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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]

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]

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: 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]

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]

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]