The respiratory nitrate reductase from Paracoccus denitrificans has been purified in the non-ionic detergent Nonidet P-40. The enzyme comprises three polypeptides, a, p and y with estimated relative molecular masses of 127000, 61 000 and 21 000. Duroquinol or reduced-viologen compounds acted as the reducing substrates. The nitrate reductase contained a b-type cytochrome that was reduced by duroquinol and oxidised by nitrate. A preparation of the enzyme that lacked both detectable b-type cytochrome and the y subunit was obtained from a trailing peak of nitrate reductase activity collected from a gel filtration column. Absence of the y subunit correlated with failure to use duroquinol as reductant; activity with reduced viologens was retained. It is concluded that in the plasma membrane of P. denitrificans the y subunit catalyses electron transfer to the a and p subunits of nitrate reductase from ubiquinol which acts as a branch point in the respiratory chain.A new assay was introduced for both nitrate and quinol-nitrate oxidoreductase activity. Diaphorase was used to couple the oxidation of NADH to the production of duroquinol whtch acted as electron donor to nitrate reductase. Under anaerobic conditions absorbance changes at 340 nm were sensitive to nitrate concentrations in the low micromolar range. This coupled assay was used to determine that the purified enzyme had &(NO; ) of 13 pM and a K, of 470 pM for ClO;, an alternative substrate. With viologen substrates K,,,(NO;) of 283 pM and K,,,(ClO;) of 470 pM were determined; the enzymes possessed a considerably higher VmX with either NO; or ClO; than was found when duroquinol was substrate. Azide was a competitive inhibitor of nitrate reduction in either assay system (Ki = 0.55 pM) but 2-n-heptyl-4-hydroxyquinoline N-oxide was effective only with the complete three-subunit enzyme and duroquinol as substrate, consistent with a site of action for this inhibitor on the b-type cytochrome Biophys. Actu 807,. This similarity is discussed in terms of a possible requirement for a nitrate transport system. The nitrate reductase system from P. denitrificans is compared with that from Escherichia coli.Respiratory nitrate reductases have been studied from a variety of bacteria. The best characterised system is that of Escherichia coli (reviewed in [l]). This enzyme is known to consist of three polypeptides: LY (Mr = 150000), p (Mr = 60000) and y (Mr = 20000), the y subunit being a b-type cytochrome. E. coli nitrate reductase is also known to utilise both nitrate and chlorate as substrates.For E. coli the product of nitrate reduction, nitrite, is either excreted or reduced further to ammonium. In denitrifying bacteria the nitrite is reduced via nitrous oxide to nitrogen gas in the process known as denitrification. Despite the physiologicdl importance of denitrification, the nitrate reductases of organisms that catalyse this process are less well characterised than the enzyme from E. coli. Studies on the enzymes from Pseudomonas ueruginosa [2] and Bacillus lichenjormis [3] ...

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