Separation of soluble denitrifying enzymes and cytochromes from Pseudomonas perfectomarinus.

Abstract

Nitrite and nitric oxide reductases were found soluble in extracts of Pseudomonas perfectomarinus cultured anaerobically at the expense of nitrate and ruptured with the French pressure cell. Malic enzyme, transhydrogenase, and flavin reductase that provided electron flow for these reductases were soluble as well. Nitrous oxide reductase remained particle-bound. Exogenous NADH was a poor electron donor for crude extracts, but a combination of malate, NADP, and NAD served well in the reduction of nitrite and nitric oxide. Nitrite reductase activity lost on dialysis of crude extract was restored by addition of this combination. Addition of free flavins was required for reduction of nitrite and nitric oxide. A nitrite reductase complex was separated from the nitric oxide reductase by gel filtration and DEAE-cellulose chromatography. NADH was an effective electron donor for this system with flavins provided as well. A c-type cytochrome with a split-α peak (perhaps associated with a d type) and two additional c-type cytochromes were separated from the nitrite reductase fraction. One of the latter (RI) emerged oxidized, the other (RII) reduced. Only nitric oxide oxidized RII. When these cytochromes were added to reaction mixtures containing nitrite reductase, activity was increased most by the split-α fraction. After reduction with dithionite, the absorption spectrum of the split-α cytochrome was returned to the oxidized spectrum by addition of nitrite but not the other oxides. A significant amount of a c-type cytochrome remained bound to the nitric oxide reductase fraction. A combination of malic acid, NAD, and NADP was more effective than NADH as electron donor for this system with free flavins provided as well. Addition of RI increased the rate of nitric oxide reduction by this fraction.