The nitrite reductase from Pseudomonas aeruginosa: essential role of two active-site histidines in the catalytic and structural properties.
Cd(1) nitrite reductase catalyzes the conversion of nitrite to NO in denitrifying bacteria. Reduction of the substrate occurs at the d(1)-heme site, which faces on the distal side some residues thought to be essential for substrate binding and catalysis. We report the results obtained by mutating to Ala the two invariant active site histidines, His-327 and His-369, of the enzyme from Pseudomonas aeruginosa. Both mutants have lost nitrite reductase activity but maintain the ability to reduce O(2) to water. Nitrite reductase activity is impaired because of the accumulation of a catalytically inactive form, possibly because the productive displacement of NO from the ferric d(1)-heme iron is impaired. Moreover, the two distal His play different roles in catalysis; His-369 is absolutely essential for the stability of the Michaelis complex. The structures of both mutants show (i) the new side chain in the active site, (ii) a loss of density of Tyr-10, which slipped away with the N-terminal arm, and (iii) a large topological change in the whole c-heme domain, which is displaced 20 A from the position occupied in the wild-type enzyme. We conclude that the two invariant His play a crucial role in the activity and the structural organization of cd(1) nitrite reductase from P. aeruginosa.
Dipartimento di Scienze Biochimiche A. Rossi Fanelli and Centro di Biologia Molecolare del Consiglio Nazionale delle Ricerche, Università di Roma La Sapienza, P. le A. Moro 5, 00185 Rome, Italy.
F
F
Cutruzzola
K
K
Brown
E K
EK
Wilson
A
A
Bellelli
M
M
Arese
M
M
Tegoni
C
C
Cambillau
M
M
Brunori
D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D006639:Histidine / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D006639:Histidine; D009565:Nitrate Reductases; D050901:Nitrate Reductase