Hollocher T C, Garber E, Cooper A J, Reiman R E
J Biol Chem. 1980 Jun 10;255(11):5027-30.
13N- and 15N-labeling experiments were carried out with Paracoccus denitrificans, grown anaerobically on nitrate, to determine whether hyponitrite might be an obligatory intermediate in denitrification and a precursor of nitrous oxide. From experiments designed to trap [13N]- or [15N,15N]hyponitrite by dilution into authentic hyponitrite it was calculated that the intracellular concentration of a presumptive hyponitrite pool must be less than 0.4 mM. In order for a pool of this size to turn over rapidly enough to handle the flux of nitrogen during dentrifucation, the spontaneous rate of hyponitrite dehydration must be enhanced by a factor of several thousand through enzyme catalysis. Cell extracts failed to catalyze this reaction under a variety of conditions. It is concluded that hyponitrite cannot be an intermediate in dentrification. In addition, the assimilation of inorganic nitrogen was studied in P. denitrificans using 13N as tracer. At low concentrations (less than 10(-8) M) of labeled nitrate and nitrite 5 to 10% of the label was assimilated into non-volatile metabolites and 90 to 95% was reduced to N2. Similarly, with 15 mM [13N]nitrate, 5% of the label went into metabolites and 95% to N2. High pressure liquid chromatography analysis of the labeled metabolites indicated that the major pathway for assimilation of inorganic nitrogen in P. denitrificans under these conditions is through ammonia incorporation via the aspartase reaction.
利用在硝酸盐上厌氧生长的反硝化副球菌进行了¹³N和¹⁵N标记实验，以确定连二次亚硝酸是否可能是反硝化作用中的必需中间体以及一氧化二氮的前体。从旨在通过稀释到真实连二次亚硝酸中来捕获[¹³N] - 或[¹⁵N,¹⁵N]连二次亚硝酸的实验计算得出，假定的连二次亚硝酸池的细胞内浓度必须小于0.4 mM。为了使这种大小的池能够足够快地周转以处理反硝化过程中的氮通量，连二次亚硝酸脱水的自发速率必须通过酶催化提高数千倍。在各种条件下，细胞提取物都未能催化该反应。得出的结论是，连二次亚硝酸不可能是反硝化作用的中间体。此外，使用¹³N作为示踪剂研究了反硝化副球菌中无机氮的同化作用。在低浓度（小于10⁻⁸ M）的标记硝酸盐和亚硝酸盐条件下，5%至10%的标记被同化为非挥发性代谢物，90%至95%被还原为N₂。同样，对于15 mM [¹³N]硝酸盐，5%的标记进入代谢物，95%进入N₂。对标记代谢物的高压液相色谱分析表明，在这些条件下反硝化副球菌中无机氮同化的主要途径是通过天冬氨酸酶反应掺入氨。
