Involvement of a B-type cytochrome in the assimilatory nitrate reductase of Neurospora crassa.

The enzyme systems which catalyze the reduction of nitrate to nitrite have generally been classified into two broad types: (a) assimilatory nitrate reductase, which is responsible for the first step in the reduction of nitrate for the ultimate biosynthesis of nitrogen-containing cell constituents (e.g., amino acids aind purines and pyrimidines), and (b) respiratory or dissimilatory nitrate reductase, which utilizes nitrate as the terminal acceptor (in place of oxygen) in the electron transport or respiratory pathway, usually under anaerobic or partially anaerobic conditions.'-6 Comparisons of the properties of the assimilatory nitrate reductase of Neurospora with those of the respiratory nitrate reductase of E. coli have clearly indicated that the above two enzyme types share several similarities (including the possession of molybdenum) and are considerably more alike than hitherto believed.3-5 Thus far the major distinguishing feature of respiratory nitrate reductase, as originally suggested by Sato, I has been the involvement of one or more cytochromes as electron carriers in the enzymatic reduction of nitrate to nitrite, a characteristic notably absent in the assimilatory system. The reasons for the inhibitory effect of molecular oxygen on the respiratory nitrate reductase only are not entirely clear. The effect is probably due both to an inhibition of adaptive formation of the enzyme" and to a competition for electrons by the oxygen itself, perhaps by way of an accompanying cytochrome oxidase. In the course of large-scale purification of the soluble assimilatory -NLADPHnitrate reductase (NADPH: nitrate oxidoreductase, E.C. 1.6.6.2), from Neurospora crassa grown under highly aerobic conditions, a b-type cytochrome was found to be associated with the fractions having nitrate reductase activity. The present paper reports that the properties of this cytochrome associated with the Neurospora nitrate reductase (including its specific reduction and oxidation by NADPH arid nitrate, respectively) imply it to be an integral part of the electron transfer scheme involved in assimilatory nitrate reduction. The cytochrome spectrum of the partially purified enzyme is clearly different from that of cytochrome bi of the E. coli respiratory nitrate reductase, and to a lesser extent from those of several other b-type cytochromes reported in the literature, but is very similar to those of bakers' yeast lactic dehydrogenase cytochrome b2 and mammalian cytochrome b5. Despite its similarity in this and several other respects to yeast lactate dehydrogenase, the Neurospora nitrate reductase has no lactate dehydrogenase activity. Nor does the yeast lactate dehydrogenase system possess nitrate reductase activity. The previous failure to observe this hemoprotein in the assimilatory enzyme can be attributed to the relatively low yield and degree of purity heretofore obtained with Neurospora crassa nitrate reductase. Materials and Methods.-Cytochrome c (type III) and FAD were provided by the Sigma Chemical Company. NADPH was obtained from the Sigma Chemiceal