Nitrous oxide reductase from Pseudomonas stutzeri

Abstract

The oxidation-reduction and spectroscopic properties of various forms of nitrous oxide reductase from Pseudomonas stutzeri were investigated. The high-activity form I of the enzyme (purple, 8 Cu, M , 140000) was reduced by a large variety of cationic, anionic and photochemically generated agents. The blue form I11 was the only product found in these experiments under anaerobic conditions. Reductive (dithionite) and oxidative (ferricyanide) titrations showed that the conversion of the purple form I to the blue species I11 was fully reversible in the absence of dioxygen. Two kinetically different phases of the reaction of form I with a stoichiometric amount of dithionite (le--equivalent/Cu) were detected: in the fast phase (seconds), the purple chromophore with ; 1 , , , at 540 nm disappeared almost completely, whereas in the slower phase (minutes) the blue species with A, , , around 650 nm was generated. Irrespective of the nature of the reductant the blue species did not react even at large excess of reductant. It was reoxidized by ferricyanide, hydrogen peroxide and nitric oxide.A new, catalytically inactive derivative of nitrous oxide reductase (form V, 2 Cu, M , 140000) was isolated from a transposon Tn5-induced mutant with defective chromophore biosynthesis. The pink color of the mutant protein faded almost completely after addition of O.5e--equivalent/Cu. In this case no blue species was found, similar to earlier observations for the regenerated, catalytically inactive protein.Varying with the sample and the pH, 50 -80% of the total copper of form 1 was in an electron-paramagneticresonance-(EPR)-silent state as compared to 47% in the mutant protein. The broad, featureless EPR signal recorded at 9.32 GHz for the blue, reduced form I11 of nitrous oxide reductase represented approximately 20% of the total copper.For the blue species no resolution enhancement was achieved at 34 GHz. At this frequency both forms I and V showed similar EPR signals with apparent g-values at 2.16 and 1.99. At 9.32 GHz, form V had an EPR signal with glI at 2.18, All = 3.55 mT (4 or 5 lines, in contrast to form I) and gI at 2.03. Above 100 K the splitting of the g,, region into seven equidistant lines in the EPR signal of the high-activity form I and the hyperfine structure of the perpendicular transition disappeared.Carbon monoxide and nitric oxide, but not nitrous oxide, had marked effects on the spectroscopic properties of the purple form I. Marked effects were also obtained for the exogenous ligands nitrite, azide, cyanate and thiocyanate. The purple chromophore disappeared in the presence of these agents and the g,, region of the corresponding EPR spectra at 9.32 GHz broadened. No superhyperfine structure originating from the interaction between the Cu(I1) centers of nitrous oxide reductase and these ligands was detected. Nitric oxide also reacted with the reduced form I11 of the enzyme, giving a species with the spectroscopic properties of the pink form 11. A considerable amount of nitrite was generated in the reaction of nitr...

A model of the copper centres of nitrous oxide reductase (Pseudomonas stutzeri)

“…Addition of a trace of the mediator dye phenazine methosulphatc, (PMS) (I!$, = +80 mV), speeds up reduction generating a blue form absorbing at 650 nm. This is consistent with earlier reports that reduction of N,OR with sodium dithionite proceeds in 2 kinetic steps, a fast phase followed by a slower phase [4]. The second phase of reduction, catalysed by PMS, gives rise to an isosbestic point at 624 nm in the absorption spectrum.…”

“…2). The spectra of the oxidised and fully reduced forms agree well with those reported previously [3,4], the 7-line hyperfine structure of the oxidised state being clearly resolved. Integration of the signals shows that 22 and 10% of the total copper is paramagnetic in the oxidised and reduced forms, respectively.…”

“…A variety of spectroscopic methods have been applied to the study of the copper ccntres but no clear structural model has yet been proposed for them. Two oxidation levels of the enzyme have been observed previously [3,4]. The oxidised state, referred to as the 'purple' form, has EPR signals representing up to 30% of the total copper and absorption bands at 480 nm and 540 nm.…”

Formation of a cytochrome c–nitrous oxide reductase complex is obligatory for N2O reduction by Paracoccus pantotrophus

“…19 An optically monitored equilibrium redox titration of the anaerobically prepared form of the enzyme showed that, in the absence of substrate, two electrons are transferred, each in a Nernstian process at +260 mV and +60 mV, the former corresponding to the addition of one electron to Cu A and, the latter, of one electron to Cu Z . 19 The enzyme can be prepared with each monomer in a fully oxidized, one-electron reduced (in which only Cu A is reduced), 20 The steady state activities towards N 2 O reduction of all these forms of the enzyme, when assayed with HH cytochrome c are similar at 0.034 U mg −1 . 14 However, use of the artificial electron donors, benzyl viologen and methyl viologen rather than the natural electron donors, in steadystate kinetic measurements results in increased activity of the enzyme to ca.…”

The tetranuclear copper active site of nitrous oxide reductase: the CuZ center

“…Since N 2 OR was first isolated from Pseudomonas stutzeri (formerly Pseudomonas perfectomarina) [28,29], it has been purified and characterized from other bacterial sources, such as Rhodobacter capsulatus (formerly Rhodopseudomonas capsulate) [30], Rhodobacter sphaeroides f. sp. denitrificans [31], Paracoccus denitrificans [32], Wolinella succinogenes [33], Flexibacter canadensis [34], Achromobacter cycloclastes [35], Pseudomonas aeruginosa [36], Paracoccus pantotrophus (formerly Thiosphaera pantotropha) [37], Thiobacillus denitrificans [38], Alcaligenes xylosoxidans [39,40], Pseudomonas nautica (renamed Marinobacter hydrocarbonoclausticus) [41], and more recently Hyphomicrobium denitrificans [42].…”