Abstract
The authors report the first partial purification of nitrous oxide reductase, a unique and labile enzyme of denitrifying bacteria. The procedure, which required anaerobic conditions throughout, resulted in a 60-fold purification relative to crude lysate in the case of Parococcus denitrificans . The molecular weight was estimated by gel exclusion chromatography to be about 85,000. The partially purified enzyme is inactivated rapidly by O sub(2), dithionite, and mercaptoethanol and is reversibly inhibited by moderate concentrations of common salts. Up to 80% of the original activity can be reconstituted following O sub(2) inactivation by incubating the enzyme with reduced benzyl viologen for 2 to 3 h. The V sub(max) pH profile shows a broad maximum at pH 8. The enzyme is irreversibly retained by common anion exchangers in the range pH 7 to 8 but can be eluted in acceptable yield as one of the last components from an imidazole-based anion exchange material by means of a pH gradient. This behavior implies that nitrous oxide reductase is very acidic.