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 denitri fying bacteria. Reduction of the substrate occurs at the d(1)-heme site, wh ich faces on the distal side some residues thought to be essential for subs trate 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 e nzyme from Pseudomonas aeruginosa, Both mutants have lost nitrite reductase activity but maintain the ability to reduce O-2 to water. Nitrite reductas e activity is impaired because of the accumulation of a catalytically inact ive form, possibly because the productive displacement of NO from the ferri c d(1)-heme iron is impaired. Moreover, the two distal His play different r oles in catalysis; His-369 is absolutely essential for the stability of the Michaelis complex. The structures of both mutants show (i) the new side ch ain in the active site, (ii) a loss of density of Tyr-10, which slipped awa y with the N-terminal arm, and (iii) a large topological change in the whol e c-heme domain, which is displaced 20 Angstrom from the position occupied in the wild-type enzyme. We conclude that the two invariant His play a cruc ial role in the activity and the structural organization of cd(1) nitrite r eductase from P. aeruginosa.