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...

Multifrequency electron paramagnetic resonance (EPR) spectra of the Cu(II) site in nitrous oxide reductase (N2OR) from Pseudomonas stutzeri confirm the assignment of the low field g value at 2.18 consistent with the seven line pattern observed at 9.31 GHz, 10 K. S‐band spectra at 20 K are better resolved than the X‐band spectra recorded at 10 K. The features observed at 2.4, 3.4, 9.31 and 35 GHz are explained by a mixed‐valence [Cu(1.5)..Cu(1.5)] S= 1/2 species with the unpaired electron delocalized between two equivalent Cu nuclei. The resemblance of the N2OR S‐band spectra to the spectra for the EPR‐detectable Cu of cytochrome c oxidase suggests that the S‐band spectrum for cytochrome c oxidase measured below 30 K may also contain hyperfine splittings from two approximately equivalent Cu nuclei.

In mammals, lactation performance may influence future reproduction of a mother and her young. We measured the quantity and quality of milk produced by female house mice (Mus musculus) during a period of 28 days after birth of a litter. We aimed to analyse how females cope with the energy demands of different sized litters as a tradeoff between current and future reproduction. Litter sizes examined were small (six young), intermediate (7.3 0–7), and large (12 young). Females met the energy demand of a growing litter both by increasing the amount of milk given to the young and by improving the quality (through an increase in total solid and fat concentrations) to a peak during days 9–16. At the onset of weaning (day 17), milk production decreased and young shifted to solid food. The main energy source in the milk of house mice was fat, which provided more than 80% of the energy to the suckling young. Average lipid concentrations were 20%, and peak values of 33% were reached during days 13–16. Protein concentrations were 6–8% and carbohydrate (lactose) concentrations 3–4%. With larger litters, females increased both the amount of milk and the absolute amount of major nutrients (lipids, proteins, lactose). However, regulation was imperfect. When litter size was doubled from six to 12, amount of milk produced rose by 44%, and energy content only rose by 30%. The reduction in milk supply for individual young in larger litters was reflected in slower growth and lower weaning weight. The efficiency of conversion of milk energy into biomass of young was highest for intermediate litters (51%; for small litters, 43%; for large ones, 36%). During days 5–16, a single pup in an intermediate litter needed less energy for metabolism and growth than pups in the other litter sizes examined. Although individual young of small litters have a relatively high weaning weight‐which might improve their future reproduction‐females gain higher reproductive success by dividing the energy available per litter between the largest number of young they can raise to a weaning weight of on average 9 g. For the time‐lifen reproductive success of a female house mouse‐at least for the strain used in this study‐an intermediate litter size of seven seems to be best because of a favourable ratio of energy cost of lactation to number and size of young produced.

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The single conserved Cys165 outside of the Cu, domain of nitrous oxide reductase (N,OR) from Pseudomonas stutzeri was mutated to glycine to test its presumed function in metal coordination of the catalytic site, Cu,. The point mutation reduced the cellular level of N,OR 5-10-fold compared to the level of the control strain. In the mutant, the activity and the Cu content of the enzyme, as well as the transcript level of the N,OR structural gene, nos2, remained unaffected. The mutant enzyme was processed and exported into the periplasm like the wild-type enzyme. Chemical analysis for sulfhydryl groups gave about nine -SH groupdmonomer of the apoenzyme prepared from the wild-type enzyme, in accordance with the nine cysteine residues of the derived amino acid sequence. Eight -SH groups were found to form disulfide bridges in the holoenzyme dimer. We propose that in the native state of the enzyme Cys165 does not bind to Cu,, but may be part of a disulfide bridge essential for the stability of N,OR. Immediately downstream of the genes nosDFx encoding the components for Cu incorporation into the reductase, we have identified the open reading frame, ORFL, whose derived product has the signature of a protein disulfide isomerase.Keywords: nitrous oxide reductase ; copper protein ; disulfide bond; site-directed mutagenesis ; protein disulfide-isomerase.Nitrous oxide reductase (the nos2 product) is a periplasmic multicopper enzyme. It undergoes Cu insertion and maturation as part of, or subsequent to, the export process [l]. The catalytic site of N,O reductase (N,OR) is thought to be a binuclear type-3 copper, Cu, [2, 31. Studies of the electronic absorbance, EPR and magnetic circular dichroism spectra of the oxidized, partially reduced and reduced states of the enzyme, led to the conclusion that Cu, contains at least one cysteine ligand and cycles between a diamagnetic oxidized Cu(II)/Cu(II) and an S = 1/2, mixed-valence state, [Cu,(l .5 The identification of genes that affect protein maturation has emphasized the importance of protein disulfide isomerases for the folding process in the periplasm. Several ancillary components for protein folding in this compartment have been identified [7, 81. Evidence for disulfide bonds in the periplasmic N,OR can reveal indirectly the necessity of a protein disulfide isomerase as part of the posttranslocational maturation process.Disulfide and sulfhydryl groups have not been determined by direct chemical means for any of the eight purified N,O reductases. Here, we identify a new gene which is part of the nos cluster [9] and has the putative function of a disulfide isomerase. Pseudomonas stutzeri N,OR carries at position 16.5 the only cysteine residue that is conserved outside the Cu, domain. We have investigated, by site-directed mutagenesis, whether or not this residue is an active ligand of Cu,. The substitution of Cys16.5 for glycine destabilized N,OR (manifested by a strong decrease in its cellular level) but retained its catalytic activity. These findings indicate that Cys165 is no...