The Nitrite Reductase from Pseudomonas Aeruginosa: Essential Role of Two Active-Site Histidines in the Catalytic and Structural Properties
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The nitrite reductase from Pseudomonas aeruginosa: Essential role of two active-site histidines in the catalytic and structural properties Francesca Cutruzzola` *, Kieron Brown†, Emma K. Wilson*, Andrea Bellelli*, Marzia Arese*, Mariella Tegoni†, Christian Cambillau†, and Maurizio Brunori*‡ *Dipartimento di Scienze Biochimiche ‘‘A. Rossi Fanelli’’ and Centro di Biologia Molecolare del Consiglio Nazionale delle Ricerche, Universita`di Roma ‘‘La Sapienza,’’ 00185 Rome, Italy; and †Architecture et Fonction des Macromole´cules Biologiques, Unite´Mixte de Recherche 6098, Centre National de la Recherche Scientifique and Universite´s 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) Cd1 nitrite reductase catalyzes the conversion of nitrite to NO in essential for NIR activity (NIRV) but have no effect on the oxygen denitrifying bacteria. Reduction of the substrate occurs at the d1- reductase activity. The 3D structures of both mutants shows that (i) heme site, which faces on the distal side some residues thought to be Ala replaces His in the distal d1-heme pocket of both mutants; (ii) essential for substrate binding and catalysis. We report the results Tyr-10 slips away together with the N-terminal arm; and (iii) the obtained by mutating to Ala the two invariant active site histidines, c-heme domain experiences a large topological change relative to His-327 and His-369, of the enzyme from Pseudomonas aeruginosa. the d1-heme domain, which is unmodified. Our results allow us to Both mutants have lost nitrite reductase activity but maintain the propose a mechanism for catalysis of nitrite reduction, based largely ability to reduce O2 to water. Nitrite reductase activity is impaired on the essential role of the electrostatic potential imposed by the because of the accumulation of a catalytically inactive form, possibly invariant His in assisting the dissociation of the product NO from because the productive displacement of NO from the ferric d1-heme the ferric d1-heme iron. The proposed mechanism may be of iron is impaired. Moreover, the two distal His play different roles in significance for heme proteins involved in the metabolism and catalysis; His-369 is absolutely essential for the stability of the Michae- transport of NO, all of which have to avoid being trapped in a lis complex. The structures of both mutants show (i) the new side ‘‘dead-end’’ complex with the reduced heme iron. 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 Materials and Methods the whole c-heme domain, which is displaced 20 Å from the position Mutagenesis and Protein Purification. Mutagenesis of His-327 to Ala occupied in the wild-type enzyme. We conclude that the two invari- was carried out as in ref. 8; His-369 was mutated to Ala with the use ant His play a crucial role in the activity and the structural organization of a U.S.E. Mutagenesis Kit (Amersham Pharmacia). Subcloning, of cd1 nitrite reductase from P. aeruginosa. expression in Pseudomonas putida, and purification were obtained as described (9, 10). In the P. putida expression system, the protein Ϫ he conversion of nitrite (NO2 ) to nitric oxide (NO) is is synthesized with the c-heme, but no d1-heme; this semiapo-NIR Tcatalyzed in denitrifying bacteria by the periplasmic nitrite is then reconstituted in vitro with the d1-heme extracted from reductases (NIRs), which are either copper- or heme-containing wild-type (wt) Pa-NIR as detailed in ref. 9. enzymes (1, 2). Heme NIRs are homodimers of two 60-kDa subunits, each containing one covalently bound c-heme and one General Characterization. Reduced derivatives were obtained by d1-heme. These enzymes catalyze not only the one-electron adding anaerobically excess ascorbate to oxidized NIR. Cyto- Ϫ reduction of NO2 to NO but also the four-electron reduction of chrome oxidase activity was assessed at 20°C in 50 mM sodium O2 to2H2O. Extensive spectroscopic and functional studies (3) phosphate buffer (pH 7.0) by measuring the rate of oxidation of have been carried out on cd1NIR from Pseudomonas aeruginosa reduced P. aeruginosa cytochrome c551 (1–20 M) as described (Pa-NIR); the c-heme domain is the electron’s entry site, in ref. 11. NIR activity was measured anaerobically at 27°C in 50 whereas catalysis occurs at the level of the d1-heme. mM sodium phosphate buffer (pH 6.2), either after oxidation of The three-dimensional (3D) structure of NIR from P. aeruginosa reduced azurin (12) or during amperometric measurement of has been solved (by x-ray diffraction) for the oxidized, reduced, and NO production with a NO electrode (ISO-NO; World Precision reduced NO-bound forms of the enzyme (4, 5). The overall Instruments, Sarasota, FL). A typical experiment at the elec- structure of this enzyme is similar to that published by Fulop et al. trode was carried out at 25°C in the presence of ascorbate (13 (6) for cd1 NIR from Paracoccus pantotrophus (formerly called mM) and N,N,N,N-tetramethyl-p-phenylenediamine (0.1 mM) Thiosphaera pantotropha; ref. 7). In both enzymes each subunit is as electron donors and initiated by adding different concentra- organized in two structurally distinct domains: an N-terminal tions of nitrite (10–1,200 M). ␣-helical domain containing the c-heme and a C-terminal eight- Stopped-flow experiments were carried out anaerobically with  blade -propeller domain with the d1-heme binding site. The distal the use of a TN6500 (Tracor Northern, Madison, WI) multidiode side of the d1-heme pocket is lined up with several important residues that include Tyr-10 (coming from the N-terminal arm of the other monomer) and two invariant histidine residues, His-327 This paper was submitted directly (Track II) to the PNAS office. and His-369 (Fig. 1A), presumably involved in substrate binding Abbreviations: NIR, nitrite reductase; Pa-NIR, Pseudomonas aeruginosa NIR; wt, wild type; and͞or protonation. Reduction of Pa-NIR leads to conformational 3D, three-dimensional. changes (5), involving motion of a loop in the c-heme domain, Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.rcsb.org (PDB ID codes 1HZU and 1HZV). rotation of Tyr-10 away from the d1-heme site, and loss of the distal ‡To whom reprint requests should be addressed at: Dipartimento di Scienze Biochimiche d1-heme iron ligand, which in oxidized Pa-NIR is a hydroxyl (4). ‘‘A. Rossi Fanelli,’’ Universita`di Roma ‘‘La Sapienza,’’ P.le A. Moro 5, 00185 Rome, Italy. To elucidate the role of the two invariant distal residues His-327 E-mail: [email protected]. and His-369 (Fig. 1A) in the catalytic mechanism of Pa-NIR, we The publication costs of this article were defrayed in part by page charge payment. This have prepared and characterized two site-directed mutants, H369A article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. and H327A. The kinetic data show that these two histidines are §1734 solely to indicate this fact. 2232–2237 ͉ PNAS ͉ February 27, 2001 ͉ vol. 98 ͉ no. 5 www.pnas.org͞cgi͞doi͞10.1073͞pnas.041365298 Downloaded by guest on September 27, 2021 Fig. 1. The active site of cd1 NIR from P. aeruginosa (Pa-NIR). The model of the d1-heme pocket of the wt reduced enzyme complexed with nitrite is shown in Ϫ A. The stereochemistry of NO2 was simulated starting from the coordinates of the NO adduct of reduced Pa-NiR (5). Among the key amino acid side chains shown here, notice that Tyr-10 comes from the other monomer (identified as sub. B), as a result of a domain swapping across the 2-fold axis of the homodimer (4). The 3D structure of the d1-heme pocket of the two mutants in the oxidized state is shown in the same orientation in B (for H369A) and C (for H327A). The FoϪFc Sigma A negative electron density map is also represented at the place of the missing side chain; this map is contoured at Ϫ3 for B and Ϫ4 for C. array spectrometer coupled to a Gibson–Durrum stopped-flow maps. Furthermore, a two-body molecular replacement procedure apparatus. Reduced NIR (4–8 M before mixing) in degassed 50 failed to yield the position of the c-heme domain. We therefore mM sodium phosphate buffer (pH 8.0) was mixed with nitrite decided to apply multiple wavelength anomalous diffraction (0.02–1 mM) in the presence of 1 mM ascorbate at 25°C. The time (MAD) techniques to the anomalous signal of the 2 Fe ions. Three course of the reaction was followed in the wavelength range of 380 MAD data sets were collected on BM14 (ESRF) at 3.8-Å resolu- to 650 nm; analysis of the experimental data was carried out as tion. Finally, the complete structure was determined by a combi- described (13). nation of MAD techniques and phase combination, with the use of the molecular replacement results with the d1-heme domain. The Crystallization and Structure Determination of the H369A and H327A resolution was extended to 2.8 Å with the use of a data set already Mutants. A detailed description of the crystallization procedures collected on ID14-EH2 (ESRF) at a single wavelength. The refined and of the structures has yet to be published (K.B., V. Roig- model has a Rwork of 22.9% and a Rfree of 28.5%. Zamboni, F.C., E.K.W., A.B., M.A., D.