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Pardoux, R., A. Fiévet, C. Carreira, C. Brochier-Armanet, O. Valette, Z. Dermoun, B. Py, A. Dolla, S. R. Pauleta, and C. Aubert. "The bacterial MrpORP is a novel Mrp/NBP35 protein involved in iron-sulfur biogenesis." Scientific Reports. 9.1 (2019). AbstractWebsite
Nóbrega, C. S., and S. R. Pauleta Reduction of hydrogen peroxide in gram-negative bacteria – bacterial peroxidases. Vol. 74. Advances in Microbial Physiology, 74., 2019. AbstractWebsite
Pauleta, S. R., M. S. P. Carepo, and I. Moura. "Source and reduction of nitrous oxide." Coordination Chemistry Reviews. 387 (2019): 436-449. AbstractWebsite
Carreira, C., O. Mestre, R. F. Nunes, I. Moura, and S. R. Pauleta. "Genomic organization, gene expression and activity profile of Marinobacter hydrocarbonoclasticus denitrification enzymes." PeerJ. 2018.9 (2018). AbstractWebsite
Nóbrega, C. S., B. Devreese, and S. R. Pauleta. "YhjA - An Escherichia coli trihemic enzyme with quinol peroxidase activity." Biochimica et Biophysica Acta - Bioenergetics. 1859.6 (2018): 411-422. AbstractWebsite
Maiti, B. K., L. B. Maia, S. R. Pauleta, I. Moura, and J. J. Moura. "Protein-Assisted Formation of Molybdenum Heterometallic Clusters: Evidence for the Formation of S2MoS2-M-S2MoS2 Clusters with M = Fe, Co, Ni, Cu, or Cd within the Orange Protein." Inorg Chem (2017). AbstractWebsite

The Orange Protein (ORP) is a small bacterial protein, of unknown function, that harbors a unique molybdenum/copper (Mo/Cu) heterometallic cluster, [S2MoVIS2CuIS2MoVIS2]3-, noncovalently bound. The apo-ORP is able to promote the formation and stabilization of this cluster, using CuII- and MoVIS42- salts as starting metallic reagents, to yield a Mo/Cu-ORP that is virtually identical to the native ORP. In this work, we explored the ORP capability of promoting protein-assisted synthesis to prepare novel protein derivatives harboring molybdenum heterometallic clusters containing iron, cobalt, nickel, or cadmium in place of the "central" copper (Mo/Fe-ORP, Mo/Co-ORP, Mo/Ni-ORP, or Mo/Cd-ORP). For that, the previously described protein-assisted synthesis protocol was extended to other metals and the Mo/M-ORP derivatives (M = Cu, Fe, Co, Ni, or Cd) were spectroscopically (UV-visible and electron paramagnetic resonance (EPR)) characterized. The Mo/Cu-ORP and Mo/Cd-ORP derivatives are stable under oxic conditions, while the Mo/Fe-ORP, Mo/Co-ORP, and Mo/Ni-ORP derivatives are dioxygen-sensitive and stable only under anoxic conditions. The metal and protein quantification shows the formation of 2Mo:1M:1ORP derivatives, and the visible spectra suggest that the expected {S2MoS2MS2MoS2} complexes are formed. The Mo/Cu-ORP, Mo/Co-ORP, and Mo/Cd-ORP are EPR-silent. The Mo/Fe-ORP derivative shows an EPR S = 3/2 signal (E/D approximately 0.27, g approximately 5.3, 2.5, and 1.7 for the lower M= +/-1/2 doublet, and g approximately 5.7 and 1.7 (1.3 predicted) for the upper M = +/-3/2 doublet), consistent with the presence of either one S = 5/2 FeIII antiferromagnetically coupled to two S = 1/2 MoV or one S = 3/2 FeI and two S = 0 MoVI ions, in both cases in a tetrahedral geometry. The Mo/Ni-ORP shows an EPR axial S = 1/2 signal consistent with either one S = 1/2 NiI and two S = 0 MoVI or one S = 1/2 NiIII antiferromagnetically coupled to two S = 1/2 MoV ions, in both cases in a square-planar geometry. The Mo/Cu-ORP and Mo/Cd-ORP are described as {MoVI-CuI-MoVI} and {MoVI-CdII-MoVI}, respectively, while the other derivatives are suggested to exist in at least two possible electronic structures, {MoVI-MI-MoVI} <--> {MoV-MIII-MoV}.

Nóbrega, C. S., M. Raposo, G. Van Driessche, B. Devreese, and S. R. Pauleta. "Biochemical characterization of the bacterial peroxidase from the human pathogen Neisseria gonorrhoeae." Journal of Inorganic Biochemistry. 171 (2017): 108-119. AbstractWebsite
Carreira, C., S. R. Pauleta, and I. Moura. "The catalytic cycle of nitrous oxide reductase — The enzyme that catalyzes the last step of denitrification." Journal of Inorganic Biochemistry. 177 (2017): 423-434. AbstractWebsite
Ramos, S., R. M. Almeida, C. M. Cordas, JJG Moura, S. R. Pauleta, and I. Moura. "Insights into the recognition and electron transfer steps in nitric oxide reductase from Marinobacter hydrocarbonoclasticus." Journal of Inorganic Biochemistry. 177 (2017): 402-411. AbstractWebsite
E. Johnston, C. Carreira, Dell'Acqua Dey Sofia Pauleta Moura Solomon S. S. R. I. "Spectroscopic Definition of the CuZ0 Intermediate in Turnover of Nitrous Oxide Reductase and Molecular Insight into the Catalytic Mechanism." JACS (2017).
Almeida, R. M., S. Dell'acqua, I. Moura, S. R. Pauleta, and JJG Moura CHAPTER 11: Electron Transfer and Molecular Recognition in Denitrification and Nitrate Dissimilatory Pathways. Eds. I. Moura, JJG Moura, L. B. Maia, C. D. Garner, and S. R. Pauleta. Vol. 2017-January. RSC Metallobiology, 2017-January. Royal Society of Chemistry, 2017. Abstract

The electron transfer pathways for the enzymes involved in the four sequential steps of the denitrification pathway are reviewed. In addition, brief information on the electron transfer events is also provided on two enzymes that participate in the dissimilatory nitrate reduction to ammonia. The two main aspects discussed are the intra- and inter-molecular electron transfer pathways and the molecular recognition processes involving the redox partners. When available, information on the residues that are involved in these pathways is given, and their role in electron transfer and/or the formation of the transient electron transfer complexes is discussed. © The Royal Society of Chemistry 2017.

Moura, I., L. B. Maia, S. R. Pauleta, and JJG Moura CHAPTER 1: A Bird's Eye View of Denitrification in Relation to the Nitrogen Cycle. Eds. I. Moura, JJG Moura, L. B. Maia, C. D. Garner, and S. R. Pauleta. Vol. 2017-January. RSC Metallobiology, 2017-January. Royal Society of Chemistry, 2017. Abstract

This book is devoted to denitrification, an anaerobic process that is used by a wide range of bacteria for energy generation. The overall process involves nitrate, which is present in soil or water, being reduced to gaseous dinitrogen. This initial chapter aims to place denitrification in the larger context of the nitrogen biogeochemical cycle (a bird's eye view). Detailed topics are developed through the many following contributions. Denitrification is a landscape for probing the structures, functions and mechanisms of action of a wide range of highly specialised metalloenzymes. These carry out, sequentially, four oxo-transfer reactions: NO3 - → NO2 - → NO → N2O → N2. The environmental implications of these processes are of particular relevance. Nitrate accumulation and the release of nitrous oxide into the atmosphere due to the excessive use of fertilisers in agriculture are examples of two environmental problems in which denitrification plays a central role. © The Royal Society of Chemistry 2017.

Pauleta, S. R., C. Carreira, and I. Moura CHAPTER 7: Insights into Nitrous Oxide Reductase. Eds. I. Moura, JJG Moura, L. B. Maia, C. D. Garner, and S. R. Pauleta. Vol. 2017-January. RSC Metallobiology, 2017-January. Royal Society of Chemistry, 2017. Abstract

Nitrous oxide reductase is the enzyme that catalyses the last step of the denitrification pathway, reducing nitrous oxide to dinitrogen gas. This enzyme is a functional homodimer with two copper centres, CuA and a "CuZ centre", located in different domains. The CuA centre is the electron transferring centre, while the catalytic centre is the "CuZ centre", a unique metal centre in biology - a tetranuclear copper centre with a μ4-bridging sulphide. The enzyme has been isolated with the "CuZ centre" in two different forms, CuZ(4Cu2S) and CuZ∗(4Cu1S), with the first presenting an additional μ2-sulphur atom as a bridging ligand between CuI and CuIV of the "CuZ centre", whereas the second form was identified as a water-derived molecule. Spectroscopic analysis of CuZ∗(4Cu1S), together with computational studies, indicated that there is a hydroxide bound to CuI. Genomic analysis has identified the presence of two different types of nitrous oxide reductase, the typical and "atypical", with a single member of the last group having been isolated to date, from Wolinella succinogenes. Thus, here the structure of the "typical" nitrous oxide reductase with either CuZ(4Cu2S) or CuZ∗(4Cu1S), as well as its spectroscopic and catalytic properties, will be discussed. © The Royal Society of Chemistry 2017.

Maiti, B. K., I. Moura, J. J. Moura, and S. R. Pauleta. "The small iron-sulfur protein from the ORP operon binds a [2Fe-2S] cluster." Biochim Biophys Acta. 1857 (2016): 1422-9. AbstractWebsite

A linear cluster formulated as [S2MoS2CuS2MoS2](3-), a unique heterometallic cluster found in biological systems, was identified in a small monomeric protein (named as Orange Protein). The gene coding for this protein is part of an operon mainly present in strict anaerobic bacteria, which is composed (in its core) by genes coding for the Orange Protein and two ATPase proposed to contain Fe-S clusters. In Desulfovibrio desulfuricans G20, there is an ORF, Dde_3197 that encodes a small protein containing several cysteine residues in its primary sequence. The heterologously produced Dde_3197 aggregates mostly in inclusion bodies and was isolated by unfolding with a chaotropic agent and refolding by dialysis. The refolded protein contained sub-stoichiometric amounts of iron atoms/protein (0.5+/-0.2), but after reconstitution with iron and sulfide, high iron load contents were detected (1.8+/-0.1 or 3.4+/-0.2) using 2- and 4-fold iron excess. The visible absorption spectral features of the iron-sulfur clusters in refolded and reconstituted Dde_3197 are similar and resemble the ones of [2Fe-2S] cluster containing proteins. The refolded and reconstituted [2Fe-2S] Dde_3197 are EPR silent, but after reduction with dithionite, a rhombic signal is observed with gmax=2.00, gmed=1.95 and gmin=1.92, consistent with a one-electron reduction of a [2Fe-2S](2+) cluster into a [2Fe-2S](1+) state, with an electron spin of S=(1/2). The data suggests that Dde_3197 can harbor one or two [2Fe-2S] clusters, one being stable and the other labile, with quite identical spectroscopic properties, but stable to oxygen.

Carepo, M. S., C. Carreira, R. Grazina, M. E. Zakrzewska, A. Dolla, C. Aubert, S. R. Pauleta, J. J. Moura, and I. Moura. "Orange protein from Desulfovibrio alaskensis G20: insights into the Mo-Cu cluster protein-assisted synthesis." J Biol Inorg Chem. 21 (2016): 53-62. AbstractWebsite

A novel metalloprotein containing a unique [S2MoS2CuS2MoS2](3-) cluster, designated as Orange Protein (ORP), was isolated for the first time from Desulfovibrio gigas, a sulphate reducer. The orp operon is conserved in almost all sequenced Desulfovibrio genomes and in other anaerobic bacteria, however, so far D. gigas ORP had been the only ORP characterized in the literature. In this work, the purification of another ORP isolated form Desulfovibrio alaskensis G20 is reported. The native protein is monomeric (12443.8 +/- 0.1 Da by ESI-MS) and contains also a MoCu cluster with characteristic absorption bands at 337 and 480 nm, assigned to S-Mo charge transfer bands. Desulfovibrio alaskensis G20 recombinant protein was obtained in the apo-form from E. coli. Cluster reconstitution studies and UV-visible titrations with tetrathiomolybdate of the apo-ORP incubated with Cu ions indicate that the cluster is incorporated in a protein metal-assisted synthetic mode and the protein favors the 2Mo:1Cu stoichiometry. In Desulfovibrio alaskensis G20, the orp genes are encoded by a polycistronic unit composed of six genes whereas in Desulfovibrio vulgaris Hildenborough the same genes are organized into two divergent operons, although the composition in genes is similar. The gene expression of ORP (Dde_3198) increased 6.6 +/- 0.5 times when molybdate was added to the growth medium but was not affected by Cu(II) addition, suggesting an involvement in molybdenum metabolism directly or indirectly in these anaerobic bacteria.

Nobrega, C. S., I. H. Saraiva, C. Carreira, B. Devreese, M. Matzapetakis, and S. R. Pauleta. "The solution structure of the soluble form of the lipid-modified azurin from Neisseria gonorrhoeae, the electron donor of cytochrome c peroxidase." Biochim Biophys Acta. 1857 (2016): 169-76. AbstractWebsite

Neisseria gonorrhoeae colonizes the genitourinary track, and in these environments, especially in the female host, the bacteria are subjected to low levels of oxygen, and reactive oxygen and nitrosyl species. Here, the biochemical characterization of N. gonorrhoeae Laz is presented, as well as, the solution structure of its soluble domain determined by NMR. N. gonorrhoeae Laz is a type 1 copper protein of the azurin-family based on its spectroscopic properties and structure, with a redox potential of 277+/-5 mV, at pH7.0, that behaves as a monomer in solution. The globular Laz soluble domain adopts the Greek-key motif, with the copper center located at one end of the beta-barrel coordinated by Gly48, His49, Cys113, His118 and Met122, in a distorted trigonal geometry. The edge of the His118 imidazole ring is water exposed, in a surface that is proposed to be involved in the interaction with its redox partners. The heterologously expressed Laz was shown to be a competent electron donor to N. gonorrhoeae cytochrome c peroxidase. This is an evidence for its involvement in the mechanism of protection against hydrogen peroxide generated by neighboring lactobacilli in the host environment.

Almeida, R. M., S. Dell'acqua, L. Krippahl, J. J. Moura, and S. R. Pauleta. "Predicting Protein-Protein Interactions Using BiGGER: Case Studies." Molecules. 21 (2016). AbstractWebsite

The importance of understanding interactomes makes preeminent the study of protein interactions and protein complexes. Traditionally, protein interactions have been elucidated by experimental methods or, with lower impact, by simulation with protein docking algorithms. This article describes features and applications of the BiGGER docking algorithm, which stands at the interface of these two approaches. BiGGER is a user-friendly docking algorithm that was specifically designed to incorporate experimental data at different stages of the simulation, to either guide the search for correct structures or help evaluate the results, in order to combine the reliability of hard data with the convenience of simulations. Herein, the applications of BiGGER are described by illustrative applications divided in three Case Studies: (Case Study A) in which no specific contact data is available; (Case Study B) when different experimental data (e.g., site-directed mutagenesis, properties of the complex, NMR chemical shift perturbation mapping, electron tunneling) on one of the partners is available; and (Case Study C) when experimental data are available for both interacting surfaces, which are used during the search and/or evaluation stage of the docking. This algorithm has been extensively used, evidencing its usefulness in a wide range of different biological research fields.

Neca, A. J., R. Soares, M. S. Carepo, and S. R. Pauleta. "Resonance assignment of DVU2108 that is part of the Orange Protein complex in Desulfovibrio vulgaris Hildenborough." Biomol NMR Assign. 10 (2016): 117-20. AbstractWebsite

We report the 94 % assignment of DVU2108, a protein belonging to the Orange Protein family, that in Desulfovibrio vulgaris Hildenborough forms a protein complex named the Orange Protein complex. This complex has been shown to be implicated in the cell division of this organism. DVU2108 is a conserved protein in anaerobic microorganisms and in Desulfovibrio gigas the homologous protein was isolated with a novel Mo-Cu cluster non-covalently attached to the polypeptide chain. However, the heterologously produced DVU2108 did not contain any bound metal. These assignments provide the means to characterize the interaction of DVU2108 with the proteins that form the Orange Protein complex using NMR methods.

Johnston, E. M., S. Dell'acqua, S. R. Pauleta, I. Moura, and E. I. Solomon. "Protonation state of the Cu4S2 CuZ site in nitrous oxide reductase: redox dependence and insight into reactivity." Chem Sci. 6 (2015): 5670-5679. AbstractWebsite

Spectroscopic and computational methods have been used to determine the protonation state of the edge sulfur ligand in the Cu4S2 CuZ form of the active site of nitrous oxide reductase (N2OR) in its 3CuICuII (1-hole) and 2CuI2CuII (2-hole) redox states. The EPR, absorption, and MCD spectra of 1-hole CuZ indicate that the unpaired spin in this site is evenly delocalized over CuI, CuII, and CuIV. 1-hole CuZ is shown to have a mu2-thiolate edge ligand from the observation of S-H bending modes in the resonance Raman spectrum at 450 and 492 cm-1 that have significant deuterium isotope shifts (-137 cm-1) and are not perturbed up to pH 10. 2-hole CuZ is characterized with absorption and resonance Raman spectroscopies as having two Cu-S stretching vibrations that profile differently. DFT models of the 1-hole and 2-hole CuZ sites are correlated to these spectroscopic features to determine that 2-hole CuZ has a mu2-sulfide edge ligand at neutral pH. The slow two electron (+1 proton) reduction of N2O by 1-hole CuZ is discussed and the possibility of a reaction between 2-hole CuZ and O2 is considered.

Maiti, B. K., L. B. Maia, C. M. Silveira, S. Todorovic, C. Carreira, M. S. Carepo, R. Grazina, I. Moura, S. R. Pauleta, and J. J. Moura. "Incorporation of molybdenum in rubredoxin: models for mononuclear molybdenum enzymes." J Biol Inorg Chem. 20 (2015): 821-9. AbstractWebsite

Molybdenum is found in the active site of enzymes usually coordinated by one or two pyranopterin molecules. Here, we mimic an enzyme with a mononuclear molybdenum-bis pyranopterin center by incorporating molybdenum in rubredoxin. In the molybdenum-substituted rubredoxin, the metal ion is coordinated by four sulfurs from conserved cysteine residues of the apo-rubredoxin and two other exogenous ligands, oxygen and thiol, forming a Mo((VI))-(S-Cys)4(O)(X) complex, where X represents -OH or -SR. The rubredoxin molybdenum center is stabilized in a Mo(VI) oxidation state, but can be reduced to Mo(IV) via Mo(V) by dithionite, being a suitable model for the spectroscopic properties of resting and reduced forms of molybdenum-bis pyranopterin-containing enzymes. Preliminary experiments indicate that the molybdenum site built in rubredoxin can promote oxo transfer reactions, as exemplified with the oxidation of arsenite to arsenate.

Saponaro, A., C. Donadoni, S. R. Pauleta, F. Cantini, M. Matzapetakis, G. Thiel, L. Banci, B. Santoro, and A. Moroni. "HCN Channels: The Molecular Basis for their cAMP-TRIP8b Regulation." Biophysical Journal. Vol. 108. Biophys J, 108. 2015. 366a. Abstract
Saponaro, A., S. R. Pauleta, F. Cantini, M. Matzapetakis, C. Hammann, C. Donadoni, L. Hu, G. Thiel, L. Banci, B. Santoro, and A. Moroni. "Structural basis for the mutual antagonism of cAMP and TRIP8b in regulating HCN channel function." Proc Natl Acad Sci U S A. 111 (2014): 14577-82. AbstractWebsite

cAMP signaling in the brain mediates several higher order neural processes. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels directly bind cAMP through their cytoplasmic cyclic nucleotide binding domain (CNBD), thus playing a unique role in brain function. Neuronal HCN channels are also regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit that antagonizes the effects of cAMP by interacting with the channel CNBD. To unravel the molecular mechanisms underlying the dual regulation of HCN channel activity by cAMP/TRIP8b, we determined the NMR solution structure of the HCN2 channel CNBD in the cAMP-free form and mapped on it the TRIP8b interaction site. We reconstruct here the full conformational changes induced by cAMP binding to the HCN channel CNBD. Our results show that TRIP8b does not compete with cAMP for the same binding region; rather, it exerts its inhibitory action through an allosteric mechanism, preventing the cAMP-induced conformational changes in the HCN channel CNBD.

Moura, I., C. Carreira, S. Pauleta, R. F. Nunes, J. J. Moura, S. Ramos, S. Dell'acqua, and O. Einsle. "INSIGHTS INTO THE CATALYTICCYCLE OF Pseudomonas nautica NITROUS OXIDE REDUCTASE." Journal of Biological Inorganic Chemistry. Vol. 19. J Biol Inorg Chem, 19. 2014. S104. Abstract