MONOMERIC PSEUDOMONAS-AERUGINOSA NITRITE REDUCTASE - PREPARATION, CHARACTERIZATION, AND KINETIC-PROPERTIES

Monomeric nitrite reductase in an active form has been prepared by con trolled succinylation of the dimeric native enzyme of Pseudomonas aeru ginosa and subsequent purification. The monomeric enzyme has an optica l spectrum indistinguishable from that of the native enzyme. On the ot her hand, circular dichroic spectra in the heme and peptide absorption regions show differences with respect to the dimer that indicate that the chemical modification and/or the dissociation into monomers somew hat perturb the chromophores' environment and the secondary structure. The (negatively charged) monomer is unable to oxidize its physiologic al substrates, azurin and cytochrome c(551). This loss of activity is not due to monomerization, but is linked to the total net charge of th e succinylated molecule, which interestingly enough acquires the abili ty to oxidize efficiently eukaryotic cytochrome c (which is not a subs trate of the native dimeric enzyme). Stopped-flow studies show that th e reduced monomer reacts with oxygen with a kinetic pattern similar to that shown by the dimeric enzyme. However, a higher reaction rate in the bimolecular binding of oxygen and a much higher oxygen affinity th an for the native enzyme are observed. The evidence reported in this p aper indicates that the dimeric state of Pseudomonas nitrite reductase is not a prerequisite for the ferrocytochrome c-oxygen oxidoreductase activity of this enzyme.