Tagged: NapAB

Kinetics of substrate inhibition of periplasmic nitrate reductase

J Jacques, B Burlat, P Arnoux, M Sabaty, B Guigliarelli, C Léger, D Pignol, V Fourmond

Biochim. Biophys. Acta Bioenerg. E pub June 1st, 2014. doi: 10.1016/j.bbabio.2014.05.357

Periplasmic nitrate reductase catalyzes the reduction of nitrate into nitrite using a mono- nuclear 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 iso- lated 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 their dependence on electrode potential and sub- strate concentration to learn about the properties of the active and inactive forms of the enzyme. We conclude that the substrate-inhibited enzyme never significantly accu- mulates 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.

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

JG Jacques; V Foumond; P Arnoux; M Sabaty; E Etienne; S Grosse; F Biaso; P Bertrand; D Pignol; C Léger; B Guigliarelli; B Burlat

In 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.

The prokaryotic Mo/W-bisPGD enzymes family: A catalytic workhorse in bioenergetic.

Grimaldi S, Schoepp-Cothenet B, Ceccaldi P, Guigliarelli B, Magalon A.

Over the past two decades, prominent importance of molybdenum-containing enzymes in prokaryotes has been put forward by studies originating from different fields. Proteomic or bioinformatic studies underpinned that the list of molybdenum-containing enzymes is far from being complete with to date, more than fifty different enzymes involved in the biogeochemical nitrogen, carbon and sulfur cycles. In particular, the vast majority of prokaryotic molybdenum-containing enzymes belong to the so-called dimethylsulfoxide reductase family. Despite its extraordinary diversity, this family is characterized by the presence of a Mo/W-bis(pyranopterin guanosine dinucleotide) cofactor at the active site. This review highlights what has been learned about the properties of the catalytic site, the modular variation of the structural organization of these enzymes, and their interplay with the isoprenoid quinones. In the last part, this review provides an integrated view of how these enzymes contribute to the bioenergetics of prokaryotes. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.

DFT Investigation of the Molybdenum Cofactor in Periplasmic Nitrate Reductases : Structure of the Mo(V) EPR-Active Species

Biaso F., Burlat B., Guigliarelli B

Inorg Chem. 2012 Mar 19;51(6):3409-19. doi: 10.1021/ic201533p. Epub 2012 Mar 7.

The periplasmic nitrate reductase NAP belongs to the DMSO reductase family that regroups molybdoenzymes housing a bis-molybdopterin cofactor as the active site. Several forms of the Mo(V) state, an intermediate redox state in the catalytic cycle of the enzyme, have been evidenced by EPR spectroscopy under various conditions, but their structure and catalytic relevance are not fully understood. On the basis of structural data available from the literature, we built several models that reproduce the first coordination sphere of the molybdenum cofactor and used DFT methods to make magneto-structural correlations on EPR-detected species. “High-g” states, which are the most abundant Mo(V) species, are characterized by a low-anisotropy g tensor and a high gmin value. We assign this signature to a six-sulfur coordination sphere in a pseudotrigonal prismatic geometry with a partial disulfide bond. The “very high-g” species is well described with a sulfido ion as the sixth ligand. The “low-g” signal can be successfully associated to a Mo(V) sulfite–oxidase-type active site with only one pterin moiety coordinated to the molybdenum ion with an oxo or sulfido axial ligand. For all these species we investigate their catalytic activity using a thermodynamic point of view on the molybdenum coordination sphere. Beyond the periplasmic nitrate reductase case, this work provides useful magneto-structural correlations to characterize EPR-detected species in mononuclear molybdoenzymes.

Reassessing the strategies for trapping catalytic intermediates during nitrate reductase turnover

Fourmond V, Sabaty M, Arnoux D, Bertrand P, Pignol D, Léger C.

J Phys Chem B. 2010 Mar 11;114(9):3341-7. doi: 10.1021/jp911443y.

We examined the kinetics of nitrate reduction by periplasmic nitrate reductase (Nap) by using protein film voltammetry and solution assays. We demonstrate that, under turnover conditions, the enzyme exists as a mixture of active and inactive forms which interconvert on a time scale that is much slower than turnover. The dead-end species accumulates under mildly reducing conditions and at high nitrate concentration, resulting in substrate inhibition and in an uncommon hysteresis in the voltammetric signature. Solution assays with two electron donors having different reduction potentials fully support the electrochemical results. This illustrates the consequences of the high flexibility of the active site molybdenum coordination sphere and questions the conclusions from earlier studies in which attempts were made to trap catalytic intermediates of Nap in experiments carried out under turnover conditions at very high substrate concentration.

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

Fourmond V, Burlat B, Dementin S, Sabaty M, Arnoux D, Etienne E, Guigliarelli B, Bertrand P, Pignol D, Léger C

Biochemistry. 2010 Mar 23;49(11):2424-32. doi: 10.1021/bi902140e.

Rhodobacter sphaeroides periplasmic nitrate reductase (Rs NapAB) is one of the enzymes whose assays give odd results: in spectrophotometric assays with methyl viologen as the electron donor, the activity increases as the reaction progresses, whereas the driving force provided by the soluble redox partner decreases; in protein film voltammetry (PFV), whereby the enzyme directly exchanges electrons with an electrode, the activity of NapAB decreases at large overpotential, whereas a monotonic increase is expected [Elliott, S. J., et al. (2002) Biochim. Biophys. Acta 1555, 54−59]. The relations between these phenomena and the catalytic mechanism are still debated. By studying NapAB mutants, we found that the peculiar dependences of electrochemical and solution activities on driving force are greatly affected by substituting certain amino acids that are located in the vicinity of the active site (M153, Q384, R392); this led us to establish and discuss the relation between the experimental parameters of the electrochemical and spectrophotometric assays: we show that the rate of reduction of the enzyme (which depends on the electrode potential or on the concentration of reduced MV) modulates the activity of the enzyme, but the “solution potential” does not. Our results also support the view that the complex profiles of activity versus potential are fingerprints of the active site chemistry, rather than direct consequences of changes in the redox states of relays that are remote from the active site.

Correcting for electrocatalyst desorption and inactivation in chronoamperometry experiments

Fourmond V, Lautier T, Baffert C, Leroux F, Liebgott PP, Dementin S, Rousset M, Arnoux P, Pignol D, Meynial-Salles I, Soucaille P, Bertrand P, Léger C

Anal Chem. 2009 Apr 15;81(8):2962-8. doi: 10.1021/ac8025702.

Chronoamperometric experiments with adsorbed electrocatalysts are commonly performed either for analytical purposes or for studying the catalytic mechanism of a redox enzyme. In the context of amperometric sensors, the current may be recorded as a function of time while the analyte concentration is being increased to determine a linearity range. In mechanistic studies of redox enzymes, chronoamperometry proved powerful for untangling the effects of electrode potential and time, which are convoluted in cyclic voltammetric measurements, and for studying the energetics and kinetics of inhibition. In all such experiments, the fact that the catalyst’s coverage and/or activity decreases over time distorts the data. This may hide meaningful features, introduce systematic errors, and limit the accuracy of the measurements. We propose a general and surprisingly simple method for correcting for electrocatalyst desorption and inactivation, which greatly increases the precision of chronoamperometric experiments. Rather than subtracting a baseline, this consists in dividing the current, either by a synthetic signal that is proportional to the instant electroactive coverage or by the signal recorded in a control experiment. In the latter, the change in current may result from film loss only or from film loss plus catalyst inactivation. We describe the different strategies for obtaining the control signal by analyzing various data recorded with adsorbed redox enzymes: nitrate reductase, NiFe hydrogenase, and FeFe hydrogenase. In each case we discuss the trustfulness and the benefit of the correction. This method also applies to experiments where electron transfer is mediated, rather than direct, providing the current is proportional to the time-dependent concentration of catalyst.

Major Mo(V) EPR signature of Rhodobacter sphaeroides periplasmic nitrate reductase arising froms a dead-end species that activates upon reduction. Relation to other molybdoenzymes from the DMSO reductase family.

Fourmond V, Burlat B, Dementin S, Arnoux P, Sabaty M, Boiry S, Guigliarelli B, Bertrand P, Pignol D, Léger C

Enzymes of the DMSO reductase family use a mononuclear Mo-bis(molybdopterin) cofactor (MoCo) to catalyze a variety of oxo-transfer reactions. Much functional information on nitrate reductase, one of the most studied members of this family, has been gained from EPR spectroscopy, but this technique is not always conclusive because the signature of the MoCo is heterogeneous, and which signals correspond to active species is still unsure. We used site-directed mutagenesis, EPR and protein film voltammetry to demonstrate that the MoCo in R. sphaeroides periplasmic nitrate reductase (NapAB) is subject to an irreversible reductive activation process whose kinetics we precisely define. This activation quantitatively correlates with the disappearance of the so-called “Mo(V) high-g” EPR signal, but this reductive process is too slow to be part of the normal catalytic cycle. Therefore, in NapAB, this most intense and most commonly observed signature of the MoCo arises from a dead-end, inactive state that gives a catalytically competent species only after reduction. This activation proceeds, even without substrate, according to a reduction followed by an irreversible nonredox step, both of which are pH independent. An apparently similar process occurs in other nitrate reductases (both assimilatory and membrane bound), and this also recalls the redox cycling procedure, which activates periplasmic DMSO reductases and simplifies their spectroscopic signatures. Hence we propose that heterogeneity at the active site and reductive activation are common properties of enzymes from the DMSO reductase family. Regarding NapAB, the fact that we could detect no Mo EPR signal upon reoxidizing the fully reduced enzyme suggests that the catalytically active form of the Mo(V) is thermodynamically unstable, as is the case for other enzymes of the DMSO reductase family. Our original approach, which combines spectroscopy and protein film voltammetry, proves useful for discriminating the forms of the active site on the basis of their catalytic properties. This could be applied to other enzymes for which the question arises as to the catalytic relevance of certain long-lived, spectroscopically characterized species.

Effects of slow substrate binding and release in redox enzymes: theory and application to periplasmic nitrate reductase

Bertrand P, Frangioni B, Dementin S, Sabaty M, Arnoux P, Guigliarelli B, Pignol D, Léger C

For redox enzymes, the technique called protein film voltammetry makes it possible to determine the entire profile of activity against driving force by having the enzyme exchanging directly electrons with the rotating-disc electrode onto which it is adsorbed. Both the potential location of the catalytic response and its detailed shape report on the sequence of catalytic events, electron transfers and chemical steps, but the models that have been used so far to decipher this signal lack generality. For example, it was often proposed that substrate binding to multiple redox states of the active site may explain that turnover is greater in a certain window of electrode potential, but no fully analytical treatment has been given. Here, we derive (i) the general current equation for the case of reversible substrate binding to any redox states of a two-electron active site (as exemplified by flavins and Mo cofactors), (ii) the quantitative conditions for an extremum in activity to occur, and (iii) the expressions from which the substrate-concentration dependence of the catalytic potential can be interpreted to learn about the kinetics of substrate binding and how this affects the reduction potential of the active site. Not only does slow substrate binding and release make the catalytic wave shape highly complex, but we also show that it can have important consequences which will escape detection in traditional experiments:  the position of the wave (this is the driving force that is required to elicit catalysis) departs from the reduction potential of the active site even at the lowest substrate concentration, and this deviation may be large if substrate binding is irreversible. This occurs in the reductive half-cycle of periplasmic nitrate reductase where irreversibility lowers the driving force required to reduce the active site under turnover conditions and favors intramolecular electron transfer from the proximal [4Fe4S]+ cluster to the active site MoV.

Access to the active site of periplasmic nitrate reductase: insights from site-directed mutagenesis and zinc inhibition studies.

Dementin S, Arnoux P, Frangioni B, Grosse S, Léger C, Burlat B, Guigliarelli B, Sabaty M, Pignol D.

The periplasmic nitrate reductase (NapAB), a member of the DMSO reductase superfamily, catalyzes the first step of the denitrification process in bacteria. In this heterodimer, a di-heme NapB subunit is associated to the catalytic NapA subunit that binds a [4Fe-4S] cluster and a bis(molybdopterin guanine dinucleotide) cofactor. Here, we report the kinetic characterization of purified mutated heterodimers from Rhodobacter sphaeroides. By combining site-directed mutagenesis, redox potentiometry, EPR spectroscopy, and enzymatic characterization, we investigate the catalytic role of two conserved residues (M153 and R392) located in the vicinity of the molybdenum active site. We demonstrate that M153 and R392 are involved in nitrate binding:  the Vm measured on the M153A and R392A mutants are similar to that measured on the wild-type enzyme, whereas the Km for nitrate is increased 10-fold and 200-fold, respectively. The use of an alternative enzymatic assay led us to discover that NapAB is uncompetitively inhibited by Zn2+ ions (Ki‘ = 1 μM). We used this property to further probe the active site access in the mutant enzymes. It is proposed that R392 acts as a filter by preventing a direct reduction of the Mo atom by small reducing molecules and partially protecting the active site against zinc inhibition. In addition, we show that M153 is a key residue mediating this inhibition likely by coordinating Zn2+ ions via its sulfur atom. This residue is not conserved in the DMSO reductase superfamily while it is conserved in the periplasmic nitrate reductase family. Zinc inhibition is therefore likely to be specific and restricted to periplasmic nitrate reductases.