Monomeric Pseudomonas aeruginosa nitrite reductase: preparation, characterization, and kinetic properties.
Monomeric nitrite reductase in an active form has been prepared by controlled succinylation of the dimeric native enzyme of Pseudomonas aeruginosa and subsequent purification. The monomeric enzyme has an optical spectrum indistinguishable from that of the native enzyme. On the other 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 somewhat perturb the chromophores' environment and the secondary structure. The (negatively charged) monomer is unable to oxidize its physiological substrates, azurin and cytochrome c551. This loss of activity is not due to monomerization, but is linked to the total net charge of the succinylated molecule, which interestingly enough acquires the ability to oxidize efficiently eukaryotic cytochrome c (which is not a substrate of the native dimeric enzyme). Stopped-flow studies show that the 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 than for the native enzyme are observed. The evidence reported in this paper indicates that the dimeric state of Pseudomonas nitrite reductase is not a prerequisite for the ferrocytochrome c-oxygen oxidoreductase activity of this enzyme.
Silvestrini
M C
MC
Dipartimento di Scienze Biochimiche, Università di Roma La Sapienza, Italia.
Tordi
M G
MG
Citro
G
G
Vecchini
P
P
Brunori
M
M
D002942:Circular Dichroism; D007700:Kinetics; D009572:Nitrite Reductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013387:Succinic Anhydrides / Q000737:chemistry
D013387:Succinic Anhydrides; C031801:succinic anhydride; D009572:Nitrite Reductases; D010100:Oxygen