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2001
Pettigrew, G., C. Goodhew, S. Pauleta, C. Costa, I. Moura, J. Moura, N. Palma, L. Krippahl, K. Jumel, S. Harding, and A. Cooper. "Cytochrome c peroxidase and its redox partners - binary and ternary complexes." Journal of Inorganic Biochemistry. 86 (2001): 86. AbstractWebsite
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Pauleta, S. R., C. Costa, A. Cooper, I. Moura, and G. W. Pettigrew. "Cytochrome c peroxidase as a model system to study electron transfer complexes." Journal of Inorganic Biochemistry. 86 (2001): 374. AbstractWebsite
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Qiu, Y., S. R. Pauleta, Y. Lu, C. F. Goodhew, I. Moura, G. W. Pettigrew, and J. A. Shelnutt. "Structural changes associated with calcium-dependent activation of the di-heme cytochrome c peroxidase of Paracoccus pantotrophus." Journal of Inorganic Biochemistry. 86 (2001): 386. AbstractWebsite
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Pauleta, S. R., Y. Lu, C. F. Goodhew, I. Moura, G. W. Pettigrew, and J. A. Shelnutt. "Calcium-dependent conformation of a heme and fingerprint peptide of the diheme cytochrome c peroxidase from Paracoccus pantotrophus." Biochemistry. 40 (2001): 6570-6579. AbstractWebsite

The structural changes in the heme macrocycle and substituents caused by binding of Ca2+ to the diheme cytochrome c peroxidase from Paracoccus pantotrophus were clarified by resonance Raman spectroscopy of the inactive fully oxidized form of the enzyme. The changes in the macrocycle vibrational modes are consistent with a Ca2+-dependent increase in the out-of-plane distortion of the low-potential heme, the proposed peroxidatic heme. Most of the increase in out-of-plane distortion occurs when the high-affinity site I is occupied, but a small further increase in distortion occurs when site II is also occupied by Ca2+ or Mg2+. This increase in the heme distortion explains the red shift in the Soret absorption band that occurs upon Ca2+ binding. Changes also occur in the low-frequency substituent modes of the heme, indicating that a structural change in the covalently attached fingerprint pentapeptide of the LP heme occurs upon Ca2+ binding to site I. These structural changes may lead to loss of the sixth ligand at the peroxidatic heme in the semireduced form of the enzyme and activation.

2002
Pauleta, S. R., Y. Lu, C. F. Goodhew, Y. Qiu, I. Moura, G. W. Pettigrew, and J. A. Shelnutt. "Structural changes in the calcium-dependent activation of the di-heme cytochrome c peroxidase of Paracoccus pantotrophus." Biophysical Journal. 82 (2002): 14A. AbstractWebsite
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2003
Pettigrew, G. W., S. R. Pauleta, C. F. Goodhew, A. Cooper, M. Nutley, K. Jumel, S. E. Harding, C. Costa, L. Krippahl, I. Moura, and J. Moura. "Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans containing more than one cytochrome." Biochemistry. 42 (2003): 11968-11981. AbstractWebsite

According to the model proposed in previous papers [Pettigrew, G. W., Prazeres, S., Costa, C., Palma, N., Krippahl, L., and Moura, J. J. (1999) The structure of an electron-transfer complex containing a cytochrome c and a peroxidase, J. Biol. Chem. 274, 11383-11389; Pettigrew, G. W., Goodhew, C. F., Cooper, A., Nutley, M., Jumel, K., and Harding, S. E. (2003) Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans, Biochemistry 42, 2046-2055], cytochrome c peroxidase of Paracoccus denitrificans can accommodate horse cytochrome c and Paracoccus cytochrome c(550) at different sites on its molecular surface. Here we use H-1 NMR spectroscopy, analytical ultracentrifugation, molecular docking simulation, and microcalorimetry to investigate whether these small cytochromes can be accommodated simultaneously in the formation of a ternary complex. The pattern of perturbation of heme methyl and methionine methyl resonances in binary and ternary solutions shows that a ternary complex can be formed, and this is confirmed by the increase in the sedimentation coefficient upon addition of horse cytochrome c to a solution in which cytochrome c(550) fully occupies its binding site on cytochrome c peroxidase. Docking experiments in which favored binary solutions of cytochrome, c(550) bound to cytochrome c peroxidase act as targets for horse cytochrome c and the reciprocal experiments in which favored binary solutions of horse cytochrome c bound to cytochrome c peroxidase act as targets for cytochrome c(550) show that the enzyme can accommodate both cytochromes at the same time on adjacent sites. Microcalorimetric titrations are difficult to interpret but are consistent with a weakened binding of horse cytochrome c to a binary complex of cytochrome c peroxidase and cytochrome c(550) and binding of cytochrome c(550) to the cytochrome c peroxidase that is affected little by the presence of horse cytochrome c in the other site. The presence of a substantial capture surface for small cytochromes on the cytochrome c peroxidase has implications for rate enhancement mechanisms which ensure that the two electrons required for re-reduction of the enzyme after reaction with hydrogen peroxide are delivered efficiently.

2004
Pauleta, S. R., A. Cooper, M. Nutley, N. Errington, S. Harding, F. Guerlesquin, C. F. Goodhew, I. Moura, JJG Moura, and G. W. Pettigrew. "A copper protein and a cytochrome bind at the same site on bacterial cytochrome c peroxidase." Biochemistry. 43 (2004): 14566-14576. AbstractWebsite

Pseudoazurin binds at a single site on cytochrome c peroxidase from Paracoccus pantotrophus with a K-d of 16.4 muM at 25 degreesC, pH 6.0, in an endothermic reaction that is driven by a large entropy change. Sedimentation velocity experiments confirmed the presence of a single site, although results at higher pseudoazurin concentrations are complicated by the dimerization of the protein. Microcalorimetry, ultracentrifugation, and H-1 NMR spectroscopy studies in which cytochrome c550, pseudoazurin, and cytochrome c peroxidase were all present could be modeled using a competitive binding algorithm. Molecular docking simulation of the binding of pseudoazurin to the peroxidase in combination with the chemical shift perturbation pattern for pseudoazurin in the presence of the peroxidase revealed a group of solutions that were situated close to the electron-transferring heme with Cu-Fe distances of about 14 Angstrom. This is consistent with the results of H-1 NMR spectroscopy, which showed that pseudoazurin binds closely enough to the electron - transferring heme of the peroxidase to perturb its set of heme methyl resonances. We conclude that cytochrome c550 and pseudoazurin bind at the same site on the cytochrome c peroxidase and that the pair of electrons required to restore the enzyme to its active state after turnover are delivered one-by-one to the electron-transferring heme.

Pauleta, S. R., F. Guerlesquin, C. F. Goodhew, B. Devreese, J. VanBeeumen, AS Pereira, I. Moura, and G. W. Pettigrew. "Paracoccus pantotrophus pseudoazurin is an electron donor to cytochrome c peroxidase." Biochemistry. 43 (2004): 11214-25. AbstractWebsite

The gene for pseudoazurin was isolated from Paracoccus pantotrophus LMD 52.44 and expressed in a heterologous system with a yield of 54.3 mg of pure protein per liter of culture. The gene and protein were shown to be identical to those from P. pantotrophus LMD 82.5. The extinction coefficient of the protein was re-evaluated and was found to be 3.00 mM(-1) cm(-1) at 590 nm. It was confirmed that the oxidized protein is in a weak monomer/dimer equilibrium that is ionic-strength-dependent. The pseudoazurin was shown to be a highly active electron donor to cytochrome c peroxidase, and activity showed an ionic strength dependence consistent with an electrostatic interaction. The pseudoazurin has a very large dipole moment, the vector of which is positioned at the putative electron-transfer site, His81, and is conserved in this position across a wide range of blue copper proteins. Binding of the peroxidase to pseudoazurin causes perturbation of a set of NMR resonances associated with residues on the His81 face, including a ring of lysine residues. These lysines are associated with acidic residues just back from the rim, the resonances of which are also affected by binding to the peroxidase. We propose that these acidic residues moderate the electrostatic influence of the lysines and so ensure that specific charge interactions do not form across the interface with the peroxidase.

2006
Pettigrew, G. W., A. Echalier, and S. R. Pauleta. "Structure and mechanism in the bacterial dihaem cytochrome c peroxidases." Journal of Inorganic Biochemistry. 100 (2006): 551-567. AbstractWebsite

The bacterial cytochroine c peroxidases contain an electron-transferring haem c (E) and a peroxidatic haem c (P). Many are isolated in an inactive oxidised state. Reduction of the E baem promotes Ca2+-dependent spin state and coordination changes at the P haem rendering it accessible to ligand. Recent crystallographic work on the oxidised and mixed valence enzymes has suggested a mechanism by which an electron entering the E haem remotely triggers this activation of the P haem. Binding of hydrogen peroxide at the activated P haem leads to an intermediate catalytic form containing two oxidising equivalents, one of which is a ferryl oxene. This form of the enzyme is then reduced by two single electron transfers to the E haem delivered by small redox proteins such as cytochromes or cupredoxins. The binding of these small redox proteins is dominated by global electrostatic forces but the interfaces of the electron transfer complexes that are formed are largely hydrophobic and relatively non-specific. These features allow very high electron transfer rates in the steady state. (c) 2006 Elsevier Inc. All rights reserved.

Auchere, F., S. R. Pauleta, P. Tavares, I. Moura, and JJG Moura. "Kinetics studies of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and superoxide reductases." Journal of Biological Inorganic Chemistry. 11 (2006): 433-444. AbstractWebsite

In this work we present a kinetic study of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and members of the three different classes of superoxide reductases (SORs). SORs from the sulfate-reducing bacteria Desulfovibrio vulgaris (Dv) and D. gigas (Dg) were chosen as prototypes of classes I and II, respectively, while SOR from the syphilis spyrochete Treponema pallidum (Tp) was representative of class III. Our results show evidence for different behaviors of SORs toward electron acceptance, with a trend to specificity for the electron donor and acceptor from the same organism. Comparison of the different k (app) values, 176.9 +/- 25.0 min(-1) in the case of the Tp/Tp electron transfer, 31.8 +/- 3.6 min(-1) for the Dg/Dg electron transfer, and 6.9 +/- 1.3 min(-1) for Dv/Dv, could suggest an adaptation of the superoxide-mediated electron transfer efficiency to various environmental conditions. We also demonstrate that, in Dg, another iron-sulfur protein, a desulforedoxin, is able to transfer electrons to SOR more efficiently than rubredoxin, with a k (app) value of 108.8 +/- 12.0 min(-1), and was then assigned as the potential physiological electron donor in this organism.

2007
Pauleta, S. R., A. G. Duarte, M. S. Carepo, AS Pereira, P. Tavares, I. Moura, and J. J. Moura. "NMR assignment of the apo-form of a Desulfovibrio gigas protein containing a novel Mo-Cu cluster." Biomol NMR Assign. 1 (2007): 81-3. AbstractWebsite

We report the 98% assignment of the apo-form of an orange protein, containing a novel Mo-Cu cluster isolated from Desulfovibrio gigas. This protein presents a region where backbone amide protons exchange fast with bulk solvent becoming undetectable. These residues were assigned using 13C-detection experiments.

de Sousa, P. M. P., S. R. Pauleta, M. L. S. Goncalves, G. W. Pettigrew, I. Moura, M. M. C. dos Santos, and JJG Moura. "Mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by P-pantotrophus pseudoazurin: kinetics of intermolecular electron transfer." Journal of Biological Inorganic Chemistry. 12 (2007): 691-698. AbstractWebsite

This work reports the direct electrochemistry of Paracoccus pantotrophus pseudoazurin and the mediated catalysis of cytochrome c peroxidase from the same organism. The voltammetric behaviour was examined at a gold membrane electrode, and the studies were performed in the presence of calcium to enable the peroxidase activation. A formal reduction potential, E (0)', of 230 +/- 5 mV was determined for pseudoazurin at pH 7.0. Its voltammetric signal presented a pH dependence, defined by pK values of 6.5 and 10.5 in the oxidised state and 7.2 in the reduced state, and was constant up to 1 M NaCl. This small copper protein was shown to be competent as an electron donor to cytochrome c peroxidase and the kinetics of intermolecular electron transfer was analysed. A second-order rate constant of 1.4 +/- 0.2 x 10(5) M(-1) s(-1) was determined at 0 M NaCl. This parameter has a maximum at 0.3 M NaCl and is pH-independent between pH 5 and 9.

2008
de Sousa, P. M. P., S. R. Pauleta, D. Rodrigues, M. L. S. Goncalves, G. W. Pettigrew, I. Moura, JJG Moura, and M. M. C. dos Santos. "Benefits of membrane electrodes in the electrochemistry of metalloproteins: mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by horse cytochrome c: a case study." Journal of Biological Inorganic Chemistry. 13 (2008): 779-787. AbstractWebsite

A comparative study of direct and mediated electrochemistry of metalloproteins in bulk and membrane-entrapped solutions is presented. This work reports the first electrochemical study of the electron transfer between a bacterial cytochrome c peroxidase and horse heart cytochrome c. The mediated catalysis of the peroxidase was analysed both using the membrane electrode configuration and with all proteins in solution. An apparent Michaelis constant of 66 +/- 4 and 42 +/- 5 mu M was determined at pH 7.0 and 0 M NaCl for membrane and bulk solutions, respectively. The data revealed that maximum activity occurs at 50 mM NaCl, pH 7.0, with intermolecular rate constants of (4.4 +/- 0.5) x 10(6) and (1.0 +/- 0.5) x 10(6) M(-1) s(-1) for membrane-entrapped and bulk solutions, respectively. The influence of parameters such as pH or ionic strength on the mediated catalytic activity was analysed using this approach, drawing attention to the fact that careful analysis of the results is needed to ensure that no artefacts are introduced by the use of the membrane configuration and/or promoters, and therefore the dependence truly reflects the influence of these parameters on the (mediated) catalysis. From the pH dependence, a pK of 7.5 was estimated for the mediated enzymatic catalysis.

Pauleta, S. R., Y. Lu, C. F. Goodhew, I. Moura, G. W. Pettigrew, and J. A. Shelnutt. "Calcium-dependent heme structure in the reduced forms of the bacterial cytochrome c peroxidase from Paracoccus pantotrophus." Biochemistry. 47 (2008): 5841-5850. AbstractWebsite

This work reports for the first time a resonance Raman study of the mixed-valence and fully reduced forms of Paracoccus pantotrophus bacterial cytochrome c peroxidase. The spectra of the active mixed-valence enzyme show changes in the structure of the ferric peroxidatic heme compared to the fully oxidized enzyme; these differences are observed upon reduction of the electron-transferring heme and upon full occupancy of the calcium site. For the mixed-valence form in the absence of Ca2+, the peroxidatic heme is six-coordinate and low-spin on the basis of the frequencies of the structure-sensitive Raman lines: the enzyme is inactive. With added Ca2+, the peroxidatic heme is five-coordinate high-spin and active. The calcium-dependent spectral differences indicate little change in the conformation of the ferrous electron-transferring heme, but substantial changes in the conformation of the ferric peroxidatic heme. Structural changes associated with Ca2+ binding are indicated by spectral differences in the structure-sensitive marker lines, the out-of-plane low-frequency macrocyclic modes, and the vibrations associated with the heme substituents of that heme. The Ca2+-dependent appearance of a strong gamma(15) saddling-symmetry mode for the mixed-valence form is consistent with a strong saddling deformation in the active peroxidatic heme, a feature seen in the Raman spectra of other peroxidases. For the fully reduced form in the presence of Ca2+, the resonance Raman spectra show that the peroxidatic heme remains high-spin.

Moura, I., S. R. Pauleta, and JJG Moura. "Enzymatic activity mastered by altering metal coordination spheres." Journal of Biological Inorganic Chemistry. 13 (2008): 1185-1195. AbstractWebsite

Metalloenzymes control enzymatic activity by changing the characteristics of the metal centers where catalysis takes place. The conversion between inactive and active states can be tuned by altering the coordination number of the metal site, and in some cases by an associated conformational change. These processes will be illustrated using heme proteins (cytochrome c nitrite reductase, cytochrome c peroxidase and cytochrome cd(1) nitrite reductase), non-heme proteins (superoxide reductase and [ NiFe]-hydrogenase), and copper proteins (nitrite and nitrous oxide reductases) as examples. These examples catalyze electron transfer reactions that include atom transfer, abstraction and insertion.

Dell'acqua, S., S. R. Pauleta, E. Monzani, AS Pereira, L. Casella, JJG Moura, and I. Moura. "Electron transfer complex between nitrous oxide reductase and cytochrome c(552) from Pseudomonas nautica: Kinetic, nuclear magnetic resonance, and docking studies." Biochemistry. 47 (2008): 10852-10862. AbstractWebsite

The multicopper enzyme nitrous oxide reductase (N2OR) catalyzes the final step of denitrification, the two-electron reduction of N2O to N-2. This enzyme is a functional homodimer containing two different multicopper sites: CuA and CuZ. CuA is a binuclear copper site that transfers electrons to the tetranuclear copper sulfide CuZ, the catalytic site. In this study, Pseudomonas nautica cytochrome C-552 was identified as the physiological electron donor. The kinetic data show differences when physiological and artificial electron donors are compared [cytochrome vs methylviologen (MV)]. In the presence of cytochrome c(552), the reaction rate is dependent on the ET reaction and independent of the N2O concentration. With MV, electron donation is faster than substrate reduction. From the study of cytochrome c(552) concentration dependence, we estimate the following kinetic parameters: K-mc512 = 50.2 +/- 9.0 mu M and V-maxc551 1.8 +/- 10.6 units/mg. The N2O concentration dependence indicates a K-mN2O of 14.0 +/- 2.9 mu M using MV as the electron donor. The pH effect on the kinetic parameters is different when MV or cytochrome c(552) is used as the electron donor (pK(a) = 6.6 or 8.3, respectively). The kinetic study also revealed the hydrophobic nature of the interaction, and direct electron transfer studies showed that CuA is the center that receives electrons from the physiological electron donor. The formation of the electron transfer complex was observed by H-1 NMR protein-protein titrations and was modeled with a molecular docking program (BiGGER). The proposed docked complexes corroborated the ET studies giving a large number of solutions in which cytochrome c(552) is placed near a hydrophobic patch located around the CuA center.

2009
Najmudin, S., C. Bonifacio, A. G. Duarte, S. R. Pauleta, I. Moura, JJG Moura, and MJ Romao. "Crystallization and crystallographic analysis of the apo form of the orange protein (ORP) from Desulfovibrio gigas. (vol F65, pg 730, 2009)." Acta Crystallographica Section F-Structural Biology and Crystallization Communications. 65 (2009): 856. AbstractWebsite
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Almeida, R., S. Pauleta, I. Moura, and JJG Moura. "The electron transfer complex between D. gigas Superoxide Reductase and Rubredoxin." Febs Journal. 276 (2009): 128. AbstractWebsite
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Moura, J., L. Krippahl, S. Pauleta, R. Almeida, and S. Del Acqua. "Molecular interactions/electron transfer protein complexes using Docking algorithms, spectroscopy (NMR) and site direct mutagenesis." Febs Journal. 276 (2009): 11. AbstractWebsite
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Najmudin, S., C. Bonifacio, A. G. Duarte, S. R. Pauleta, I. Moura, J. J. Moura, and MJ Romao. "Crystallization and crystallographic analysis of the apo form of the orange protein (ORP) from Desulfovibrio gigas." Acta Crystallogr Sect F Struct Biol Cryst Commun. 65 (2009): 730-2. AbstractWebsite

The orange-coloured protein (ORP) from Desulfovibrio gigas is a 12 kDa protein that contains a novel mixed-metal sulfide cluster of the type [S(2)MoS(2)CuS(2)MoS(2)]. Diffracting crystals of the apo form of ORP have been obtained. Data have been collected for the apo form of ORP to 2.25 A resolution in-house and to beyond 2.0 A resolution at ESRF, Grenoble. The crystals belonged to a trigonal space group, with unit-cell parameters a = 43, b = 43, c = 106 A.

Rivas, M. G., C. S. Mota, S. R. Pauleta, M. S. P. Carepo, F. Folgosa, S. L. A. Andrade, G. Fauque, AS Pereira, P. Tavares, JJ Calvete, I. Moura, and JJG Moura. "Isolation and characterization of a new Cu-Fe protein from Desulfovibrio aminophilus DSM12254." Journal of Inorganic Biochemistry. 103 (2009): 1314-1322. AbstractWebsite

The isolation and characterization of a new metalloprotein containing Cu and Fe atoms is reported. The as-isolated Cu-Fe protein shows an UV-visible spectrum with absorption bands at 320 nm, 409 nm and 615 nm. Molecular mass of the native protein along with denaturating electrophoresis and mass spectrometry data show that this protein is a multimer consisting of 14 +/- 1 subunits of 15254.3 +/- 7.6 Da. Mossbauer spectroscopy data of the as-isolated Cu-Fe protein is consistent with the presence of [2Fe-2S](2+) centers. Data interpretation of the dithionite reduced protein suggest that the metallic cluster could be constituted by two ferromagnetically coupled [2Fe-2S](+) spin delocalized pairs. The biochemical properties of the Cu-Fe protein are similar to the recently reported molybdenum resistance associated protein from Desulfovibrio, D. alaskensis. Further-more, a BLAST search from the DNA deduced amino acid sequence shows that the Cu-Fe protein has homology with proteins annotated as zinc resistance associated proteins from Desulfovibrio, D. alaskensis, D. vulgaris Hildenborough, D. piger ATCC 29098. These facts suggest a possible role of the Cu-Fe protein in metal tolerance. (C) 2009 Published by Elsevier Inc.

Xie, X., R. G. Hadt, S. R. Pauleta, P. J. Gonzalez, S. Un, I. Moura, and E. I. Solomon. "A variable temperature spectroscopic study on Paracoccuspantotrophus pseudoazurin: protein constraints on the blue Cu site." J Inorg Biochem. 103 (2009): 1307-13. AbstractWebsite

The blue or Type 1 (T1) copper site of Paracoccuspantotrophus pseudoazurin exhibits significant absorption intensity in both the 450 and 600 nm regions. These are sigma and pi S(Cys) to Cu(2+) charge transfer (CT) transitions. The temperature dependent absorption, EPR, and resonance Raman (rR) vibrations enhanced by these bands indicate that a single species is present at all temperatures. This contrasts the temperature dependent behavior of the T1 center in nitrite reductase [S. Ghosh, X. Xie, A. Dey, Y. Sun, C. Scholes, E. Solomon, Proc. Natl. Acad. Sci. 106 (2009) 4969-4974] which has a thioether ligand that is unconstrained by the protein. The lack of temperature dependence in the T1 site in pseudoazurin indicates the presence of a protein constraint similar to the blue Cu site in plastocyanin where the thioether ligand is constrained at 2.8 A. However, plastocyanin exhibits only pi CT. This spectral difference between pseudoazurin and plastocyanin reflects a coupled distortion of the site where the axial thioether in pseudoazurin is also constrained, but at a shorter Cu-S(Met) bond length. This leads to an increase in the Cu(2+)-S(Cys) bond length, and the site undergoes a partial tetragonal distortion in pseudoazurin. Thus, its ground state wavefunction has both sigma and pi character in the Cu(2+)-S(Cys) bond.

Almeida, R. M., S. R. Pauleta, I. Moura, and JJG Moura. "Rubredoxin as a paramagnetic relaxation-inducing probe." Journal of Inorganic Biochemistry. 103 (2009): 1245-1253. AbstractWebsite

The paramagnetic effect due to the presence of a metal center with unpaired electrons is no longer considered a hindrance in protein NMR spectroscopy. In the present work, the paramagnetic effect due to the presence of a metal center with impaired electrons was used to map the interface of an electron transfer complex. Desulfovibrio gigas cytochrome c(3) was chosen as target to study the effect of the paramagnetic probe, Fe-rubredoxin, which produced specific line broadening in the heme IV methyl resonances M2(1) and M18(1). The rubredoxin binding surface in the complex with cytochrome c(3) was identified in a heteronuclear 2D NMR titration. The identified heme methyls on cytochrome c(3) are involved in the binding interface of the complex, a result that is in agreement with the predicted complexes obtained by restrained molecular docking, which shows a cluster of possible solutions near heme IV. The use of a paramagnetic probe in (1)HNMR titration and the mapping of the complex interface, in combination with a molecular simulation algorithm proved to be a valuable strategy to study electron transfer complexes involving non-heme iron proteins and cytochromes. (C) 2009 Elsevier Inc. All rights reserved.

2010
Dell'acqua, S., S. R. Pauleta, P. M. P. de Sousa, E. Monzani, L. Casella, JJG Moura, and I. Moura. "A new CuZ active form in the catalytic reduction of N2O by nitrous oxide reductase from Pseudomonas nautica." Journal of Biological Inorganic Chemistry. 15 (2010): 967-976. AbstractWebsite

The final step of bacterial denitrification, the two-electron reduction of N2O to N-2, is catalyzed by a multi-copper enzyme named nitrous oxide reductase. The catalytic centre of this enzyme is a tetranuclear copper site called CuZ, unique in biological systems. The in vitro reconstruction of the activity requires a slow activation in the presence of the artificial electron donor, reduced methyl viologen, necessary to reduce CuZ from the resting non-active state (1Cu(II)/3Cu(I)) to the fully reduced state (4Cu(I)), in contrast to the turnover cycle, which is very fast. In the present work, the direct reaction of the activated form of Pseudomonas nautica nitrous oxide reductase with stoichiometric amounts of N2O allowed the identification of a new reactive intermediate of the catalytic centre, CuZA degrees, in the turnover cycle, characterized by an intense absorption band at 680 nm. Moreover, the first mediated electrochemical study of Ps. nautica nitrous oxide reductase with its physiological electron donor, cytochrome c-552, was performed. The intermolecular electron transfer was analysed by cyclic voltammetry, under catalytic conditions, and a second-order rate constant of (5.5 +/- A 0.9) x 10(5) M-1 s(-1) was determined. Both the reaction of stoichiometric amounts of substrate and the electrochemical studies show that the active CuZA degrees species, generated in the absence of reductants, can rearrange to the resting non-active CuZ state. In this light, new aspects of the catalytic and activation/inactivation mechanism of the enzyme are discussed.

Cameron, D. L., J. Jakus, S. R. Pauleta, G. W. Pettigrew, and A. Cooper. "Pressure Perturbation Calorimetry and the Thermodynamics of Noncovalent Interactions in Water: Comparison of Protein-Protein, Protein-Ligand, and Cyclodextrin-Adamantane Complexes." Journal of Physical Chemistry B. 114 (2010): 16228-16235. AbstractWebsite

Pressure perturbation calorimetry measurements on a range of cyclodextrin adamantane, protein ligand (lysozyme-(GlcNac)(3) and ribonuclease-2'CMP) and protein-protein (cytochrome c peroxidase-pseudoazurin) complexes in aqueous solution show consistent reductions in thermal expansibilities compared to the uncomplexed molecules. Thermodynamic data for binding, obtained by titration calorimetry, are also reported. Changes in molar expansibilities can be related to the decrease in solvation during complexation. Although reasonable estimates for numbers of displaced water molecules may be obtained in the case of rigid cyclodextrin-adamantane complexes, protein expansibility data are less easily reconciled. Comparison of data from this wide range of systems indicates that effects are not simply related to changes in solvent-accessible surface area, but may also involve changes in macromolecular dynamics and flexibility. This adds to the growing consensus that understanding thermodynamic parameters associated with noncovalent interactions requires consideration of changes in internal macromolecular fluctuations and dynamics that may not be related to surface area-related solvation effects alone.