyzing the entire shapes of catalytic voltammograms, the energetics of the catalytic cycles (reduction potentials and acidity constants of the active states), including the detailed profiles of activity against pH and the sequences of proton and electron transfers, have been determined, and these are discussed with respect to the mechanism. PFV, which probes rates as a continuous function of the electrochemical potential (i.e., in the "potential domain"), is proven to be an invaluable tool for determining the redox properties of an active site in the presence of its substrate, at room temperature, and during turnover. This is especially relevant in the case of the active states of hydrogenase, since one of its substrates (the proton) is always present at significant levels in the titration medium at physiological pH values.

Binding Sites↗

PubMed2002

Detection and interpretation of redox potential optima in the catalytic activity of enzymes.

It is no surprise that the catalytic activity of electron-transport enzymes may be optimised at certain electrochemical potentials in ways that are analogous to observations of pH-rate optima. This property is observed clearly in experiments in which an enzyme is adsorbed on an electrode surface which can supply or receive electrons rapidly and in a highly controlled manner. In such a way, the rate of catalysis can be measured accurately as a function of the potential (driving force) that is applied. In this paper, we draw attention to a few examples in which this property has been observed in enzymes that are associated with membrane-bound respiratory chains, and we discuss its possible origins and implications for in vivo regulation.

Animals↗