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

Reductive activation in periplasmic nitrate reductase involves chemical modifications of the Mo-cofactor beyond the first coordination sphere of the metal ion

International audienceIn Rhodobacter sphaeroides periplasmic nitrate reductase NapAB, the major Mo(V) form (the “high g” species) in air-purified samples is inactive and requires reduction to irreversibly convert into a catalytically competent form (Fourmond et al., J. Phys. Chem., 2008). In the present work, we study the kinetics of the activation process by combining EPR spectroscopy and direct electrochemistry. Upon reduction, the Mo (V) “high g” resting EPR signal slowly decays while the other redox centers of the protein are rapidly reduced, which we interpret as a slow and gated (or coupled) intramolecular electron transfer between the [4Fe–4S] center and the Mo cofactor in the inactive enzyme. Besides, we detect spin–spin interactions between the Mo(V) ion and the [4Fe–4S]1 + cluster which are modified upon activation of the enzyme, while the EPR signatures associated to the Mo cofactor remain almost unchanged. This shows that the activation process, which modifies the exchange coupling pathway between the Mo and the [4Fe–4S]1 + centers, occurs further away than in the first coordination sphere of the Mo ion. Relying on structural data and studies on Mo-pyranopterin and models, we propose a molecular mechanism of activation which involves the pyranopterin moiety of the molybdenum cofactor that is proximal to the [4Fe–4S] cluster. The mechanism implies both the cyclization of the pyran ring and the reduction of the oxidized pterin to give the competent tricyclic tetrahydropyranopterin form

Pseudomonas aeruginosa expresses a functional human natriuretic peptide receptor ortholog: involvement in biofilm formation

International audienceConsiderable evidence exists that bacteria detect eukaryotic communication molecules and modify their virulence accordingly. In previous studies, it has been demonstrated that the increasingly antibiotic resistant pathogen Pseudomonas aeruginosa can detect the human hormones Brain Natriuretic peptide (BNP) and C-type Natriuretic Peptide (CNP) at micromolar concentrations. In response, the bacterium modifies its behavior to adapt to the host physiology, increasing its overall virulence. Identifying the bacterial sensor for these hormones and interfering with this sensing mechanism offers an exciting opportunity to directly affect the infection process. Here, we show that BNP and CNP strongly decrease P. aeruginosa biofilm formation. Isatin, an antagonist of human natriuretic peptide receptors (NPR), prevents this effect. Furthermore, the human NPR-C receptor agonist cANF4-23 mimics the effects of natriuretic peptides on P. aeruginosa while sANP, the NPR-A receptor agonist, appears weakly active. We show in silico that NPR-C, a preferential CNP receptor, and the P. aeruginosa protein AmiC have similar 3D structures, and that both CNP and isatin bind to AmiC. We demonstrate that CNP acts as an AmiC agonist, enhancing the expression of the ami operon in P. aeruginosa.Binding of CNP and NPR-C agonists to AmiC was confirmed by microscale thermophoresis. Finally, using an amiC mutant strain, we demonstrate that AmiC is essential for CNP effects on biofilm formation. In conclusion, the AmiC bacterial sensor possesses similar structural and pharmacological profiles to the human NPR-C receptor and appears to be a bacterial receptor for human hormones that enables P. aeruginosa to modulate biofilm expression