Sofia Pauleta

Iron-sulfur clusters are ubiquitous and ancient prosthetic groups that are present in all kingdoms of life. In the 1960s, they were recognized to play a role in electron-transfer reactions, but since then several other functions were identified, which can be attributed to their flexible coordination and redox properties. In here, the canonical iron-sulfur clusters, as well as the ones with other coordinating ligands will be described. The chapter has also been updated to account for the advances in the knowledge of complex iron-sulfur clusters of nitrogenase and hydrogenases. In addition, the role of iron-sulfur clusters in metabolic regulation, as sensors of gases (nitric oxide, oxygen), iron and cellular content of iron-sulfur clusters, cellular redox status, and redox cycling compounds, as well as their role in DNA processing enzymes, and their involvement in catalysis of a wide range of reactions will be described. Iron-sulfur clusters also participate in their biosynthetic and repair pathways. The knowledge in this field as evolved tremendously in recent years, which would require a complete chapter devoted to it by itself, reason why the authors have decided not to include this subject in this chapter. The chapter is an update of the one published in the previous edition, focusing on the recent advances mostly on the iron-sulfur clusters involved in new catalytic functions, sensor mechanisms and DNA processing.

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.

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.

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.

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.

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.

Two cyclen-derived Gd probes, [Gd-DOTAM](3+) and [Gd-DOTP](5-) (DOTAM = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetamide; DOTP = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylenephosphonate)), were assessed as paramagnetic relaxation enhancement (PRE)-inducing probes for characterization of protein-protein interactions. Two proteins, Desulfovibrio gigas rubredoxin and Desulfovibrio gigas cytochrome c(3), were used as model partners. In a (1)H NMR titration it was shown that [Gd-DOTP](5-) binds to cytochrome c(3) near heme IV, causing pronounced PREs, characterized by line width broadenings of the heme methyl resonances at ratios as low as 0.08. A K(d) of 23 +/- 1 muM was calculated based on chemical shift perturbation of selected heme methyl resonances belonging to three different heme groups, caused by allosteric effects upon [Gd-DOTP](5-) binding to cytochrome c(3) at a molar ratio of 2. The other probe, [Gd-DOTAM](3+), caused PREs on a well-defined patch near the metal center of rubredoxin (especially the patch constituted by residues D19-G23 and W37-S45, which broaden beyond detection). This effect was partially reversed for some resonances (C6-Y11, in particular) when cytochrome c(3) was added to this system. Both probes were successful in causing reversible PREs at the partner binding site, thus showing to be good probes to identify partners' binding sites and since the interaction is reversible to structurally characterize protein complexes by better defining the complex interface.

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.

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.

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.

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.

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.

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.

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.

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.