Aerssens E, Tiedje J M, Averill B A
J Biol Chem. 1986 Jul 25;261(21):9652-6.
The mechanism of the denitrification and nitrosation reactions catalyzed by the heme cd-containing nitrite reductase from Pseudomonas stutzeri JM 300 has been studied with whole cell suspensions using H2(18)O, 15NO, and 15NO-2. The extent of H2(18)O exchange with the enzyme-bound nitrosyl intermediate, as determined by the 18O content of product N2O, decreased with increasing nitrite concentration, which is consistent with production of N2O by sequential reaction of two nitrite ions with the enzyme. Reaction of NO with whole cells in H2(18)O gave amounts of 18O in the N2O product consistent with equilibration of nitric oxide with a small pool of free nitrite. Using 15NO and NH2OH, competition between denitrification and nitrosation reactions was demonstrated, as is required if the enzyme-nitrosyl complex is an intermediate in both nitrosation and denitrification reactions. The first evidence for exchange of 18O between H2(18)O and a nitrosation intermediate occurring after the enzyme-nitrosyl complex, presumably an enzyme-bound nitrosamine, has been obtained. The collective results are most consistent with denitrification N2O originating via attack of NO-2 on a coordinated nitrosyl, as proposed earlier (Averill, B. A., and Tiedje, J. M. (1982) FEBS Lett. 138, 8-11).
利用H₂¹⁸O、¹⁵NO和¹⁵NO₂，通过全细胞悬浮液研究了来自施氏假单胞菌JM 300的含血红素cd的亚硝酸还原酶催化的反硝化和亚硝化反应机制。根据产物N₂O的¹⁸O含量确定，H₂¹⁸O与酶结合的亚硝酰中间体的交换程度随亚硝酸盐浓度的增加而降低，这与两个亚硝酸盐离子与该酶的顺序反应产生N₂O一致。在H₂¹⁸O中，NO与全细胞反应生成的N₂O产物中的¹⁸O量与一氧化氮与少量游离亚硝酸盐池的平衡一致。使用¹⁵NO和NH₂OH，证明了反硝化和亚硝化反应之间的竞争，如果酶-亚硝酰复合物是亚硝化和反硝化反应的中间体，则需要这种竞争。已经获得了关于H₂¹⁸O与酶-亚硝酰复合物之后（可能是酶结合的亚硝胺）的亚硝化中间体之间进行¹⁸O交换的首个证据。总体结果与如前所述（Averill, B. A., and Tiedje, J. M. (1982) FEBS Lett. 138, 8 - 11）通过NO₂⁻攻击配位的亚硝酰产生反硝化N₂O最为一致。
