Functional and structural comparison of nitric oxide reductases from denitrifying fungi Cylindrocarpon tonkinense and Fusarium oxysporum

Abstract

Two isozymes of nitric oxide reductase (Nor) from the denitrifying fungus Cylindrocarpon tonkinense (c.Nor1 and c.Nor2) are the heme-enzyme cytochrome P-450's (Usuda et al. (1995) Appl. Environ. Microbiol. 61, 883–889). However, they catalyze the NO reduction to N 2O, but not the monooxygenation reaction using O 2. We kinetically and spectrophotometrically studied the reactions of the two Nor's with NO and electron donor, NAD(P)H, using flash-photolysis and stopped-flow rapid scan methods. The enzyme in the Fe 3+ state can bind NO to yield the Fe 3+NO complex. When the resultant Fe 3+NO complex reacted with the electron donor, it was converted to the Fe 3+ enzyme via a transient formation of the characteristic intermediate ( I). The spectroscopic results were essentially the same as those of the Nor from another denitrifying fungus Fusarium oxysporum (f.Nor), which we previously reported (Shiro et al. (1995) J. Biol. Chem. 270, 1617–1623), suggesting that these fungal Nor's catalyze the NO reduction by the same mechanism. Most probably, the Fe 3+NO complex of the Nor is reduced with two-electrons directly transferred from NAD(P)H to yield the intermediate I, and then the I reacts with another NO to generate N 2O and the Fe 3+ enzyme. However, the kinetic measurements showed that the reaction rate constant of each step was variable depending on the combination of the Nor and the electron donor; i.e., c.Nor1+NADH, c.Nor2+NADPH, c.Nor2+NADH and f.Nor+NADH. In particular, the rate constant for the electron transfer step from the electron donor to the Fe 3+NO enzyme is dramatically different among these systems. On the other hand, we also measured paramagnetically shifted 1H-NMR spectra of c.Nor2 and f.Nor in the ferrous (reduced) state, where the iron-bound Cys β-CH 2 signal was observed at the same position (≈270 ppm) for c.Nor2 and f.Nor, indicating that the Cys thiolate (S −) coordinates to the heme iron in the same fashion in the Nor's. However, the heme peripheral proton signals were subtly but significantly different in their positions between the two enzymes. On the basis of these kinetic and spectroscopic data, we suggested that the Fe-S − binding character is not essential for the NO reduction reactivity, but that the subtle difference in interaction of their hemes with the surroundings is possibly responsible for the difference in the Nor reactivity, especially in the electron transfer step from NAD(P)H to the Fe 3+NO moiety.