The respiratory nitrate reductase from Paracoccus denitrificans. Molecular characterisation and kinetic properties

Abstract

The respiratory nitrate reductase from Paracoccus denitrificans has been purified in the non-ionic detergent Nonidet P-40. The enzyme comprises three polypeptides, .alpha., .beta. and .gamma. with estimated relative molecular masses of 127000, 61000 and 21000. 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 .gamma. subunit was obtained from a trailing peak of nitrate reductase activity collected from a gel filtration column. Absence of the .gamma. 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 .gamma. subunit catalyses electron transfer to the .alpha. and .beta. 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 which 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 .**GRAPHIC**. of 13 .mu.M and a Km of 470 .mu.M for .**GRAPHIC**. an alternative substrate. With viologen substrates .**GRAPHIC**. of 283 .mu.M and .**GRAPHIC**. of 470 .mu.M were determined: the enzymes possessed a considerably higher Vmax with either .**GRAPHIC**. or .**GRAPHIC**. than was found when duroquinol was substrate. Azide was a competitive inhibitor of nitrate reduction in either assay system (Ki = 0.55 .mu.M) but 2-n-heptyl-4-hydroxyquinoline N-oxide was effective only with the complete three-subunit enyzme and duroquinol as substrate, consistent with a site of action for this inhibitor on the b-type cytochrome. The low Km for nitrate observed in the duriquinol assay is comparable with the apparent .**GRAPHIC**. recently reported for intact cells of P. denitrificans [Parsonage, D., Greenfield, A.J. and Ferguson, S.J. (1985) Biochem. Biophys. Acta 807, 81-95]. 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.