Purification, properties and limited proteolysis of nitrate reductase from Pseudomonas denitrificans

Abstract

The membrane-bound nitrate reductase (nitrate: (acceptor) oxidoreductase, EC 1.7.99.4) of Pseudomonas denitrificans ATCC 13867 solubilized either by treatment with sodium deoxycholate plus ammonium sulfate (nitrate reductase I) or by neutral heat treatment (nitrate reductase II) was purified to homogeneity. The molecular weights of nitrate reductase I ( s 20,w = 11.2 S) measured by gel filtration and sedimentation equilibrium methods were 220 000 and 230 000, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicates that nitrate reductase I is composed of three different subunits with molecular weights of 136 000, 55 000 and 19 000. Nitrate reductase I was found to contain (in relative molar amounts): 1.0 heme, 0.74 molybdenum, 13 non-heme iron, and 12 acid-labile sulfide. Neither FAD nor FMN was detected. The molecular weights of nitrate reductase II ( s 20,w = 9.6 S) measured by gel filtration and sedimentation equilibrium methods were 190 000 and 200 000, respectively. Nitrate reductase II was shown electrophoretically to be composed of two different subunits with molecular weights of 136 000 and 55 000. Nitrate reductase I was converted to nitrate reductase II by heat treatment in alkaline solution or by aging. The composition of nitrate reductase II was essentiatlly the same as that of nitrate reductase I, except that heme was not detected. Some enzymological properties are reported. The enzyme was shown to contain the molybdenum cofactor. Oxidation products of the molybdenum cofactor gave pterin-6-carboxylic acid and 6-carboxypterin-7-sulfonic acid upon alkaline KMnO 4 oxidation. During storage of 4°C for about a month, a purified preparation of nitrate reductase II was converted to a new electrophoretic form, the molecular weight of which was approx. 169 000. Electrophoretic analysis of freshly prepared and stored enzyme indicated conversion of the small subunit ( M r 55 000) to a 43 kDa polypeptide and partial cleavage of the large subunit ( M r 136 000) to 87 kDa and 47 kDa fragments. Similar polypeptide fragments were obtained by incubation of the enzyme with trypsin. The trypsin-treated enzyme ( M r 169 000) retained full enzymatic activity. Despite the tryptic cleavage, the three fragments appeared associated upon gel chromatography.