Sofia Pauleta

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.

In this work it is demonstrated that the characterization of c-type haem containing proteins by electrochemical techniques needs to be cautiously performed when using pyrolytic graphite electrodes. An altered form of the cytochromes, which has a redox potential 300 mV lower than that of the native state and displays peroxidatic activity, can be induced by interaction with the pyrolytic graphite electrode. Proper control experiments need to be performed, as altered conformations of the enzymes containing c-type haems can show activity towards the enzyme substrate. The work was focused on the study of the activation mechanism and catalytic activity of cytochrome c peroxidase from Paracoccus pantotrophus. The results could only be interpreted with the assignment of the observed non-turnover and catalytic signals to a non-native conformation state of the electron-transferring haem. The same phenomenon was detected for Met-His monohaem cytochromes (mitochondrial cytochrome c and Desulfovibrio vulgaris cytochrome c-553), as well as for the bis-His multihaem cytochrome c(3) from Desulfovibrio gigas, showing that this effect is independent of the axial coordination of the c-type haem protein. Thus, the interpretation of electrochemical signals of c-type (multi)haem proteins at pyrolytic graphite electrodes must be carefully performed, to avoid misassignment of the signals and incorrect interpretation of catalytic intermediates.

Marinobacter hydrocarbonoclasticus is a marine bacterium widespread in the Mediterranean sea and Atlantic Ocean, and growing at temperate temperatures. This bacterium can perform complete denitrification, reducing nitrate to molecular nitrogen under anaerobic conditions. Here the nitrite concentration, pH and nitrous oxide reductase activity was monitored during bacterial growth, showing that this bacterium can also perform complete denitrification under low oxygen tension, using lactate as carbon source, in the presence of nitrate as alternative electron acceptor. Nitrous oxide reductase activity was observed after 7 h of growth under low oxygen tensions, and is maintained constant after 48h. Nitrite concentration reaches its maximum at mid-exponential phase and in the stationary phase, at lower oxygen tensions is almost non-existent.

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.

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.

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.

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.

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.

Nitrous oxide is a potent greenhouse gas, whose atmospheric concentration has been increasing since the introduction of the Haber Bosch process led to the widespread use of nitrogenous fertilizers. One of the pathways to its destruction is reduction to molecular nitrogen by the enzyme nitrous oxide reductase found in denitrifying bacteria. This enzyme catalyzes the last step of the denitrification pathway. It has two copper centers, a binuclear CuA center, similar to the one found in cytochrome c oxidase, and the CuZ center, a unique tetranuclear copper center now known to possess either one or two sulfide bridges. Nitrous oxide reductase has been isolated in different forms, depending on the oxidation state and molecular forms of its Cu centers. Recently, the structure of a purple form, which has both centers in the oxidized state, revealed that the CuZ center has the form [Cu4S2]. This review summarizes the biogenesis and regulation of nitrous oxide reductase, and the spectroscopic and kinetic properties of nitrous oxide reductase. The proposed activation and catalytic mechanism, as well as, electron transfer pathways are discussed in the light of the various structures of the CuZ center. (C) 2012 Published by Elsevier B.V.

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 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.

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.

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 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.