A study on the transport and dissimilatory reduction of nitrate in Paracoccus denitrificans using viologen dyes as electron donors

Abstract

The controversial subject of nitrate transport into the denitrifying cells of Paracoccus denitrificans was studied employing methyl or benzyl viologen cation radicals as electron donors for the respiratory nitrate reductase. In intact cells, the oxidation of methyl viologen by nitrate was sensitive to low concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone ( I 50 = 0.8 μM). Treatment of cells with 0.1%v Triton X-100 (1) completely relieved the inhibition by the uncoupler, (2) raised the values of K m(NO 3 −) and V m from 23 ± 5 μM and 6.4 ± 0.3 nmol MV s −1mg dw −1 to 120 ± 10 μM and 13.5 ± 0.5 nmol MV s −1mg dw −1, respectively, and (3) changed the type of inhibition by azide from noncompetitive ( K i = 0.18 ± 0.01 μM) to competitive ( K i = 0.18 ± 0.02 μM) with respect to nitrate. On the other hand, permeabilisation had little effects on the activation energy ( ≈ 57 kJ mol −1 ) and on the maximal degree of inhibition by 2-heptyl-4-hydroxyquinoline- N-oxide (≈ 50%). Observations (1) and (2) suggest an active transport mechanism for nitrate, whereas (3) can be best explained by a simultaneous leakage of the accumulayed nitrate from the cells. Results from a HPLC analysis of cellular nitrate levels and their changes elicited by azide (an increase) or uncoupler (a decrease) are compatible with the occurrence of a cytoplasmic nitrate pool linking up nitrate transporter and nitrate reductase.