PCV - Programme interdiciplinaire en physique et chimie du vivant

Metalloproteins involved in oxidative stress defence studied by Far-IR Terahertz spectroscopy and theoretical methods – ProMeTHz

Submission summary

Metals play fundamental role in biology and they are involves in the catalysis of a large variety of reactions in more than one third of the proteins. In metalloenzymes, metal-site reactivity results from a fine balance between the chemical nature and spatial distribution of the ligands. It is the whole protein which tunes the strength of the metal-ligand bonds. It is therefore of prime interest to study into the details the parameters that govern the reactivity of metal active sites in proteins. Vibrational spectroscopy is particularly well suited to give essential information on metal-ligand bond properties. Up to now, only resonance Raman spectroscopy has been widely used. It is however strictly limited to analyze metal sites presenting absorption transitions in the UV-Vis domain. Infrared difference spectroscopy, which allows to directly investigating vibrational modes from metal sites with high sensitivity, is very promising. In contrast to resonance Raman spectroscopy, no absorption in the UV-Vis is required, and all metal sites and redox states are in principle accessible by this technique. However, up to now, difference spectroscopy in the Far-IR to THz domain, where metal-ligand bond are expected to contribute, has almost not be used to analyse metalloproteins. This is mainly due to limitations concerning the setups and the interpretation of the data. Presently, we are in position to overcome these difficulties. The aim of this project is to bring together an interdisciplinary consortium of biochemists, physicists, and theoretical chemists, to develop Far-IR to THz difference spectroscopy, for the analysis of metal-ligand bond strengths in metalloproteins. This unprecedented development implies the realization of new experimental set-ups. Novel measuring cells will be conceived for at-home spectrometers and adapted to work with the highly brilliant Far-IR to THz beam line AILES of the SOLEIL Synchrotron. These set-ups will allow working with purged optics, giving the advantage of high sensitivity and spectral resolution, which is necessary for the precise analysis of metal-ligand vibrations in proteins. This project also involves systematic combination of experimental and modelling strategies. Theoretical calculations will involve the analysis of small size model clusters, using methods considering electrons explicitly (DFT and post HF) and mixed methods based on QM (for the active metal site) and MM (for the protein environment), to model the whole protein. This modelling approach, confronted with the experimental data, will allow normal mode prediction for the metal-ligand vibrations. It will also define the role of structural motifs at distance from the metal, on the strength of these bonds. The method based on DFT has been recently validated by one of the teams of this project on Cu-histamine complexes. These developments as a whole will be validated on the well-studied Cu-metalloprotein, azurin. The new approach for far-IR difference spectroscopy will be used to study two metalloproteins with crucial role in cell protection against oxidative stress, and notably for the elimination of the highly toxic superoxide radical species. Detoxification of reactive oxygen species, including superoxide, is essential to the survival of the organisms during aerobic life. The dysfunction or the overcome of these antioxidant systems can lead to heavy pathologies in humans, cellular degeneration, and have notable contribution in cell ageing. The far-IR developments will be applied to study two antioxidant enzymes: superoxide reductase (SOR), which has been recently unravelled by one of the teams involved in this project, and superoxide dismutase (SOD), for which mutations have been associated to human pathologies including Familial Amyotrophic Lateral Sclerosis. For SOD, the far-IR studies will allow to establish a correlation between the strength of the Cu histidine bonds and the interactions formed by Cu with exogenous ligands, and thus to evaluate the affinity of Cu for superoxide. For SOR, the Far-IR approach will allow us to determine the prime role of the protein environment of the iron active site ligands on the reactivity towards superoxide. This project will allow us to develop a spectral domain, still almost unexploited, for the analysis of metalloproteins, and to demonstrate its significance, by the identification of structural properties essential to understand the reactivity of two enzymes involved in cell protection against oxidative stress.

Project coordination

Catherine BERTHOMIEU (COMMISSARIAT A L'ENERGIE ATOMIQUE (C.E.A.) - CENTRE D'ETUDE NUCLEAIRE DE CADARACHE)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

COMMISSARIAT A L'ENERGIE ATOMIQUE (C.E.A.) - CENTRE D'ETUDE NUCLEAIRE DE CADARACHE
SYNCHROTRON SOLEIL

Help of the ANR 500,000 euros
Beginning and duration of the scientific project: - 36 Months

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