The nitrite reductase from Pseudomonas aeruginosa : Essential role of two active-site histidines in the catalytic and structural properties

doi:10.1073/pnas.041365298

The nitrite reductase from Pseudomonas aeruginosa: Essential role of two active-site histidines in the catalytic and structural properties

^*Dipartimento di Scienze Biochimiche “A. Rossi Fanelli” and Centro di Biologia Molecolare del Consiglio Nazionale delle Ricerche, Università di Roma “La Sapienza,” 00185 Rome, Italy; and ^†Architecture et Fonction des Macromolécules Biologiques, Unité Mixte de Recherche 6098, Centre National de la Recherche Scientifique and Universités de Marseille I and II, 13402 Marseille Cedex 20, France

Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved December 18, 2000 (received for review August 3, 2000)

Abstract

Cd₁ nitrite reductase catalyzes the conversion of nitrite to NO in denitrifying bacteria. Reduction of the substrate occurs at the d₁-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 O₂ 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 d₁-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 Å 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 cd₁ nitrite reductase from P. aeruginosa.

Footnotes

↵ ‡ To whom reprint requests should be addressed at: Dipartimento di Scienze Biochimiche “A. Rossi Fanelli,” Università di Roma “La Sapienza,” P.le A. Moro 5, 00185 Rome, Italy. E-mail: maurizio.brunori{at}uniroma1.it.
This paper was submitted directly (Track II) to the PNAS office.
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.rcsb.org (PDB ID codes 1HZU and 1HZV).
Abbreviations:

NIR,

nitrite reductase;

Pa-NIR,

Pseudomonas aeruginosa NIR;

wt,

wild type;

3D,

three-dimensional