Stéphane Besson

A novel type of catalytic copper cluster in nitrous oxide reductase

Nitrous oxide (N2O) is a greenhouse gas, the third most significant contributor to global warming. As a key process for N2O elimination from the biosphere, N2O reductases catalyze the two-electron reduction of N2O to N2. These 2 × 65 kDa copper enzymes are thought to contain a CuA electron entry site, similar to that of cytochrome c oxidase, and a CuZ catalytic center. The copper anomalous signal was used to solve the crystal structure of N2O reductase from Pseudomonas nautica by multiwavelength anomalous dispersion, to a resolution of 2.4 Å. The structure reveals that the CuZ center belongs to a new type of metal cluster, in which four copper ions are liganded by seven histidine residues. N2O binds to this center via a single copper ion. The remaining copper ions might act as an electron reservoir, assuring a fast electron transfer and avoiding the formation of dead-end products.

A cytochrome c peroxidase from Pseudomonas nautica 617 active at high ionic strength: expression, purification and characterization.

Cytochrome c peroxidase was expressed in cells of Pseudomonas nautica strain 617 grown under microaerophilic conditions. The 36.5 kDa dihaemic enzyme was purified to electrophoretic homogeneity in three chromatographic steps. N-terminal sequence comparison showed that the Ps. nautica enzyme exhibits a high similarity with the corresponding proteins from Paracoccus denitrificans and Pseudomonas aeruginosa. UV-visible spectra confirm calcium activation of the enzyme through spin state transition of the peroxidatic haem. Monohaemic cytochrome c(552) from Ps. nautica was identified as the physiological electron donor, with a half-saturating concentration of 122 microM and allowing a maximal catalytic centre activity of 116,000 min(-1). Using this cytochrome the enzyme retained the same activity even at high ionic strength. There are indications that the interactions between the two redox partners are mainly hydrophobic in nature.

Measuring the cytochrome C nitrite reductase activity-practical considerations on the enzyme assays.

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.

Electron transfer and docking between cytochrome cd1 nitrite reductase and different redox partners - A comparative study.

Cytochrome cd1 nitrite reductases (cd1NiRs) 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 cd1NiR (Mhcd1NiR) 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) c552 providing the highest value. In situations where cyt c552 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 Mhcd1NiR and cyt c552 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 c552 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 Mhcd1NiR and different types of redox mediators, efficient ET is only observed with the putative physiological electron donor cyt c552.

Spectrophotometric confirmation for oxygen ligation to the reduced haem d 1 of Pseudomonas nautica nitrite reductase

Cytochrome cd 1 was discovered in the Gram-negative bacterium Pseudomonas (Ps.) aeruginosa by Horio and coworkers [1]. It was tagged a cytochrome oxidase on the base of its capacity to reduce oxygen. Only a few years later was its main, if not only, physiological function as a nitrite reductase made clear [2]. Numerous articles have since been published on the spectrophotometry of cytochromes cd 1 isolated from various bacteria incuding Ps. aeruginosa (see [3] for an overview) or Ps. nautica [4]. It was earlier believed that the nature of the electron donor was responsible for the considerable discrepancies observed between visible spectra of the same cytochrome cd 1, in the regions of reduced haem d 1 absorption peaks [5]. However, Shimada and Orii [6] demonstrated that an oxygenated-haem d 1 reaction intermediate was the main reason for these discrepancies. Optical spectra of Ps. nautica cytochrome cd 1 recorded under anaerobic conditions still exhibited differences and an investigation was decided to assess and compare respective effects of pH, buffer nature and level of oxygen contamination.

Spectroscopic Characterization of the Nitrate Reductase from Pseudomonas Nautica 617

The anaerobic electron transfer system of the marine denitrifying bacterium Pseudomonas (Ps.) nautica strain 617 has been studied in detail [1] and several soluble metalloproteins have been isolated and characterized including monohemic cytochromes [2, 3], a dihemic cytochrome c 549 [4], a cytochrome cd 1—type nitrite reductase [5] and a 7Fe-containing ferredoxin [6].

Spectroscopic Properties of the Cytochrome CD1 from the Marine Denitrifier Pseudomonas Nautica

Previous studies on the anaerobic electron transfer system of the marine denitrifier Pseudomonas (Ps.) nautica strain 617 allowed the purification and the characterization of three major soluble proteins: a 7 Fe — containing ferredoxin [1], the monohemic cytochrome c 552 [2] and the dihemic cytochrome c 549 [3].