Functional study of the [Ni-Fe] hydrogenase (approach by EPR spectroscopy and photochemistry)

Les hydrogénases sont les métalloenzymes, issues de micro-organismes anaérobies, qui catalysent l'oxydation réversible de l'hydrogène moléculaire, suivant la réaction : H2 2H+ +2e-. Notre objectif est de progresser dans la compréhension du mécanisme réactionnel de ces enzymes, à savoir : le processus d'activation de H2 au niveau du centre catalytique à Ni-Fe et les voies de transfert des électrons et des protons issus de l'oxydation de H2. Nous utilisons une approche combinant la spectroscopie RPE et la photochimie pour étudier à températures cryogéniques, les propriétés de photosensibilité d'un intermédiaire clé du mécanisme catalytique, l'état Ni-C. Nous étudions en détail le profil énergétique de la photoconversion, la cinétique des processus de recombinaison, et l'influence de différents facteurs comme la température, les effets isotopiques H/D et la fixation d'inhibiteur (CO), dans le cas de plusieurs hydrogénases à [Ni-Fe-] et à [Ni-Fe-Se] de bactéries sulfato-réductrices ou d'organismes hyperthermophiles (A. aeolicus). L'analyse des interactions magnétiques entre le centre actif à Ni-Fe et le centre [4Fe-4S]1+ proximal par la simulation numérique des spectres RPE multifréquences est également utilisée pour sonder les changements structuraux induits par l'irradiation. Nos résultats mettent en évidence un transfert de protons photo-induits au niveau des cystéines terminales de l'ion nickel, et permettent d'identifier le résidu Glutamate impliqué dans la première étape de transfert de protons, comme le confirment nos études de mutagénèse dirigée vers ce résidu.AIX-MARSEILLE1-BU Sci.St Charles (130552104) / SudocSudocFranceF

Electron paramagnetic resonance studies of molybdenum enzymes

International audienceElectron Paramagnetic Resonance (EPR) is certainly the first and the most widely used spectroscopic technique for studying structure and function of Mo and W enzymes. Although only Mo(v) and W(v) states can be detected, a considerable wealth of data was provided since the seminal EPR works performed on xanthine oxidase and nitrate reductase more than 55 years ago. In this chapter, we give a comprehensive overview of the various applications of EPR on the ubiquitous Mo-enzymes, which exhibit such an extraordinary diversity of substrates and catalyzed reactions. Elucidating the nature of Mo(v) intermediates is a considerable challenge to progress in understanding these processes. The g-tensor analyses are helpful in that aim. But it is essentially thanks to the advances in pulsed EPR methods like ENDOR, ESEEM and HYSCORE, combined with efficient isotopic enrichment strategies, that the measurements of hyperfine couplings of Mo-cofactor with neighbouring magnetic nuclei have brought the most interesting data. Thus, we illustrate how the analysis of hyperfine parameters associated with computational chemistry methods is becoming a powerful way to provide high-resolution structural data on Mo(v) species and enzyme mechanisms. In addition, EPR study of spin–spin couplings between Mo-cofactor and other paramagnetic centres appears as a promising way to gain long-range structural data in these systems

Mechanism of inhibition of NiFe hydrogenase by nitric oxide

International audienceHydrogenases reversibly catalyze the oxidation of molecular hydrogen and are inhibited by several small molecules including O2, CO and NO. In the present work, we investigate the mechanism of inhibition by NO of the oxygen-sensitive NiFe hydrogenase from Desulfovibrio fructosovorans by coupling site-directed mutagenesis, protein film voltammetry (PFV) and EPR spectroscopy. We show that micromolar NO strongly inhibits NiFe hydrogenase and that the mechanism of inhibition is complex, with NO targeting several metallic sites in the protein. NO reacts readily at the NiFe active site according to a two-step mechanism. The first and faster step is the reversible binding of NO to the active site followed by a slower and irreversible transformation at the active site. NO also induces irreversible damage of the iron–sulfur centers chain. We give direct evidence of preferential nitrosylation of the medial [3Fe–4S] to form dinitrosyl–iron complexes

The Megavirus Chilensis Cu,Zn-Superoxide Dismutase: the First Viral Structure of a Typical Cellular Copper Chaperone-Independent Hyperstable Dimeric Enzyme

International audienceUnlabelled: Giant viruses able to replicate in Acanthamoeba castellanii penetrate their host through phagocytosis. After capsid opening, a fusion between the internal membranes of the virion and the phagocytic vacuole triggers the transfer in the cytoplasm of the viral DNA together with the DNA repair enzymes and the transcription machinery present in the particles. In addition, the proteome analysis of purified mimivirus virions revealed the presence of many enzymes meant to resist oxidative stress and conserved in the Mimiviridae. Megavirus chilensis encodes a predicted copper, zinc superoxide dismutase (Cu,Zn-SOD), an enzyme known to detoxify reactive oxygen species released in the course of host defense reactions. While it was thought that the metal ions are required for the formation of the active-site lid and dimer stabilization, megavirus chilensis SOD forms a very stable metal-free dimer. We used electron paramagnetic resonance (EPR) analysis and activity measurements to show that the supplementation of the bacterial culture with copper and zinc during the recombinant expression of Mg277 is sufficient to restore a fully active holoenzyme. These results demonstrate that the viral enzyme's activation is independent of a chaperone both for disulfide bridge formation and for copper incorporation and suggest that its assembly may not be as regulated as that of its cellular counterparts. A SOD protein is encoded by a variety of DNA viruses but is absent from mimivirus. As in poxviruses, the enzyme might be dispensable when the virus infects Acanthamoeba cells but may allow megavirus chilensis to infect a broad range of eukaryotic hosts. Importance: Mimiviridae are giant viruses encoding more than 1,000 proteins. The virion particles are loaded with proteins used by the virus to resist the vacuole's oxidative stress. The megavirus chilensis virion contains a predicted copper, zinc superoxide dismutase (Cu,Zn-SOD). The corresponding gene is present in some megavirus chilensis relatives but is absent from mimivirus. This first crystallographic structure of a viral Cu,Zn-SOD highlights the features that it has in common with and its differences from cellular SODs. It corresponds to a very stable dimer of the apo form of the enzyme. We demonstrate that upon supplementation of the growth medium with Cu and Zn, the recombinant protein is fully active, suggesting that the virus's SOD activation is independent of a copper chaperone for SOD generally used by eukaryotic SODs

Kinetics of substrate inhibition of periplasmic nitrate reductase

International audiencePeriplasmic nitrate reductase catalyzes the reduction of nitrate into nitrite using a mononuclear molybdenum cofactor that has nearly the same structure in all enzymes of the DMSO reductase family. In previous electrochemical investigations, we found that the enzyme exists in several inactive states, some of which may have been previously isolated and mistaken for catalytic intermediates. In particular, the enzyme slowly and reversibly inactivates when exposed to high concentrations of nitrate. Here, we study the kinetics of substrate inhibition and its dependence on electrode potential and substrate concentration to learn about the properties of the active and inactive forms of the enzyme. We conclude that the substrate-inhibited enzyme never significantly accumulates in the EPR-active Mo(+ V) state. This conclusion is relevant to spectroscopic investigations where attempts are made to trap a Mo(+ V) catalytic intermediate using high concentrations of nitrate

Is engineering O2 tolerant hydrogenase just a matter of reproducing the active site of O2-resistant enzymes ?

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Dependence of catalytic activity on driving force in solution assays and protein film voltammetry: insights from the comparison of nitrate reductase mutants.

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Is engineering O2-tolerant hydrogenases just a matter of reproducing the active sites of the naturally occurring O2-resistant enzymes?

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Evidence for [2Fe-2S] 2+ and Linear [3Fe-4S] 1+ Clusters in a Unique Family of Glycine/Cysteine-Rich Fe-S Proteins from Megavirinae Giant Viruses

International audienceWe have discovered a protein with an amino acid composition exceptionally rich in glycine and cysteine residues in the giant virus mimivirus. This small 6 kDa protein is among the most abundant proteins in the icosahedral 0.75 μm viral particles; it has no predicted function but is probably essential for infection. The aerobically purified red-brownish protein overproduced inEscherichia coli contained both iron and inorganic sulfide. UV/vis, EPR, and Mössbauer studies revealed that the viral protein, coined GciS, accommodated two distinct Fe-S clusters: a diamagnetic S = 0 [2Fe-2S]2+ cluster and a paramagnetic S = 5/2 linear [3Fe-4S]1+ cluster, a geometry rarely stabilized in native proteins. Orthologs of mimivirus GciS were identified within all clades of Megavirinae, a Mimiviridae subfamily infecting Acanthamoeba, including the distantly related tupanviruses, and displayed the same spectroscopic features. Thus, these glycine/cysteine-rich proteins form a new family of viral Fe-S proteins sharing unique Fe-S cluster binding properties