Export 67 results:
Sort by: Author Title Type [ Year  (Desc)]
., Submitted. Abstract
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}.

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
Carepo, M. S., S. R. Pauleta, A. G. Wedd, J. J. Moura, and I. Moura. "Mo-Cu metal cluster formation and binding in an orange protein isolated from Desulfovibrio gigas." J Biol Inorg Chem. 19 (2014): 605-14. AbstractWebsite

The orange protein (ORP) isolated from the sulfate-reducing bacterium Desulfovibrio gigas (11.8 kDa) contains a mixed-metal sulfide cluster of the type [S2MoS2CuS2MoS2](3-) noncovalently bound to the polypeptide chain. The D. gigas ORP was heterologously produced in Escherichia coli in the apo form. Different strategies were used to reconstitute the metal cluster into apo-ORP and obtain insights into the metal cluster synthesis: (1) incorporation of a synthesized inorganic analogue of the native metal cluster and (2) the in situ synthesis of the metal cluster on the addition to apo-ORP of copper chloride and tetrathiomolybdate or tetrathiotungstate. This latter procedure was successful, and the visible spectrum of the Mo-Cu reconstituted ORP is identical to the one reported for the native protein with absorption maxima at 340 and 480 nm. The (1)H-(15)N heteronuclear single quantum coherence spectra of the reconstituted ORP obtained by strategy 2, in contrast to strategy 1, exhibited large changes, which required sequential assignment in order to identify, by chemical shift differences, the residues affected by the incorporation of the cluster, which is stabilized inside the protein by both electrostatic and hydrophobic interactions.

Maiti, B. K., T. Aviles, I. Moura, S. R. Pauleta, and JJG Moura. "Synthesis and characterization of [S2MoS2Cu(n-SPhF)](2-) (n = o, m, P) clusters: Potential F-19-NMR structural probes for Orange Protein." Inorg Chem Commun. 45 (2014): 97-100. AbstractWebsite

Three fluorinated Mo-Cu-thiolate isomers,[Ph4Ph[S2MoS2Cu(n-SPhF)], [n-SPhF = 2-fluorothiophenol (la)], 3-fluorothiophenol (lb), and 4-fluorothiophenol (1c)] were synthesized and spectroscopically characterized. The F-19-NMR signal of the fluorine atom in the.benzene has different chemical shift for each isomer, which is highly influenced by the local environment that can be manipulated by different solvents and solutes. The fluorine-19 chemical shift is an advantageous NMR structural probe in alternative to H-1-NMR [B.K. Maiti, T. Aviles, M. Matzapetakis, I. Moura, S.R. Pauleta, JJ.G. Moura, Eur. J. Inorg. Chem. (2012) 4159.], that can be used to provide local information on the pocket of the metal cluster in the Orange Protein (ORP). (C) 2014 Elsevier B.V. All rights reserved.

Saponaro, A. C., M. Matzapetakis, B. Santoro, S. R. Pauleta, and A. Moroni. "The Auxiliary Subunit TRIP8B Inhibits the Binding of CAMP to HCN2 Channels Through an Allosteric Mechanism." Biophysical Journal. Vol. 106. Biophys J, 106. 2014. 758a. Abstract
Johnston, E. M., S. Dell'acqua, S. Ramos, S. R. Pauleta, I. Moura, and E. I. Solomon. "Determination of the active form of the tetranuclear copper sulfur cluster in nitrous oxide reductase." J Am Chem Soc. 136 (2014): 614-7. AbstractWebsite

N2OR has been found to have two structural forms of its tetranuclear copper active site, the 4CuS Cu(Z)* form and the 4Cu2S Cu(Z) form. EPR, resonance Raman, and MCD spectroscopies have been used to determine the redox states of these sites under different reductant conditions, showing that the Cu(Z)* site accesses the 1-hole and fully reduced redox states, while the Cu(Z) site accesses the 2-hole and 1-hole redox states. Single-turnover reactions of N2OR for Cu(Z) and Cu(Z)* poised in these redox states and steady-state turnover assays with different proportions of Cu(Z) and Cu(Z)* show that only fully reduced Cu(Z)* is catalytically competent in rapid turnover with N2O.

Maiti, B. K., L. B. Maia, K. Pal, B. Pakhira, T. Aviles, I. Moura, S. R. Pauleta, J. L. Nunez, A. C. Rizzi, CD Brondino, S. Sarkar, and J. J. Moura. "One electron reduced square planar bis(benzene-1,2-dithiolato) copper dianionic complex and redox switch by O2/HO(-)." Inorg Chem. 53 (2014): 12799-808. AbstractWebsite

The complex [Ph4P]2[Cu(bdt)2] (1(red)) was synthesized by the reaction of [Ph4P]2[S2MoS2CuCl] with H2bdt (bdt = benzene-1,2-dithiolate) in basic medium. 1(red) is highly susceptible toward dioxygen, affording the one electron oxidized diamagnetic compound [Ph4P][Cu(bdt)2] (1(ox)). The interconversion between these two oxidation states can be switched by addition of O2 or base (Et4NOH = tetraethylammonium hydroxide), as demonstrated by cyclic voltammetry and UV-visible and EPR spectroscopies. Thiomolybdates, in free or complex forms with copper ions, play an important role in the stability of 1(red) during its synthesis, since in its absence, 1(ox) is isolated. Both 1(red) and 1(ox) were structurally characterized by X-ray crystallography. EPR experiments showed that 1(red) is a Cu(II)-sulfur complex and revealed strong covalency on the copper-sulfur bonds. DFT calculations confirmed the spin density delocalization over the four sulfur atoms (76%) and copper (24%) atom, suggesting that 1(red) has a "thiyl radical character". Time dependent DFT calculations identified such ligand to ligand charge transfer transitions. Accordingly, 1(red) is better described by the two isoelectronic structures [Cu(I)(bdt2, 4S(3-,)*)](2-) <--> [Cu(II)(bdt2, 4S(4-))](2-). On thermodynamic grounds, oxidation of 1(red) (doublet state) leads to 1(ox) singlet state, [Cu(III)(bdt2, 4S(4-))](1-).

Johnston, E. M., S. Dell'acqua, S. Gorelsky, S. R. Pauleta, I. Moura, and E. I. Solomon. "Electronic structure and reactivities of resting and intermediate forms of the tetranuclear copper cluster in nitrous oxide reductase." Abstracts of Papers of the American Chemical Society. Vol. 248. Abstr Pap Am Chem S, 248. 2014. Abstract
Moura, JJG, B. K. Maiti, C. Carreira, L. B. Maia, S. P. Carepo, S. R. Pauleta, and I. Moura. "Metal substituted rubredoxins: a sulfur rich coordination site as models for metalloenzymes." Journal of Biological Inorganic Chemistry. Vol. 19. J Biol Inorg Chem, 19. 2014. S731. Abstract
Almeida, R. M., P. Turano, I. Moura, J. J. Moura, and S. R. Pauleta. "Superoxide reductase: different interaction modes with its two redox partners." ChemBioChem. 14 (2013): 1858-66. AbstractWebsite

Anaerobic organisms have molecular systems to detoxify reactive oxygen species when transiently exposed to oxygen. One of these systems is superoxide reductase, which reduces O2 (.-) to H2 O2 without production of molecular oxygen. In order to complete the reduction of superoxide anion, superoxide reductase requires an electron, delivered by its redox partners, which in Desulfovibrio gigas are rubredoxin and/or desulforedoxin. In this work, we characterized the interaction of Desulfovibrio gigas superoxide reductase with both electron donors by using steady-state kinetics, 2D NMR titrations, and backbone relaxation measurements. The rubredoxin surface involved in the electron transfer complex with superoxide reductase comprises the solvent-exposed hydrophobic residues in the vicinity of its metal center (Cys9, Gly10, Cys42, Gly43, and Ala44), and a Kd of 3 muM at 59 mM ionic strength was estimated by NMR. The ionic strength dependence of superoxide-mediated rubredoxin oxidation by superoxide reductase has a maximum kapp of (37 +/- 12) min(-1) at 157 mM. Relative to the electron donor desulforedoxin, its complex with superoxide reductase was not detected by chemical shift perturbation, though this protein is able to transfer electrons to superoxide reductase with a maximum kapp of (31 +/- 7) min(-1) at an ionic strength of 57 mM. Competition experiments using steady-state kinetics and NMR spectroscopy (backbone relaxation measurements and use of a paramagnetic relaxation enhancement probe) with Fe-desulforedoxin in the presence of (15) N-Zn-rubredoxin showed that these two electron donors compete for the same site on the enzyme surface, as shown in the model structure of the complex generated by using restrained molecular docking calculations. These combined strategies indicate that the two small electron donors bind in different manners, with the desulforedoxin complex being a short lived electron transfer complex or more dynamic, with many equivalent kinetically competent orientations.

Nóbrega, C. S., M. Matzapetakis, and S. R. Pauleta. "(1)H, (13)C and (15)N resonance assignment of the soluble form of the Lipid-modified Azurin from Neisseria gonorrhoeae." Biomol NMR Assign. 7 (2013): 311-4. AbstractWebsite

Lipid-modified azurin (Laz) from Neisseria gonorrhoeae is a type 1 copper protein proposed to be the electron donor to several enzymes involved in the resistance mechanism to reactive oxygen and nitrogen species. Here we report the backbone and side-chain resonance assignment of Laz in the reduced form, which has been complete at 97 %. The predicted secondary structure indicates that this protein belongs to the azurin subfamily of type 1 copper proteins.