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Accueil > Les équipes > Biophysique des métalloprotéines - BIP07 > Membres de l’équipe > Pages personelles > Pierre Dorlet

Pierre Dorlet




Directeur de Recherche CNRS

E-mail : pdorlet "at" imm.cnrs.fr
Phone : +33 4 91 16 46 10





My research interests deal with metalloprotein systems that I study at the molecular level mostly using various Electron Paramagnetic Resonance (EPR) techniques. EPR is a powerful spectroscopy to study paramagnetic species such as transition metal centers or radicals. It is particularly suited in bioinorganic chemistry for the understanding of enzymatic systems using metal ions at their active site. After working for many years in Photosynthesis, dealing with the oxygen evolving complex of Photosystem II as well as related inorganic model complexes for artificial photosynthesis, I have moved toward systems involved in oxidative stress and detoxification. My current main research projects are presented below.

You can find a summary of my CV and my list of publications by following the ORCID ID link above.


NO-SYNTHASES



Nitric Oxide as signaling molecule

In 1998, the Nobel Prize was awarded to three American scientists for their contribution to unveiling the roles of a new biological mediator, Nitric Oxide (NO). Following its discovery in the 1980’s as a signaling molecule in the cardiovascular system, NO became the focus of many research fields and was found to play critical roles in an increasing number of (patho)physiological processes in mammals. NO is a ubiquitous biological messenger involved along two different registers : on one side, NO is involved in key-signaling processes such as the regulation of the vascular tone and of the neuronal influx via activation of soluble guanylate cyclase (sGC). On the other side, NO is a cytotoxic molecule via its chemical reactivity. This latter activity is commonly associated with the development of several major pathologies including cardiovascular, inflammatory and neurodegenerative diseases The ability for NO to exert different biological activities is a consequence of its high reactivity. Indeed, the biological chemistry of NO is extremely complex and leads to the co-existence of several reactive nitrogen and oxygen activated species (ROS/RNOS) that each exhibits a different chemical reactivity. Among the most important reactions, NO can form adducts with the metal centers of metalloproteins, or with dioxygen to form NO2 and N2O3 involved in nitration and nitrosation of thiols and DNA, and with superoxide to form peroxynitrite (ONOO-) involved in nitration, nitrosation and oxidation of virtually all bio-molecules. As a consequence, the balance between beneficial (signaling) and harmful effects of NO is linked to its ability to (in)activate its biological target or to form side-products that will react nonspecifically.

The NO-synthase Family

In mammals, NO is synthesized by a family of enzymes called NO-Synthases (NOSs). All mammalian NOSs (mNOSs) have been isolated and cloned in the early 90’s. Each biological activity of NO is associated with a distinct isoform of NOSs : endothelial NOS (eNOS) is involved in blood pressure regulation, angiogenesis, or anti-adhesion of platelets and neutrophils. Neuronal NOS (nNOS) is involved in learning processes, synaptic plasticity, or in the regulation of the gastro-intestinal tractus. The inducible NOS (iNOS) is expressed in non-specific immune responses against tumors, viruses or bacteria. Although they all synthesize the same molecules and share between 50 and 60% protein sequence homology, the three isoforms have distinct biological functions due to their specific cellular and subcellular localization in brain, cardiovascular or immune systems.
With the development of genome sequencing, NOSs have been identified in hundreds of organisms throughout the living kingdoms. This has raised the questions on the exact role of NOS proteins. Indeed, the knowledge on NOSs mostly comes from the study of the canonical proteins from mammals, the molecular mechanism of which has not been fully determined yet and is still under debate. In addition to that, a few bacterial NOSs have also been studied but their exact role remains largely unknown. Significant differences in the catalytic mechanism for the second step have also been revealed between bacterial and mammalian NOSs. Recently, despite the total absence of NOS-like proteins in the genome of higher plants, full length NOSs have been discovered in green microalgae. The first NOS from the plant kingdom was discovered in Ostreococcus tauri and characterized by spectroscopy. This has triggered renewed interest in the NOS family whose roles now extend further than what was characterized in the mammalian enzymes. A new NOS from cyanobacteria Synechococcus PCC 7335, exhibiting an additional globin domain, has just been characterized.
NOS enzymes are multidomain proteins that are functional as dimers. The domain composition of NOS varies depending on phyla (Figure 1).

Figure 1. Domain organization of NO-synthases. NOSoxy : oxygenase domain (contains the heme and the biopterin cofactor), Zn : zinc binding site, Cam : calmodulin binding domain, NOSred : reductase domain (contains FMN, FAD and NAD sites), Globin : additional heme domain.

The heme oxygenase domain (NOSox) at the N-terminal extremity contains the active site that catalyses the oxidation of L-arginine (L-Arg) in L-citrulline and releases nitric oxide (NO) (Figure 2). The main features of this highly complex reaction have been unraveled in the last 30 years. The structure of the heme oxygenase domain has been solved and shows some similarities with cytochrome P450, albeit many differences exist. The active site contains the two cofactors heme and H4B that are in close vicinity (see Figure 2). The H4B cofactor has long been supposed to play a structural role by stabilizing the dimer, but also plays a direct redox role during catalysis. In the first step the Nw-nitrogen of L-Arg guanidinium group is hydroxylated leading to the formation of NOHA. The second step corresponds to the oxidation of the hydroxylguanidinium group into the corresponding urea (L-citrulline). The overall reaction consumes three exogenous electrons (1.5 NADPH molecules) and two O2 molecules and liberates two water molecules and one NO molecule. The catalytic sites of the heme oxygenase domain, where the formation of NO occurs, have very similar amino acid composition in the three main isoforms, a selection pressure likely explained by the same reaction catalysed by the three isoforms.

Figure 2 : Top : Reactions catalyzed by NOSs. The two-step reaction catalysed by nitric oxide synthases. In the first step L-Arg is hydroxylated to form Nw-hydroxy L-Arg (NOHA). In the second step, NOHA is converted to citrulline and nitric oxide (NO). Bottom : Structure of the active site in the oxygenase domain (pdb code 2G6M, Rat nNOS).

The reductase domain (NOSred) (FMN and FAD domains, analogue to other diflavin enzymes such as CPR, cytochrome P450 reductase) is responsible for the supply of heme with electrons following the flow : NADPH->FAD->FMN->heme suggesting the FMN domain swings between FAD domain and oxygenase during catalysis. The electron transfer from FMN to heme is known to be the rate-limiting step of the reaction. Because of the many steps, side reactions may exist under suboptimal conditions and can lead to several uncoupling reactions and the release of superoxide, hydrogen peroxide and other reactive nitrogen or oxygen species. Notably the electron transfer occurs in trans, from the FMN group of monomer A to the heme group in the monomer B of the dimeric NOS. Thus the inactivity of monomeric NOS can be explained by the impossibility of FMN to give its electrons to the heme group from the same monomer, possibly for steric reasons. In contrast to the interface of the heme oxygenase dimer which is highly hydrophobic, the FMN/FAD interdomain interface is dominated by electrostatic interactions. In addition, the structure of the heme oxygenase displays a large positively charged surface which likely facilitate interactions with the negatively charged FMN domain during catalysis.

NO-Synthase and Diatoms

A recent publication has shown the presence of NOSs in diatoms (Bacillariophyceae) that are one of the most diverse and important group of phytoplankton. They are central to aquatic environments and account for about 20% of global productivity. Beside their ecological role, they are attractive models for biotechnology since they have a silicified cell wall that is used as a model for nanotechnology. They also produce diverse molecules of biotechnological interest such as pigments or lipids that can be used as biofuels. Diatom genomes contain a variety of genes acquired from different sources during their evolutionary history. As a result, their metabolic properties are very different from those of the much better studied green algae and land plants. NO• has been estimated at nM range in seawaters and shown to protect marine phytoplankton against abiotic stresses. An ever growing list of genome sequences available allowed us to identify NOS-encoding genes in many diatoms. Interestingly, the phylogenetic analysis of the resulting NOS sequences leads to an updated tree (Figure 3) that encompasses the different groups of NOSs characterized so far. It divides into two main clades, one closer to the mammalian, bacterial and plant NOSs and the other one closer to the cyanobacterial proteins. However, NOS genes are not present in all diatoms while all diatoms can produce NO• via reductive processes (i.e. nitrite reductase).

Figure 3. Maximum likelihood phylogenetic tree of diatom NOSs aligned with a series of NOSs representative of bacteria (brown), mammals (red), microalgae (green) and cyanobacteria (blue) phyla. The full length enzyme sequences were used to build this tree. However, a similar tree is obtained by using the oxygenase domain only sequences.

In collaboration with Dr. Brigitte Gontero-Meunier and the team BIP02, we have chosen two model organisms (one of each clade) to study the NOS family in Diatoms. This project will help elucidate the evolution of this family of proteins. It will also improve the understanding of the role of NO• signalling in the physiology and ecology of this important group of algae and more generally, be relevant for the evolution of NO• signalling.


A NOVEL PERIPLASMIC PROTEIN INVOLVED IN COPPER RESISTANCE


The team of Dr. Soufian Ouchane (Department of Microbiology, I2BC, Gif-sur-Yvette) has discovered a new protein involved in copper resistance in the purple photosynthetic bacterium Rubrivivax gelatinosus. This bacterium can grow in a medium containing up to 1.2 mM in copper ions. Under these conditions, it naturally overexpresses a protein, CopI, located in the periplasm. I have been collaborating with Anne Durand, in charge of the CopI project at I2BC, for a few years now in order to gain insights into this enzyme by using biophysical methods. EPR results show that there are two Cu(II) sites per protein (Figure 4), one of which being a green type cupredoxin. We are currently characterizing both sites to determine their ligands, structural and electronic properties. Since the function of this new protein is unknown, we are also trying to determine possible catalysis.

Figure 4. EPR spectrum of a frozen solution of purified CopI protein complemented in Cu(II) (bottom). The two top spectra show the deconvolution into both components, a green cupredoxin type signal (top) and a square planar site (middle) that we suppose located in the N terminal His rich portion of the protein. Grey lines represent simulated spectra.