Expression of <i>Pseudomonas aeruginosa</i> nitrite reductase in <i>Pseudomonas putida</i> and characterization of the recombinant protein

Abstract

Nitrite reductase from Pseudomonas aeruginosa has been successfully expressed in Pseudomonas putida. The purified recombinant enzyme contains haem c but no haem d1. Nonetheless, like the holoenzyme from Ps. aeruginosa, it is a stable dimer (molecular mass 120 kDa), and electron transfer to oxidized azurin is biphasic and follows bimolecular kinetics (k1 = 1.5 x 10(5) and k2 = 2.2 x 10(4) M-1.s-1). Unlike the chemically produced apoenzyme, recombinant nitrite reductase containing only haem c is water-soluble, stable at neutral pH and can be quantitatively reconstituted with haem d1, yielding a holoenzyme with the same properties as that expressed by Ps. aeruginosa (namely optical and c.d. spectra, molecular mass, cytochrome c551 oxidase activity and CO-binding kinetics).