Assimilatory Nitrate Reductase of Rhodopseudomonas capsulata AD2: A Molybdo-Hemeprotein

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Abstract
The assimilatory nitrate reductase of the phototrophic bacterium R. capsulata strain AD2 was purified to homogeneity by combination of (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and isoelectric focusing (isoelectric point of 4.8). The purified enzyme was active only with reduced viologen dyes or reduced flavin as electron donors. Contrary to other bacterial assimilatory nitrate reductases, the enzyme was not inhibited by chlorate, but rather accepted this substance as an alternate substrate. The MW of the enzyme was 185,000 dalton as determined by gel filtration. Subunit analysis by sodium dodecyl sulfate (SDS) gel electrophoresis yielded a single protein band with a MW of 85,000 dalton, suggesting that the enzyme was composed of 2 identical subunits. The nitrate reductase contained 0.8 g-atoms Mo/1.85 .times. 105 g protein and exhibited absorption maxima at 418, 523 and 552 nm in the reduced state (dithionite as reductant). The nitrate reductase of R. capsulata AD2 is the 1st prokaryotic enzyme of the assimilatory type that has been shown to contain heme.