Purification and properties of ferredoxin—nitrate reductase from the cyanobacterium Plectonema boryanum

Abstract

Assimilatory ferrodoxin—nitrate reductase has been purified to homogeneity from the cyanobacterium Plectonema boryanum. The enzyme was solubilized by sonication and, following heat treatment, ammonium sulfate precipitation and chromatography on DEAE-Toyopearl, Sephadex G-150 and hydroxyapatite, was purified 19000-fold with a yield of 15%. The purified enzyme had a specific activity of 305 and 1020 μmol NO 2 − formed/min per mg protein for ferrodoxin-linked and methyl viologen-linked nitrate reductase activities, respectively. The molecular weights measured by sodium dodecyl sulfate gel electrophoresis and sedimentation equilibrium were 83 000 and 85 000, respectively, indicating that the enzyme consists of a single polypeptide chain. The purified nitrate reductase contained 0.95 atom of molybdenum, and four iron and four acid-labile sulfur atoms per molecule ( M r 85 000). The spectrum of the enzyme resembled that of iron-sulfur proteins. The CD and MCD spectra suggested the presence of an iron-sulfur cluster in the molecule. Some similarities of amino-acid composition were observed between the cyanobacterial nitrate reductase, NAD(P)H-nitrate reductase and the catalytic subunit of the dissimilatory nitrate reductase from Escherichia coli. The stoichiometry of the nitrate reductase reaction confirmed a two-electron reduction of nitrate to nitrite by reduced ferredoxin or reduced methyl viologen. The K m values of the native enzyme was 38 μM for reduced ferredoxin and 2500 μM for reduced methyl viologen. Dithionite converted the native enzyme into a modified form which had no ferredoxin-linked nitrate reductase activity but retained methyl viologen-linked activity with a pH optimum at 10.2.