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J. Biol. Chem., Vol. 282, Issue 20, 14761-14767, May 18, 2007

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Fast Dissociation of Nitric Oxide from Ferrous Pseudomonas aeruginosa cd₁ Nitrite Reductase

A NOVEL OUTLOOK ON THE CATALYTIC MECHANISM^*

Serena Rinaldo, Alessandro Arcovito, Maurizio Brunori^¶1, and Francesca Cutruzzolà^¶

From the Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and ^¶Istituto di Biologia e Patologia Molecolari del Consiglio Nazionale delle Ricerche, Università di Roma "La Sapienza", 00185 Rome and Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, 00168 Rome, Italy

The heme-containing periplasmic nitrite reductase (cd₁ NIR) is responsible for the production of nitric oxide (NO) in denitrifying bacterial species, among which are several animal and plant pathogens. Heme NIRs are homodimers, each subunit containing one covalently bound c-heme and one d₁-heme. The reduction of nitrite to NO involves binding of nitrite to the reduced protein at the level of d₁-heme, followed by dehydration of nitrite to yield NO and release of the latter. The crucial rate-limiting step in the catalytic mechanism is thought to be the release of NO from the d₁-heme, which has been proposed, but never demonstrated experimentally, to occur when the iron is in the ferric form, given that the reduced NO-bound derivative was presumed to be very stable, as in other hemeproteins. We have measured for the first time the kinetics of NO binding and release from fully reduced cd₁ NIR, using the enzyme from Pseudomonas aeruginosa and its site-directed mutant H369A. Quite unexpectedly, we found that NO dissociation from the reduced d₁-heme is very rapid, several orders of magnitude faster than that measured for b-type heme containing reduced hemeproteins. Because the rate of NO dissociation from reduced cd₁ NIR, measured in the present report, is faster than or comparable with the turnover number, contrary to expectations this event may well be on the catalytic cycle and not necessarily rate-limiting. This finding also provides a rationale for the presence in cd₁ NIR of the peculiar d₁-heme cofactor, which has probably evolved to ensure fast product dissociation.

Received for publication, January 31, 2007 , and in revised form, March 22, 2007.

^* This work was supported by Ministero della Università e Ricerca of Italy Grants RBLA03B3KC_004 and 2005050270_004 (to M. B.) and RBIN04PWNC_002 (to F. C.) and Grant L.401/90-2006 from the Ministero degli Affari Esteri of Italy (to M. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" Università di Roma "La Sapienza", P.le A. Moro, 5 00185 Roma, Italy. Tel.: 39064450291; Fax: 39064440062; E-mail: maurizio.brunori{at}uniroma1.it.

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