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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]

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]

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]

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]

• 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]