Critical role of His369 in the reactivity of Pseudomonas aeruginosa cytochrome cd1 nitrite reductase with oxygen.
In the denitrification pathway, Pseudomonas aeruginosa cytochrome cd1 nitrite reductase catalyzes the reduction of nitrite to nitric oxide; in vitro, this enzyme is also competent in the reduction of O2 to 2H2O. In this article, we present a comparative kinetic study of the O2 reaction in the wild-type nitrite reductase and in three site-directed mutants (Tyr10-->Phe, His369-->Ala and His327-->Ala/His369-->Ala) of the amino acid residues close to the d1 heme on the distal side. The results clearly indicate that His369 is the key residue in the control of reactivity, as its substitution with Ala, previously shown to affect the reduction of nitrite, also impairs the reaction with O2, affecting both the properties and lifespan of the intermediate species. Our findings allow the presentation of an overall picture for the reactivity of cytochrome cd1 nitrite reductase and extend our previous conclusion that the conserved distal histidines are essential for the binding to reduced d1 heme of different anions, whether a substrate such as nitrite, a ligand such as cyanide, or an intermediate in the O2 reduction. Moreover, we propose that His369 also exerts a protective role against degradation of the d1 heme, by preventing the formation and adverse effects of the reactive O2 species (never present in significant amounts in wild-type cytochrome cd1 nitrite reductase), a finding with clear physiological implications.
Centola
Fabio
F
Department of Biochemical Sciences A. Rossi Fanelli, University of Rome La Sapienza, Italy.
Rinaldo
Serena
S
Brunori
Maurizio
M
Cutruzzolà
Francesca
F
D002248:Carbon Monoxide / Q000378:metabolism; D003580:Cytochromes / Q000737:chemistry / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D009572:Nitrite Reductases / Q000737:chemistry / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013329:Structure-Activity Relationship
D003580:Cytochromes; D002248:Carbon Monoxide; D007501:Iron; D009572:Nitrite Reductases; C028123:nitrite reductase (NO-forming); D010100:Oxygen