BACTERIAL NITRATE REDUCTASES .I. SUBSTRATES, PARTICULATE STATE, AND INHIBITORS OF ENZYME A

In addition to nitrate chlorate is utilized as a substrate. The enzymes of A. aerogenes and M. denitrificans probably catalyze also the reduction of bromate but not that of iodate. Nitrate reductase A has a slightly greater affinity for NO3-than for ClO3-; but under saturating conditions chlorate is reduced at a sliggtly more rapid rate than nitrate: the ratio of chlorate- and nitrate reductase activities approaches the value of 1.5. Nitrate reductase A present in crude extracts appears to be in particulate form. It has been shown that enzyme A from either A. aerogenes or M. denitrificans is not inactivated when incubated in absence of its substrates in a medium made reducing by addition of reduced benzyl viologen. This property distinguishes it from the nitrate reductase of yeast Hansenula anomala. Enzyme A is very sensitive to cyanide and azide, but versene is without effect. In the case of cyanide the inhibition is not completely reversible and the kinetics observed are not characteristic for a competitive type of action. On the other hand, inhibition by azide is completely reversible and competitive. The Ki for N3-is very low approaching 0.001 mM. This indicates that the affinity for N3-is much higher than for NO3-or ClO3-: the Michaelis constants for these two substrates are in fact of order of 1 mM. These results suggest that nitrate reductase A contains a heavy metal, the identity of which is at present unknown. The competitive character of inhibition by azide indicates that this metal may be situated close to the active center and that it could have a role in association of enzyme with the substrate. The nitrate reductases A present in crude extracts of A. aerogenes and M. denitrificans are relatively resistant to thermal inactivation. In addition, the ratio of chlorate- and nitrate reductase activities remains constant irrespectively of temperature and duration of heating. This observation suggests that the two activities are associated with the same protein. © 1969 Springer-Verlag.