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First practical assay for soluble nitrous oxide reductase of denitrifying bacteria and a partial kinetic characterization.

1980; Elsevier BV; Volume: 255; Issue: 2 Linguagem: Inglês

10.1016/s0021-9258(19)86236-1

1083-351X

Jakob K. Kristjánsson, Thomas C. Hollocher,

Advanced Chemical Sensor Technologies

Lysis of spheroplasts made from denitrificationadapted Paracoccus denitrificans released a soluble nitrous oxide reductase which was assayed spectrophotometrically under anaerobic conditions by following the oxidation of methyl or benzyl viologen cation radical upon reduction of NzO to Nz.Other classes of reductants so far tested, including dithionite, could not substitute for viologen dyes.Viologen dyes, therefore, afford the first practical assay for this previously elusive enzyme of denitrifying bacteria.The assay is specifically an in vitro assay, because the dyes cannot couple with intracellular nitrous oxide reductase.The enzyme exhibited simple saturation kinetics with respect to both NzO and reduced viologen dye.The K,,, for NzO was about 5 p~ at 22°C and pH 7.1 and the apparent K,,, for reduced benzyl and methyl viologen was 0.9 and 0.5 p ~, respectively.Both dyes afforded the same V,, value.Oxidized viologen dyes were not inhibiting to 1 n m nor was Nz to 1 atm.In fresh lysates, V,, was about 1.2 pmol of NzO X min" X mg of protein" or about twice that for intact cells or spheroplasts utilizing yeast extract or lactate.Enzyme activity was observed to be labile in crude preparations under anaerobic conditions.Nitrous oxide reductase was inhibited by acetylene, CO, azide, and cyanide with Ki values of 28, 3.5, 0.35, and 0.045 p ~, respectively.All showed noncompetitive inhibition with respect to NzO.Bacterial denitrification is the dissimilatory reduction of nitrate or nitrite to NZ (1, 2).NzO is a free obligatory intermediate (3) and is reduced by a special enzyme, nitrous oxide reductase, about which little is known.The several nitrogenoxide reductases of denitrification serve as terminal electron carriers in anaerobic respiration which supports proton translocation, oxidative phosphorylation, and growth (4-6).While nitrate and nitrite reductase have been purified from denitrifying bacteria (7, 8), significant purification of nitrous oxide reductase has not been accomplished.Principal barriers to its purification were, until now, lack of a practical in vitro assay and loss of 90% or more of the electron transport-linked activity immediately upon cell breakage (9-13).In this paper, we describe a practical in vitro assay for