Characterization of the respiratory nitrate reductase of Klebsiella aerogenes as a molybdenum-containing iron-sulfur enzyme.

1. In respiratory nitrate reductase I of Klebsiella aerogenes, 0.24 atom of molybdenum, eight iron-sulfur groups and four tightly bound, non-heme iron atoms per molecule of enzyme (Mr 260 000) are found. 2. EPR spectra at 83 degrees K of oxidized and reduced nitrate reductase I show complex lines at g = 2.02 and g = 1.98, which are more intense in the reduced than in the oxidized enzyme. The resonances, the shape and intensity of which are rather temperature insensitive, are attributed to two species of paramagnetic molybdenum. In dithionite-reduced enzyme all these lines are saturated at the same microwave power of 15 mW. This is not the case in oxidized enzyme, where the resonance at g = 2.02 is hard to saturate. Addition of nitrate to dithionite-reduced reductase I decreases the intensity of the EPR lines to about that of oxidized enzyme. The participation of molybdenum in the electron transfer process has been discussed. 3. At 18 degrees K the oxidized enzyme exhibits an axial-symmetrical signal with g parallel = 2.10 and g = 2.03, and a signal with unknown symmetry at g = 2.015. Upon reduction by dithionite, a ferredoxin type of signal is observed with g values at 2.05, 1.95 and 1.88, while the g = 2.015 signal disappears. Reoxidation by nitrate causes a concomitant disappearance of the ferredoxin type of signal and reappearance of the g = 2.015 signal; hence iron-sulfur centres participate in the transfer of electrons to nitrate. 4. Nitrate reductase II, containing only two (Mr 117 000 and 57 000) of the three subunits found in nitrate reductase I and lacking the tightly bound iron, does not exhibit the axial-symmetrical signal (g = 2.10 and 2.03). Thus, it suggested that this signal in nitrate reductase I stems from an iron centre in the low-molecular weight subunit (Mr 52 000). 5. Inhibition studies confirm the participation of metals in the transfer of electrons from reduced benzylviologen to nitrate and show that the binding sites for these substrates are different.