TAILIEUCHUNG - Báo cáo khoa học: Critical role of His369 in the reactivity of Pseudomonas aeruginosa cytochrome cd1 nitrite reductase with oxygen

In the denitrification pathway, Pseudomonas aeruginosacytochrome cd1 nitrite reductase catalyzes the reduction of nitrite to nitric oxide;in vitro, this enzyme is also competent in the reduction of O2 to 2H2 O. In this art-icle, we present a comparative kinetic study of the O2 reaction in the wild-type nitrite reductase and in three site-directed mutants (Tyr10fiPhe, His369fiAla and His327fiAla⁄His369fiAla) of the amino acid resi-dues close to thed1 heme on the distal side. | ỊFEBS Journal Critical role of His369 in the reactivity of Pseudomonas aeruginosa cytochrome cd1 nitrite reductase with oxygen Fabio Centola1 Serena Rinaldo1 Maurizio Brunori1 2 and Francesca Cutruzzola1 2 1 Department of BiochemicalSciences A. Rossi Fanelli University of Rome La Sapienza Italy 2 Istituto di Biologia e Patologia Molecolari delCNR University of Rome La Sapienza Italy Keywords cytochrome cd1 nitrite reductase electron transfer intermediates oxygen Correspondence M. Brunori Dipartimento di Scienze Biochimiche A. Rossi Fanelli University di Roma La Sapienza P. le A. Moro 5 00185 Rome Italy Fax 39 06 4440062 Tel 39 06 4450291 E-mail These authors contributed equally to this work Received 23 June 2006 accepted 7 August 2006 doi 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 wildtype nitrite reductase and in three site-directed mutants Tyr10 fi Phe His369 fi Ala and His327 fi Ala His369 fi 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 .

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