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Identifying redox partners and the interaction surfaces is crucial for fully understanding electron flow in a respiratory chain. In this study, we focused on the interaction of nitrous oxide reductase (NOR), which catalyzes the final step in bacterial denitrification, with its physiological electron donor, either a c-type cytochrome or a type 1 copper protein. The comparison between the interaction of NOR from three different microorganisms, Pseudomonas nautica, Paracoccus denitrificans, and Achromobacter cycloclastes, with their physiological electron donors was performed through the analysis of the primary sequence alignment, electrostatic surface, and molecular docking simulations, using the bimolecular complex generation with global evaluation and ranking algorithm. The docking results were analyzed taking into account the experimental data, since the interaction is suggested to have either a hydrophobic nature, in the case of P. nautica NOR, or an electrostatic nature, in the case of P. denitrificans NOR and A. cycloclastes NOR . A set of well-conserved residues on the NOR surface were identified as being part of the electron transfer pathway from the redox partner to NOR (Ala495, Asp519, Val524, His566 and Leu568 numbered according to the P. nautica NOR sequence). Moreover, we built a model for Wolinella succinogenes NOR, an enzyme that has an additional c-type-heme-containing domain. The structures of the NOR domain and the c-type-heme-containing domain were modeled and the full-length structure was obtained by molecular docking simulation of these two domains. The orientation of the c-type-heme-containing domain relative to the NOR domain is similar to that found in the other electron transfer complexes. [ABSTRACT FROM AUTHOR]

Examines the nature and properties of the complex electron-transfer chain formed between flavodoxin and cytochrome c3. Nuclear magnetic resonance protein titrations; Prediction of protein structure by homology modeling; Analysis of the surface electrostatic potentials; Computer graphics modeling of the complex.

Copper-containing nitrous oxide reductase catalyzes a 2-electron reduction of the green-house gas N2O to yield N2. It contains two metal centers, the binuclear electron transfer site CuA, and the unique, tetranuclear CuZ center that is the site of substrate binding. Different forms of the enzyme were described previously, representing variations in oxidation state and composition of the metal sites. Hypothesizing that many reported discrepancies in the structural data may be due to radiation damage during data collection, we determined the structure of anoxically isolated Marinobacter nauticus N2OR from diffraction data obtained with low-intensity X-rays from an in-house rotating anode generator and an image plate detector. The data set was of exceptional quality and yielded a structure at 1.5 Å resolution in a new crystal form. The CuA site of the enzyme shows two distinct conformations with potential relevance for intramolecular electron transfer, and the CuZ cluster is present in a [4Cu:2S] configuration. In addition, the structure contains three additional types of ions, and an analysis of anomalous scattering contributions confirms them to be Ca2+, K+, and Cl–. The uniformity of the present structure supports the hypothesis that many earlier analyses showed inhomogeneities due to radiation effects. Adding to the earlier description of the same enzyme with a [4Cu:S] CuZ site, a mechanistic model is presented, with a structurally flexible CuZ center that does not require the complete dissociation of a sulfide prior to N2O binding. The [4Cu:2S] CuZ site in M. nauticus N 2O reductase. The electron density map shown is contoured at the 5 σ level, highlighting the presence of two sulfide ligands. 705x677mm (72 x 72 DPI) [ABSTRACT FROM AUTHOR]

Rubredoxins (Rds) and their derivatives have been extensively used, in the last few decades, in order to elucidate structure and functional aspects of metal sites involving rich sulphur coordination spheres. The derivatives have been designed with two main purposes: (a) replacement of selective and specific amino acid residues in native systems by site direct mutagenesis, and (b) replacement of native metal ion (iron), producing novel metal sites. We will highlight in this review the key amino acid residues, recognized in native Rds, directly involved in electron transfer mechanisms. By protein template assisted synthesis, metal-substituted Rds are used as structural probes and bio-models. The tetra-cysteinyl metal coordination site in Rd has the surprising capacity of chelating a wide variety of metal ions (other than native iron), and this chemistry has been witnessing significant growth based on two main interests: (i) metals such as 57 Fe, Zn, Co, Cd, Ga, In, and Hg were introduced and derivatives were synthesized in order to specifically probe structural determinants imposed by the protein, and (ii) Zn, Ni, Cu, as well as Mo (and most recently, W), were used with a particular interest: to model complex metal centres in sulphur-rich enzymes. The latter, in particular, is discussed in the context of Rd serving as a model complex of native enzymes, as well as the synthesis of small inorganic complexes. The review aims to bring synthetic biochemistry and synthetic inorganic chemistry together in a synergistic way. [ABSTRACT FROM AUTHOR]

The cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC 27774 is able to reduce nitrite to ammonia in a six-electron transfer reaction. Although extensively characterized from the spectroscopic and structural points-of-view, some of its kinetic aspects are still under explored. In this work the kinetic behaviour of ccNiR has been evaluated in a systematic manner using two different spectrophotometric assays carried out in the presence of different redox mediators and a direct electrochemical approach. Solution assays have proved that the specific activity of ccNiR decreases with the reduction potential of the electronic carriers and ammonium is always the main product of nitrite reduction. The catalytic parameters were discussed on the basis of the mediator reducing power and also taking into account the location of their putative docking sites with ccNiR. Due to the fast kinetics of ccNiR, electron delivering from reduced electron donors is rate-limiting in all spectrophotometric assays, so the estimated kinetic constants are apparent only. Nevertheless, this limitation could be overcome by using a direct electrochemical approach which shows that the binding affinity for nitrite decreases whilst turnover increases with the reductive driving force. [ABSTRACT FROM AUTHOR]

Abstract: 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 unpaired 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 M21 and M181. 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 1HNMR 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. [Copyright &y& Elsevier]

The kinetics of electron transfer between the Desulfovibro gigas hydrogenase and several electron-transfer proteins from Desulfovibrio species were investigated by cyclic voltammetry, square-wave voltammetry and chronoamperometry. The cytochrome C3 from Desulfovibrio vulgaris (Hildenborough). Desulfovibrio desulfuricans (Norway 4), Desulfovibrio desulfuricans (American Type Culture Collection 27774) and D. gigas (NCIB 9332) were used as redox carriers. They differ in their redox potentials and isoelectric point. Depending on the pH, all the reduced forms of these cytochromes were effective in electron exchange with hydrogenase. Other small electron-transfer proteins such as ferredoxin I, ferredoxin II and rubredoxin from D. gigas were tentatively used redox carriers. Only ferredoxin II was effective in mediating electron exchange between hydrogenase and the working electrode. The second-order rate constants k for the reaction between reduced proteins and hydrogenase were calculated based on the theory of the simplest electrocatalytic mechanism [Moreno, C., Costa, C., Moura, I., Le Gall, J., Liu, M. Y., Payne, W.J., van Dijk, C. & Moura. J.J.G. (1193) Eur. J.. Biochem. 212. 79-86] and the results obtained by cyclic voltammetry were compared with those obtained by chronoamperometry. Values for k of 105-106 M&sup-1; s&sup-1; (cytochrome c1 as electron carrier) and 104M&sup-1; s&sup-1; (ferredoxin II as the electron carrier) were determined. The rate-constant values are discussed in terms of the existence of an electrostatic interaction between the electrode surface and the redox carrier and between the redox carrier and a positively charged part of the enzyme. [ABSTRACT FROM AUTHOR]

The electron-transfer kinetics between three different mediators and the hexahemic enzyme nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774) were investigated by cyclic voltammetry and by chronoamperometry. The mediators, methyl viologen, Desulfovibrio vulgaris (Hildenborough) cytochrome c3 and D. desulfuricans (ATCC 27774) cytochrome c3 differ in structure, redox potential and charge. The reduced form of each mediator exchanged electrons with nitrite reductase. Second-order rate constants, k, were calculated on the basis of the theory for a simple catalytic mechanism and the results, obtained by cyclic voltammetry, were compared with those obtained by chronoamperometry. Values for k are in the range 106-108 M-1 s-1 and increase in the direction D. desulfuricans cytochrome c3→D. vulgaris cytochrome c3→methyl viologen. An explanation is advanced on the basis of electrostatic interactions and relative orientation between the partners involved. Chronoamperometry (computer controlled) offers advantages over cyclic voltammetry in the determination of homogeneous rate constants (faster, more accurate and better reproducibility). Direct, unmediated electrochemical responses of the hexaheme nitrite reductase were also reported. [ABSTRACT FROM AUTHOR]

Highlights • A third-generation biosensor based on nitric oxide reductase (NOR) was developed. • A MWCNTs/BMIMBF 4 nanocomposite was optimized to entrap NOR. • PGE/[MWCNTs/BMIMBF 4 /NOR] direct electron transfer behavior was characterized. • High sensitivity was reached towards NO unmediated bioelectrocatalytic reduction. • The biosensor kept 79–116% of its initial response after one month. Abstract Nitric oxide (NO) has a crucial role in signaling and cellular physiology in humans. Herein, a novel third-generation biosensor based on the Marinobacter hydrocarbonoclasticus metalloenzyme (nitric oxide reductase (NOR)), responsible for the NO reduction in the denitrifying processes, was developed through the direct adsorption of a new nanocomposite (multiwalled carbon nanotubes (MWCNTs)/1 -n- butyl-3-methylimidazolium tetrafluoroborate (BMIMBF 4)/NOR) onto a pyrolytic graphite electrode (PGE) surface. The NOR direct electron transfer behavior (formal potential of -0.255 ± 0.003 V vs. Ag/AgCl) and electrocatalysis towards NO reduction (−0.68 ± 0.03 V vs. Ag/AgCl) of the PGE/[MWCNTs/BMIMBF 4 /NOR] biosensor were investigated in phosphate buffer at pH 6.0. Large enzyme loading (2.04 × 10−10 mol/cm2), acceptable electron transfer rate between NOR and the PGE surface (k s = 0.35 s-1), and high affinity for NO (K m = 2.17 μmol L-1) were observed with this biosensor composition. A linear response to NO concentration (0.23–4.76 μmol L-1) was perceived with high sensitivity (0.429 μA/μmolL-1), a detection limit of 0.07 μmol L-1, appropriate repeatability (9.1% relative standard deviations (RSD)), reproducibility (6.0–11% RSD) and 80–102% recoveries. The biosensor was stable during 1 month retaining 79–116% of its initial response. These data confirmed that NOR incorporated in the MWCNTs/BMIMBF 4 nanocomposite can efficiently maintain its bioactivity paving a new and effective way for NO biosensing. [ABSTRACT FROM AUTHOR]

Abstract Understanding the direct electron transfer processes between redox proteins and electrode surface is fundamental to understand the proteins mechanistic properties and for development of novel biosensors. In this study, nitric oxide reductase (NOR) extracted from Marinobacter hydrocarbonoclasticus bacteria was adsorbed onto a pyrolytic graphite electrode (PGE) to develop an unmediated enzymatic biosensor (PGE/NOR)) for characterization of NOR direct electrochemical behaviour and NOR electroanalytical features towards NO and O 2. Square-wave voltammetry showed the reduction potential of all the four NOR redox centers: 0.095 ± 0.002, −0.108 ± 0.008, −0.328 ± 0.001 and −0.635 ± 0.004 V vs. SCE for heme c , heme b , heme b 3 and non-heme Fe B , respectively. The determined sensitivity (−4.00 × 10−8 ± 1.84 × 10−9 A/μM and - 2.71 × 10−8 ± 1.44 × 10−9 A/μM for NO and O 2 , respectively), limit of detection (0.5 μM for NO and 1.0 μM for O 2) and the Michaelis Menten constant (2.1 and 7.0 μM for NO and O 2 , respectively) corroborated the higher affinity of NOR for its natural substrate (NO). No significant interference on sensitivity towards NO was perceived in the presence of O 2 , while the O 2 reduction was markedly and negatively impacted (3.6 times lower sensitivity) by the presence of NO. These results clearly demonstrate the high potential of NOR for the design of innovative NO biosensors. Highlights • Direct electron transfer of nitric oxide reductase (NOR) was described. • NOR bioelectrocatalytic activity towards NO and O 2 reduction was characterized. • Lower Michaelis Menten constant was obtained for NO than for O 2. • O 2 has no significant effect on NOR sensitivity towards NO reduction. • The unmediated NOR-based biosensor can be useful for NO sensing in biological events. [ABSTRACT FROM AUTHOR]

Marinobacter hydrocarbonoclasticus nitric oxide reductase, c NOR, is an integral membrane protein composed of two subunits with different roles, NorC (electron transfer) and NorB (catalytic) that receives electrons from the soluble cytochrome c 552 and reduces nitric oxide to nitrous oxide in the denitrification pathway. The solvent-exposed domain of NorC, harboring a c -type heme was heterologously produced, along with its physiological electron donor, cytochrome c 552 . These two proteins were spectroscopically characterized and shown to be similar to the native proteins, both being low-spin and Met-His coordinated, with the soluble domain of NorC presenting some additional features of a high-spin heme, which is consistent with the higher solvent accessibility of its heme and weaker coordination of the methionine axial ligand. The electron transfer complex between the two proteins has a 1:1 stoichiometry, and an upper limit for the dissociation constant was estimated by 1 H NMR titration to be 1.2 ± 0.4 μM. Electrochemical techniques were used to characterize the interaction between the proteins, and a model structure of the complex was obtained by molecular docking. The electrochemical observations point to the modulation of the NorC reduction potential by the presence of NorB, tuning its ability to receive electrons from cytochrome c 552 . [ABSTRACT FROM AUTHOR]

Cytochrome cd 1 nitrite reductases ( cd 1 NiRs) catalyze the reduction of nitrite to nitric oxide in denitrifying bacteria, such as Marinobacter hydrocarbonoclasticus . Previous work demonstrated that the enzymatic activity depends on a structural pre-activation triggered by the entry of electrons through the electron transfer (ET) domain, which houses a heme c center. The catalytic activity of M. hydrocarbonoclasticus cd 1 NiR ( Mhcd 1 NiR) was tested by mediated electrochemistry, using small ET proteins and chemical redox mediators. The rate of enzymatic reaction depends on the nature of the redox partner, with cytochrome (cyt) c 552 providing the highest value. In situations where cyt c 552 is replaced by either a biological (cyt c from horse heart) or a chemical mediator the catalytic response was only observed at very low scan rates, suggesting that the intermolecular ET rate is much slower. Molecular docking simulations with the 3D model structure of Mhcd 1 NiR and cyt c 552 or cyt c showed that hydrophobic interactions favor the formation of complexes where the heme c domain of the enzyme is the principal docking site. However, only in the case of cyt c 552 the preferential areas of contact and Fe–Fe distances between heme c groups of the redox partners allow establishing competent ET pathways. The coupling of the enzyme with chemical redox mediators was also found not to be energetically favorable. These results indicate that although low activity functional complexes can be formed between Mhcd 1 NiR and different types of redox mediators, efficient ET is only observed with the putative physiological electron donor cyt c 552 . [ABSTRACT FROM AUTHOR]

Abstract: The periplasmic nitrate reductase (NapAB) from Cupriavidus necator is a heterodimeric protein that belongs to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes and catalyzes the reduction of nitrate to nitrite. The protein comprises a large catalytic subunit (NapA, 91 kDa) containing the molybdenum active site plus one [4Fe–4S] cluster, as well as a small subunit (NapB, 17 kDa), which is a diheme c-type cytochrome involved in electron transfer. Crystals of the oxidized form of the enzyme diffracted beyond 1.5 Å at the European Synchrotron Radiation Facility. This is the highest resolution reported to date for a nitrate reductase, providing true atomic details of the protein active center, and this showed further evidence on the molybdenum coordination sphere, corroborating previous data on the related Desulfovibrio desulfuricans NapA. The molybdenum atom is bound to a total of six sulfur atoms, with no oxygen ligands or water molecules in the vicinity. In the present work, we were also able to prepare partially reduced crystals that revealed two alternate conformations of the Mo-coordinating cysteine. This crystal form was obtained by soaking dithionite into crystals grown in the presence of the ionic liquid [C4mim]Cl−. In addition, UV–Vis and EPR spectroscopy studies showed that the periplasmic nitrate reductase from C. necator might work at unexpectedly high redox potentials when compared to all periplasmic nitrate reductases studied to date. [Copyright &y& Elsevier]

The multicopper enzyme nitrous oxide reductase (N2OR) catalyzes the final step of denitrification, the two-electron reduction of N2O to N2. 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 c552 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 c552, 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 C552 concentration dependence, we estimate the following kinetic parameters: Kmc552 = 50.2 ± 9.0 4 µM and Vmaxc552 = 1.8 ± 0.6 units/mg. The N2O concentration dependence indicates a KmN2O of 14.0 ± 2.9 µM using MV as the electron donor. The pH effect on the kinetic parameters is different when MV or cytochrome c552 is used as the electron donor (pKa 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 ¹H 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 c552 is placed near a hydrophobic patch located around the CuA center. [ABSTRACT FROM AUTHOR]

Abstract: Cytochrome c nitrite reductase is a multicenter enzyme that uses a five-coordinated heme to perform the six-electron reduction of nitrite to ammonium. In the sulfate reducing bacterium Desulfovibrio desulfuricans ATCC 27774, the enzyme is purified as a NrfA2NrfH complex that houses 14 hemes. The number of closely-spaced hemes in this enzyme and the magnetic interactions between them make it very difficult to study the active site by using traditional spectroscopic approaches such as EPR or UV–Vis. Here, we use both catalytic and non-catalytic protein film voltammetry to simply and unambiguously determine the reduction potential of the catalytic heme over a wide range of pH and we demonstrate that proton transfer is coupled to electron transfer at the active site. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of the superoxide anioninto hydrogen peroxide and water.Treponema pallidum(Tp), the syphilis spirochete, expresses the gene for a superoxide reductase called neelaredoxin, having the iron protein rubredoxin as the putative electron donor necessary to complete the catalytic cycle. In this work, we present the first cloning, overexpression inEscherichia coliand purification of the Tp rubredoxin. Spectroscopic characterization of this 6 kDa protein allowed us to calculate the molar absorption coefficient of the 490 nm feature of ferric iron, e=6.9±0.4 mM-1 cm-1. Moreover, the midpoint potential of Tp rubredoxin, determined using a glassy carbon electrode, was -76±5 mV. Reduced rubredoxin can be efficiently reoxidized upon addition of Na2IrCl6-oxidized neelaredoxin, in agreement with a direct electron transfer between the two proteins, with a stoichiometry of the electron transfer reaction of one molecule of oxidized rubredoxin per one molecule of neelaredoxin. In addition, in presence of a steady-state concentration of superoxide anion, the physiological substrate of neelaredoxin, reoxidation of rubredoxin was also observed in presence of catalytic amounts of superoxide reductase, and the rate of rubredoxin reoxidation was shown to be proportional to the concentration of neelaredoxin, in agreement with a bimolecular reaction, with a calculatedkapp=180 min-1. Interestingly, similar experiments performed with a rubredoxin from the sulfate-reducing bacteriaDesulfovibrio vulgarisresulted in a much lower value ofkapp=4.5 min-1. Altogether, these results demonstrated the existence for a superoxide-mediated electron transfer between rubredoxin and neelaredoxin and confirmed the physiological character of this electron transfer reaction. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cytochrome c peroxidase (CCP) catalyses the reduction of H2O2 to H2O, an important step in the cellular detoxification process. The crystal structure of the di-heme CCP from Pseudomonas nautica 617 was obtained in two different conformations in a redox state with the electron transfer heme reduced. Form IN, obtained at pH 4.0, does not contain Ca2+ and was refined at 2.2 Å resolution. This inactive form presents a closed conformation where the peroxidatic heme adopts a six-ligand coordination, hindering the peroxidatic reaction from taking place. Form OUT is Ca2+ dependent and was crystallized at pH 5.3 and refined at 2.4 Å resolution. This active form shows an open conformation, with release of the distal histidine (His71) ligand, providing peroxide access to the active site. This is the first time that the active and inactive states are reported for a di-heme peroxidase. [Copyright &y& Elsevier]

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[SUB550] at different sites on its molecular surface. Here we use [SUB1]H NMR spectroscopy, analytical ultracentrifugation, molecular docking simulation, and microcalorimetry to investigate whether these small cytochromes can be accommodated simultaneously in the formation of a temary 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[SUB550] fully occupies its binding site on cytochrome c peroxidase. Docking experiments in which favored binary solutions of cytochrome c[SUB550] 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[SUB550] 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[SUB550] and binding of cytochrome c[SUB550] 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. [ABSTRACT FROM AUTHOR]

• Direct electrochemistry showed two signals assigned to CuA and CuZ(4Cu2S) centres. • CuA reduction potential is pH dependent in the presence of CuZ*(4Cu1S) • CuA centre is controlled by two proton-coupled electron transfer process. • "CuZ" reduction is controlled by a proton-coupled electron transfer process. • Enhanced catalytic potential consistent with the intermediate CuZ0(4Cu1S) Reduction of N 2 O to N 2 is catalysed by nitrous oxide reductase in the last step of the denitrification pathway. This multicopper enzyme has an electron transferring centre, CuA, and a tetranuclear copper-sulfide catalytic centre, "CuZ", which exists as CuZ*(4Cu1S) or CuZ(4Cu2S). The redox behaviour of these metal centres in Marinobacter hydrocarbonoclasticus nitrous oxide reductase was investigated by potentiometry and for the first time by direct electrochemistry. The reduction potential of CuA and CuZ(4Cu2S) was estimated by potentiometry to be +275 ± 5 mV and +65 ± 5 mV vs SHE, respectively, at pH 7.6. A proton-coupled electron transfer mechanism governs CuZ(4Cu2S) reduction potential, due to the protonation/deprotonation of Lys397 with a p K ox of 6.0 ± 0.1 and a p K red of 9.2 ± 0.1. The reduction potential of CuA, in enzyme samples with CuZ*(4Cu1S), is controlled by protonation of the coordinating histidine residues in a two-proton coupled electron transfer process. In the cyclic voltammograms, two redox pairs were identified corresponding to CuA and CuZ(4Cu2S), with no additional signals being detected that could be attributed to CuZ*(4Cu1S). However, an enhanced cathodic signal for the activated enzyme was observed under turnover conditions, which is explained by the binding of nitrous oxide to CuZ0(4Cu1S), an intermediate species in the catalytic cycle. [ABSTRACT FROM AUTHOR]

Cytochrome b 5 reductase is an enzyme with the ability to generate superoxide anion at the expenses of NADH consumption. Although this activity can be stimulated by cytochrome c and could participate in the bioenergetic failure accounting in apoptosis, very little is known about other molecules that may uncouple the function of the cytochrome b 5 reductase. Naphthoquinones are redox active molecules with the ability to interact with electron transfer chains. In this work, we made an inhibitor screening against recombinant human cytochrome b 5 reductase based on naphthoquinone properties. We found that 5-hydroxy-1,4-naphthoquinone (known as juglone), a natural naphthoquinone extracted from walnut trees and used historically in traditional medicine with ambiguous health and toxic outcomes, had the ability to uncouple the electron transfer from the reductase to cytochrome b 5 and ferricyanide. Upon complex formation with cytochrome b 5 reductase, juglone is able to act as an electron acceptor leading to a NADH consumption stimulation and an increase of superoxide anion production by the reductase. Our results suggest that cytochrome b 5 reductase could contribute to the measured energetic failure in the erythrocyte apoptosis induced by juglone, that is concomitant with the reactive oxygen species produced by cytochrome b 5 reductase. Unlabelled Image • Juglone is a traditional drug with ambiguous health and toxic effects. • Juglone can uncouple the activity of the recombinant human erythrocytes C b 5 R. • Juglone is also able to act as an electron acceptor for the reductase. • There is a NADH consumption burst by the reductase in the presence of juglone. • O 2 ·- production is an outcome of the NADH:Juglone reductase activity of C b 5 R. [ABSTRACT FROM AUTHOR]

Nitrous oxide reductase (N[sub2]OR) catalyzes the two-electron reduction of N[sub2]O to N[sub2] and H[sub2]O in the last step of the bacterial denitrification process. It is a dimeric protein. The recently solved crystal structure of N[sub2]OR indicates that in each subunit there is a Cu[subA] center, which is the electron-transfer site, and a Cu[subZ] center, which is the catalytic site. The neighboring Cu[subA] and Cu[subZ] centers are from different subunits. The whole Cu[subZ] cluster is coordinated by seven His ligands with a weakly bound water at the Cu[subI]/Cu[subIV] edge, which is the substrate access site.