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, Mr 140000) was reduced by a large variety of cationic, anionic and photochemically generated agents. The blue form III 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 III was fully reversible in the absence of dioxygen. Two kinetically different phases of the reaction of form I with a stoichiometric amount of dithionite (1e^--equivalent/Cu) were detected: in the fast phase (seconds), the purple chromophore with lmax at 540 nm disappeared almost completely, whereas in the slower phase (minutes) the blue species with lmax 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, Mr 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 0.5e^--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 I was in an electron- paramagnetic-resonance-(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 III 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 g_|| at 2.18, A_|| = 3.55 mT (4 or 5 lines, in contrast to form I) and g _| 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 gII region of the corresponding EPR spectra at 9.32 GHz broadened. No superhyperfine structure originating from the interaction between the Cu(II) centers of nitrous oxide reductase and these ligands was detected. Nitric oxide also reacted with the reduced form III of the enzyme, giving a species with the spectroscopic properties of the pink form II. A considerable amount of nitrite was generated in the reaction of nitric oxide and the purple form I, depending on the partial pressure and the reaction time.
When form I was mixed with hydrogen peroxide or potassium superoxide at 0°C, a blue intermediate with a broad shoulder around 640 nm was observed. The EPR spectrum of the reaction product showed the presence of type 2 Cu(II) centers with g_|| = 2.26, A_|| = 18.5 mT and g _| = 2.06.
The present results indicate that the coordination sphere of the purple Cu centers in nitrous oxide reductase are rather labile towards subtle changes in the environment such as pH and exogenous ligands. The spectroscopic properties of the blue species and its persistence in the presence of strong reductants point towards a catalytic site with Cu in a ¢reduced' state, stabilized by thiol or disulfide sulfur with substantial spin density delocalized onto sulfur.