Manon Couture

• Université Laval

To target development of bioelectrochemical systems, we developed an advanced microfluidic method to identify reaction bottlenecks in the metabolic activity of a pure‐culture Geobacter sulfurreducens electroactive biofilm (EAB). The microfluidic system was devised to include perpendicular flow orientation for improved boundary layer uniformity and...

To target development of bioelectrochemical systems, we developed an advanced microfluidic method to identify reaction bottlenecks in the metabolic activity of a pure-culture Geobacter sulfurreducens electroactive biofilm (EAB). The microfluidic system was devised to include perpendicular flow orientation for improved boundary layer uniformity and...

Inclusion of edible insects in human diets is increasingly promoted as a sustainable source of proteins with high nutritional value. While consumer acceptability remains the main challenge to their integration into Western food culture, the use of edible insects as meal and protein concentrate could decrease neophobia. The defatting of edible insec...

Mammalian acetylcholinesterase (AChE) is well studied, being important in both cholinergic brain synapses and the peripheral nervous systems and also a key drug target for many diseases. In contrast, little is known about the structures and molecular mechanism of prokaryotic acetylcholinesterases. We report here the structural and biochemical chara...

Group A flavin-dependent monooxygenases catalyze the cleavage of the oxygen-oxygen bond of dioxygen, followed by the incorporation of one oxygen atom into the substrate molecule with the aid of NADPH and FAD. These flavoenzymes play an important role in many biological processes, and their most distinct structural feature is the choreographed motio...

A common kinetic framework for studies of whole‐cell catalysis is vital for understanding and optimizing bioflow reactors. In this work, we demonstrate the applicability of a flow‐adapted version of Michaelis‐Menten kinetics to an electrocatalytic bacterial biofilm. A three‐electrode microfluidic biofilm flow reactor measured increased turnover rat...

A common kinetic framework for studies of whole-cell catalysis is vital for understanding and optimizing bioflow reactors. In this work, we demonstrate the applicability of a flow-adapted version of Michaelis-Menten kinetics to a catalytic bacterial biofilm. A three-electrode microfluidic electrochemical flow cell measured increased turnover rates...

Flavohemoglobins are microbial enzymes that counter nitrosative stress, but the details of their underlying enzymatic activities and structure-function relationships are not completely understood. These enzymes have been identified in Gram-negative bacteria, certain fungi, and the parasitic protist Giardia intestinalis (gFlHb) which, despite lackin...

The activation of the peroxynitrite anion (PN) by hemoproteins, which leads to its detoxification or, on the contrary to the enhancement of its cytotoxic activity, is a reaction of physiological importance that is still poorly understood. It has been known for some years that the reaction of hemoproteins, notably cytochrome P450, with PN leads to t...

Substrate salvage or recycling is common and important for primary metabolism in cells but is rare in secondary metabolism. Herein we report flavoenzyme CrmK-mediated shunt product recycling in the biosynthesis of caerulomycin A (CRM A 1), a 2,2′-bipyridine-containing natural product that is under development as a potent novel immunosuppressive age...

Nitric Oxide (NO) and the other reactive nitrogen species (RNOS) play crucial patho-physiological roles at the interface of oxidative stress and signaling processes. In mammals, the NO synthases (NOSs) are the source of these reactive nitrogen species, and so to understand the precise biological role of RNOS and NO requires elucidation of the molec...

As part of the machinery to acquire, internalize and utilize heme as a source of iron from the host, some bacteria possess a canonical heme oxygenase, where heme plays the dual role of substrate and cofactor, the later catalyzing the cleavage of the heme moiety using O2 and electrons, and resulting in biliverdin, carbon monoxide and ferrous non-hem...

Roseobacter denitrificans is a member of the widespread marine Roseobacter genus. We report the first characterization of a truncated hemoglobin from R. denitrificans (Rd. trHb) that was purified in the heme-bound form from heterologous expression of the protein in Escherichia coli. Rd. trHb exhibits predominantly alpha-helical secondary structure...

The protozoan intestinal parasite Giardia lamblia lacks mitochondria and the ability to make haem yet encodes several putative haem-binding proteins, including three of the cytochrome b(5) family. We cloned one of these (gCYTb5-I) and expressed it within Escherichia coli as a soluble holoprotein. UV-visible and resonance Raman spectra of gCYTb5-I r...

Residues surrounding and interacting with the heme proximal ligand are important for efficient catalysis by heme proteins. The nitric oxide synthases (NOSs) are thiolate-coordinated enzymes that catalyze the hydroxylation of l-Arg in the first of the two catalytic cycles needed to synthesize nitric oxide. In NOSs, the indole NH group of a conserved...

HPr is a central protein of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). In streptococci, HPr can be phosphorylated at His(15) at the expense of PEP by enzyme I (EI) of the PTS, producing HPr(His approximately P). HPr can also be phosphorylated at Ser(46) by the ATP-dependent HPr(Ser) kinase/phosphorylase (HprK/P), produ...

The proximal ligand of thiolate-coordinated heme proteins is crucial for the activation of the oxygen molecule and hydroxylation of substrates. In nitric oxide synthases (NOSs), the heme axial cysteine ligand forms a hydrogen bond to the side chain indole nitrogen of a tryptophan residue. Resonance Raman spectroscopy was used to probe W56F and W56Y...

For many pathogenic microorganisms, iron acquisition from host heme sources stimulates growth, multiplication, ultimately enabling successful survival and colonization. In gram-negative Escherichia coli O157:H7, Shigella dysenteriae and Yersinia enterocolitica the genes encoded within the heme utilization operon enable the effective uptake and util...

During the initial growth infection stage of Mycobacterium tuberculosis (Mtb), (*)NO produced by host macrophages inhibits heme-containing terminal cytochrome oxidases, inactivates iron/sulfur proteins, and promotes entry into latency. Here we evaluate the potential of (*)NO as an inhibitor of Mtb cytochrome P450 enzymes, as represented by CYP130,...

The crystal structure of the cyano-met form of Mt-trHbO revealed two unusual distal residues Y(CD1) and W(G8) forming a hydrogen-bond network with the heme-bound ligand [Milani, M., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 5766-5771]. W(G8) is an invariant residue in group II and group III trHbs and has no counterpart in other globins. A pr...

We report the characterization by resonance Raman spectroscopy of the oxygenated complex (FeIIO2) of nitric-oxide synthases of Staphylococcus aureus (saNOS) and Bacillus subtilis (bsNOS) saturated with Nω-hydroxy-l-arginine. The frequencies of the νFe–O and νO–O modes were 530 and 1135 cm–, respectively, in both the presence and absence of tetrahyd...

The aminoacyl-beta-ketophosphonate-adenosines (aa-KPA) are stable analogs of the aminoacyl adenylates, which are high-energy intermediates in the formation of aminoacyl-tRNA catalyzed by aminoacyl-tRNA synthetases (aaRS). We have synthesized glutamyl-beta-ketophosphonate-adenosine (Glu-KPA) and glutaminyl-beta-ketophosphonate-adenosine (Gln-KPA), a...

We report here the resonance Raman spectra of the FeIII-NO and FeII-NO complexes of the bacterial NOSs (nitric oxide synthases) from Staphylococcus aureus and Bacillus subtilis. The haem-NO complexes of these bacterial NOSs displayed Fe-N-O frequencies similar to those of the mammalian NOSs, in presence and absence of L-arginine, indicating that ha...

The crystallographic structure of oxygenated trHbN from Mycobacterium tuberculosis showed an extended heme distal site hydrogen-bonding network that includes Y(B10), Q(E11), and the bound O(2) (Milani, M., et al. (2001) EMBO J. 20, 3902-3909). In the present work, we analyze the effects that substitutions at the B10 and E11 positions exert on the h...

HPr is a protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). In Gram-positive bacteria, HPr can be phosphorylated on Ser(46) by HPr(Ser) kinase/phosphorylase (HPrK/P) and on His(15) by enzyme I (EI) of the PTS. In vitro studies have shown that phosphorylation on one residue greatly inhibits the second phosp...

Little is known about the intermediates formed during catalysis by nitric-oxide synthase (NOS). We report here the characterization by resonance Raman spectroscopy of the oxygenated complex of the NOS from Staphylococcus aureus (saNOS) as well as the kinetics of formation and decay of the complex. An oxygenated complex transiently formed after mixi...

Nitric oxide synthases (NOSs) are heme proteins that catalyze the formation of nitric oxide (NO) from L-arginine and oxygen in a sequential two-step process. Three structurally similar isoforms have been identified that deliver NO to different tissues for specific functions. An understanding of the interactions of ligands with the protein is essent...

We have used resonance Raman spectroscopy to probe the heme environment of a recently discovered NOS from the pathogenic bacterium Staphylococcus aureus, named SANOS. We detect two forms of the CO complex in the absence of L-arginine, with nu(Fe-CO) at 482 and 497 cm(-1) and nu(C-O) at 1949 and 1930 cm(-1), respectively. Similarly to mammalian NOS,...

HmuO, a heme oxygenase of Corynebacterium diphtheriae, catalyzes degradation of heme using the same mechanism as the mammalian enzyme. The oxy form of HmuO, the precursor of the catalytically active ferric hydroperoxo species, has been characterized by ligand binding kinetics, resonance Raman spectroscopy, and x-ray crystallography. The oxygen asso...

Nitric-oxide synthase (NOS) catalyzes the formation of NO and citrulline from l-arginine and oxygen. However, the NO so formed has been found to auto-inhibit the enzymatic activity significantly. We hypothesized that the NO reactivity is in part controlled by hydrogen bonding between the conserved tryptophan residue (position 409 in the neuronal is...

Neuroglobin (Ngb) is a newly discovered oxygen-binding heme protein that is primarily expressed in the brain of humans and other vertebrates. To characterize the structure/function relationships of this new heme protein, we have used resonance Raman spectroscopy to determine the structure of the heme environment in Ngb from mice. In the Fe2+CO comp...

Understanding of the chemical nature of the dioxygen moiety of oxyhemoglobin is crucial for elucidation of its physiological function. In the present work, direct Raman spectroscopic observation of both the Fe—O2 and O—O stretching modes unambiguously establishes the vibrational characteristics of the oxygen-bound heme moiety in the hemoglobins of...

A putative hemoglobin (Hb) gene, related to those previously characterized in the green alga Chlamydomonas eugametos, the ciliated protozoan Paramecium caudatum, the cyanobacterium Nostoc commune and the bacterium Mycobacterium tuberculosis, was recently discovered in the complete genome sequence of the cyanobacterium Synechocystis PCC 6803. In thi...

Small hemoproteins displaying amino acid sequences 20-40 residues shorter than (non-)vertebrate hemoglobins (Hbs) have recently been identified in several pathogenic and non-pathogenic unicellular organisms, and named 'truncated hemoglobins' (trHbs). They have been proposed to be involved not only in oxygen transport but also in other biological fu...

Chlamydomonas hemoglobin is expressed in chloroplast during active photosynthesis. Its heme pocket has an unusual structure that undergoes substantial changes when exogenous ligands bind the heme iron. In the ferrous state of the heme, oxygen binds with high affinity and is stabilized by interactions from E7-glutamine and B10-tyrosine. In the prese...

The mechanisms by which nitric-oxide synthases (NOSs) bind and activate oxygen at their P450-type heme active site in order to synthesize nitric oxide from the substrate L-arginine are mostly unknown. To obtain information concerning the structure and properties of the first oxygenated intermediate of the enzymatic cycle, we have used a rapid conti...

The mechanisms by which nitric-oxide synthases (NOSs) bind and activate oxygen at their P450-type heme active site in order to synthesize nitric oxide from the substratel-arginine are mostly unknown. To obtain information concerning the structure and properties of the first oxygenated intermediate of the enzymatic cycle, we have used a rapid contin...

The homodimeric hemoglobin (HbN) fromMycobacterium tuberculosis displays an extremely high oxygen binding affinity and cooperativity. Sequence alignment with other hemoglobins suggests that the proximal F8 ligand is histidine, the distal E7 residue is leucine, and the B10 position is occupied by tyrosine. To determine how these heme pocket residues...

We have studied the unusual heme ligand structure of the ferric forms of a recombinant Chlamydomonas chloroplast hemoglobin and its several single-amino acid mutants by EPR, optical absorbance, and resonance Raman spectroscopy. The helical positions of glutamine-84, tyrosine-63, and lysine-87 are suggested to correspond to E7, B10, and E10, respect...

Two putative hemoglobin genes, glbN and glbO, were recently discovered in the complete genome sequence of Mycobacterium tuberculosis H37Rv. Here, we show that the glbN gene encodes a dimeric hemoglobin (HbN) that binds oxygen cooperatively with very high affinity (P(50) = 0.013 mmHg at 20 degrees C) because of a fast combination (25 microM(-1).s(-1...

We report the optical and resonance Raman spectral characterization of ferrous recombinant Chlamydomonas LI637 hemoglobin. We show that it is present in three pH-dependent equilibrium forms including a 4-coordinate species at acid pH, a 5-coordinate high spin species at neutral pH, and a 6-coordinate low spin species at alkaline pH. The proximal li...

Hemoglobins (Hb), which have the important task of delivering molecular oxygen by facilitating its reversible binding to the heme, are now thought to have evolved in all groups of organisms including prokaryotes, fungi, plants and animals. Our recent finding of a light-inducible chloroplastic Hb in the green unicellular alga Chlamydomonas eugametos...

When the green unicellular alga Chlamydomonas eugametos is grown under light/dark regimes, nuclear genes are periodically activated in response to the changes in light conditions. These genetic responses are dependent upon the activation of genes associated with photosynthesis (LI616 and LI637), nonphotosynthetic photoreceptors (LI410 and LI818) an...