Purification and characterization of nitrous oxide reductase from Pseudomonas aeruginosa strain P2.
Nitrous oxide reductase, which catalyzes the reduction of N2O to N2, was purified in a largely oxidized form from Pseudomonas aeruginosa strain P2 by a simple anaerobic procedure to yield an enzyme with a peptide purity of 95-98%. For the native (dimeric) enzyme, Mr = 120,000 and for the denatured subunit, Mr = 73,000. The enzyme contained four Cu atoms/subunit, was purple in color, and exhibited a broad absorption band at 550 nm with an extinction coefficient of about 11,000 M-1 x cm-1 referenced to the dimer. It was nearly inactive as prepared but could be activated by incubation with 2-(N-cyclohexylamino)ethane sulfonate buffer, pH 10, to specific activities as high as 27 mumol of N2O x min-1 x mg-1.Km for N2O and benzyl viologen radical cation was about 2 and 4 microM, respectively, both before and after enzyme activation. Activation increased the t1/2 for turnover-dependent inactivation from about 30 s to 5-10 min. Reduction of the enzyme by dithionite was kinetically biphasic and resulted in the loss of the 550-nm band and ultimate appearance of a 670-nm band. Isoelectric focusing revealed five components with pI values from 5.2 to 5.7. The pI values did not change following activation. The copper CD spectrum of the enzyme as prepared was different from that for the activated enzyme, whereas those for the enzyme after exposure to air and the activated enzyme were similar. Because the activated enzyme is a mixture of activated and inactive species, the specific activity of the activated species must be substantially greater than the observed value. Molecular heterogeneity may also explain the decreased optical absorbance and CD amplitude that resulted from the activation process. The data overall reinforce the view that the absorption spectrum of nitrous oxide reductase is not a good predictor of absolute activity.
SooHoo
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Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
Hollocher
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D000596:Amino Acids / Q000032:analysis; D002942:Circular Dichroism; D004227:Dithionite / Q000494:pharmacology; D004789:Enzyme Activation; D007526:Isoelectric Point; D007700:Kinetics; D009609:Nitrous Oxide / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013057:Spectrum Analysis
D000596:Amino Acids; D004227:Dithionite; D010088:Oxidoreductases; C022763:nitrous oxide reductase; D009609:Nitrous Oxide