Rodrigo Arias-Cartin, Pierre Ceccaldi, Barbara Schoepp-Cothenet, Klaudia Frick, Jean-Michel Blanc, Bruno Guigliarelli, Anne Walburger, Stéphane Grimaldi, Thorsten Friedrich, Véronique Receveur-Brechot, Axel Magalon. Scientific Reports 6, Article number: 37743 (2016) http://dx.doi.org/10.1038/srep37743
Simon Duval, Joanne M. Santini, David Lemaire, Florence Chaspoul, Michael J. Russell, Stephane Grimaldi, Wolfgang Nitschke, Barbara Schoepp-Cothenet, BBA – Bioenergetics (2016) doi:10.1016/j.bbabio.2016.05.003
While the molybdenum cofactor in the majority of bisPGD enzymes goes through two consecutive 1-electron redox transitions, previous protein-film voltammetric results indicated the possibility of cooperative (n = 2) redox behavior in the bioenergetic enzyme arsenite oxidase (Aio). Combining equilibrium redox titrations, optical and EPR spectroscopies on concentrated samples obtained via heterologous expression, we unambiguously confirm this claim and quantify Aio’s redox cooperativity. The stability constant, Ks, of the MoV semi-reduced intermediate is found to be lower than 10− 3. Site-directed mutagenesis of residues in the vicinity of the Mo-cofactor demonstrates that the degree of redox cooperativity is sensitive to H-bonding interactions between the pyranopterin moieties and amino acid residues. Remarkably, in particular replacing the Gln-726 residue by Gly results in stabilization of (low-temperature) EPR-observable MoVwith KS = 4. As evidenced by comparison of room temperature optical and low temperature EPR titrations, the degree of stabilization is temperature-dependent. This highlights the importance of room-temperature redox characterizations for correctly interpreting catalytic properties in this group of enzymes.
Geochemical and phylogenetic data strongly indicate that molybdenum played an essential biocatalytic roles in early life. Molybdenum’s redox versatility and in particular the ability to show cooperative (n = 2) redox behavior provide a rationale for its paramount catalytic importance throughout the evolutionary history of life. Implications of the H-bonding network modulating Molybdenum’s redox properties on details of a putative inorganic metabolism at life’s origin are discussed.
P Ceccaldi, J Rendon, Ch Léger, R Toci, B Guigliarelli, A Magalon, S Grimaldi, V Fourmond, BBA – Bioenergetics (2015) doi:10.1016/j.bbabio.2015.06.007
Pascal Arnoux, Christian Ruppelt, Flore Oudouhou, Jérôme Lavergne, Marina I. Siponen, René Toci, Ralf R. Mendel, Florian Bittner, David Pignol, Axel Magalon & Anne Walburger
Nature Communications, vol 6, Feb 4th (2015), http://dx.doi.org/10.1038/ncomms7148
Formate dehydrogenases (FDHs) are of interest as they are natural catalysts that sequester atmospheric CO2, generating reduced carbon compounds with possible uses as fuel. FDHs activity in Escherichia coli strictly requires the sulphurtransferase EcFdhD, which likely transfers sulphur from IscS to the molybdenum cofactor (Mo-bisPGD) of FDHs. Here we show that EcFdhDbinds Mo-bisPGD in vivo and has submicromolar affinity for GDP—used as a surrogate of the molybdenum cofactor’s nucleotide moieties. The crystal structure of EcFdhD in complex with GDPshows two symmetrical binding sites located on the same face of the dimer. These binding sites are connected via a tunnel-like cavity to the opposite face of the dimer where two dynamic loops, each harbouring two functionally important cysteine residues, are present. On the basis of structure-guided mutagenesis, we propose a model for the sulphuration mechanism of Mo-bisPGD where the sulphur atom shuttles across the chaperone dimer.
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.
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.
Monique Sabaty, Sandrine Grosse, Geraldine Adryanczyk, Severine Boiry, Frederic Biaso, Pascal Arnoux and David Pignol
Background. YedY, a molybdoenzyme belonging to the sulfite oxidase family, is found in most Gram-negative bacteria. It contains a twin-arginine signal sequence that is cleaved after its translocation into the periplasm. Despite a weak reductase activity with substrates such as dimethyl sulfoxide or trimethylamine N-oxide, its natural substrate and its role in the cell remain unknown. Although sequence conservation of the YedY family displays a strictly conserved hydrophobic C-terminal residue, all known studies on Escherichia coli YedY have been performed with an enzyme containing a 6 histidine-tag at the C-terminus which could hamper enzyme activity.
Results. In this study, we demonstrate that the tag fused to the C-terminus of Rhodobacter sphaeroides YedY is detrimental to the enzyme’s reductase activity and results in an eight-fold decrease in catalytic efficiency. Nonetheless this C-terminal tag does not influence the properties of the molybdenum active site, as assayed by EPR spectroscopy. When a cleavable His-tag was fused to the N-terminus of the mature enzyme in the absence of the signal sequence, YedY was expressed and folded with its cofactor. However, when the signal sequence was added upstream of the N-ter tag, the amount of enzyme produced was approximately ten-fold higher.
Conclusion. Our study thus underscores the risk of using a C-terminus tagged enzyme while studying YedY, and presents an alternative strategy to express signal sequence-containing enzymes with an N-terminal tag. It brings new insights into molybdoenzyme maturation in R. sphaeroides showing that for some enzymes, maturation can occur in the absence of the signal sequence but that its presence is required for high expression of active enzyme.
Keywords: Molybdoenzyme; YedY; TAT machinery; Signal sequence; DMSO reductase; Rhodobacter sphaeroides; Enzyme maturation
Warelow TP, Oke M, Schoepp-Cothenet B, Dahl JU, Bruselat N, Sivalingam GN, Leimkühler S, Thalassinos K, Kappler U, Naismith JH, Santini JM.
PLoS One. 2013 Aug 30;8(8):e72535. doi: 10.1371/journal.pone.0072535. eCollection 2013.
The arsenite oxidase (Aio) from the facultative autotrophic Alphaproteobacterium Rhizobium sp. NT-26 is a bioenergetic enzyme involved in the oxidation of arsenite to arsenate. The enzyme from the distantly related heterotroph, Alcaligenes faecalis, which is thought to oxidise arsenite for detoxification, consists of a large α subunit (AioA) with bis-molybdopterin guanine dinucleotide at its active site and a 3Fe-4S cluster, and a small β subunit (AioB) which contains a Rieske 2Fe-2S cluster. The successful heterologous expression of the NT-26 Aio in Escherichia coli has resulted in the solution of its crystal structure. The NT-26 Aio, a heterotetramer, shares high overall similarity to the heterodimeric arsenite oxidase from A. faecalis but there are striking differences in the structure surrounding the Rieske 2Fe-2S cluster which we demonstrate explains the difference in the observed redox potentials (+225 mV vs. +130/160 mV, respectively). A combination of site-directed mutagenesis and electron paramagnetic resonance was used to explore the differences observed in the structure and redox properties of the Rieske cluster. In the NT-26 AioB the substitution of a serine (S126 in NT-26) for a threonine as in the A. faecalis AioB explains a −20 mV decrease in redox potential. The disulphide bridge in the A. faecalis AioB which is conserved in other betaproteobacterial AioB subunits and the Rieske subunit of the cytochrome bc1 complex is absent in the NT-26 AioB subunit. The introduction of a disulphide bridge had no effect on Aio activity or protein stability but resulted in a decrease in the redox potential of the cluster. These results are in conflict with previous data on the betaproteobacterial AioB subunit and the Rieske of the bc1 complex where removal of the disulphide bridge had no effect on the redox potential of the former but a decrease in cluster stability was observed in the latter.
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.