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. Nurizzo, M.B., C.C., and M.T., unpublished observations). Briefly, crystals of the H327A Results mutant were obtained by the vapor diffusion technique by mixing Spectral and Steady-State Kinetic Properties. The optical spectra of in a 1:1 ratio the protein and a reservoir solution containing 4.0% the two mutants, H327A and H369A, are different from that of polyethylene glycol 5000 monomethyl ether, 0.1 M sodium acetate ϫ wt NIR (Fig. 2), mainly where the d1-heme absorbs (450–500 nm (pH 5.5). The space group is P4322 with cell dimensions 70.5 and 600–700 nm). In the oxidized state, the largest changes are 70.5 ϫ 281 Å. Crystals of the H369A mutant were obtained by seen for the H327A mutant, the 641-nm peak being red-shifted mixing in a 1:1 ratio the protein and a reservoir solution containing by 8 nm; in the reduced state changes are observed for both 11.5% polyethylene glycol 6000, 0.2 M imidazole͞malate (pH 6.5). mutants with a 10-nm red-shift (from 462 to 472 nm) of the The space group is P4 2 2 with cell dimensions 94.7 ϫ 94.7 ϫ 159.9 1 1 d -heme Soret band. The spectrum of the reduced NO-bound Å. These crystal forms are different from those obtained with the 1 derivative was essentially unchanged. wt protein under similar crystallization conditions (14) and at high salt concentration (4) or at low pH (4). In contrast with these wt The most interesting feature of the two mutants relates to crystal forms, the mutant crystal forms both contain only one catalysis. As shown in Table 1, both mutants catalyze the monomer per asymmetric unit, half the functional dimeric enzyme. reduction of oxygen to water as efficiently as the wt NIR; in contrast, reduction of nitrite to NO is severely compromised for Before x-ray data collection, the crystals were soaked in 30% Ϸ ethylene glycol in mother liquor and flash-frozen to 100 K. A both mutants (the activity is 1% of wt). The same effect on the H327A mutant data set was collected at 2.7-Å resolution on nitrite reductase activity was observed for both mutants between ID14-EH2 [European Synchrotron Radiation Facility (ESRF), pH 5 and 7 (not shown). Grenoble, France], integrated with DENZO (15), and reduced with SCALA (16). The structure was solved by molecular replace- Stopped-Flow Experiments on the Nitrite Reaction. Wt NIR. This ment with the use of the d1 heme domain (residues 120–543) and reaction was followed at several nitrite concentrations and at pH AMORE (17). The c-heme domain was rigid-body placed manually 8.0, which is higher than that of the steady-state experiments (pH Ϫ in the Fo Fc electron density map with TURBO-FRODO (18). The 6.2), to better resolve the kinetics. The spectra recorded at different structure was refined with CNS (19) to values of R and free R of times are shown in Fig. 2 as the difference between each spectrum 21.7 and 28.0%, respectively. and that of the fully reduced NO bound enzyme, the final product The same procedure was used with the H369A mutant, which of the reaction. The redox state of the two hemes is followed by diffracted to 2.8-Å resolution on ID14-EH2 (ESRF). However, looking at the time evolution of the absorption peaks characteristic

after molecular replacement with the use of the d1 heme domain, of each chromophore (420 and 550 nm for the c-heme; 460 and 640 BIOCHEMISTRY Ϫ no electron density for the c-heme domain was visible in the Fo Fc nm for the d1-heme). As an example, in Fig. 2 a negative peak at

Cutruzzola` et al. PNAS ͉ February 27, 2001 ͉ vol. 98 ͉ no. 5 ͉ 2233 Downloaded by guest on September 27, 2021 Fig. 2. Static and transient optical spectra of wt and mutants Pa-NIR. (Upper) Absolute spectra of the oxidized (bold line), reduced (thin line), and reduced NO (dotted line) derivatives for the wt NIR (Left), mutant H327A (Center), and mutant H369A (Right). (Lower) Time evolution of the kinetic difference spectra observed for the same proteins, after the reduced enzyme is mixed with nitrite anaerobically. The two sets of difference spectra for each protein referto experiments carried out at the lowest (10 ␮M; A, C, and E) and the highest (0.15 or 0.5 mM; B, D, and F) nitrite concentrations. The arrow indicates the direction of the time course, from 6 ms to 245 s. To better follow the spectral evolution with time, the difference spectra at selected times (6 ms and 1, 25, and 180 s) are drawn as thick lines. The insets show the time course as followed at the maximum of reduced d1-heme (462 nm for the wt and 472 nm for the two mutants), fitted to two or three exponentials (continuous lines). Kinetic experiments were carried out in 50 mM phosphate buffer (pH 8.0) and 25°C.

550 nm is diagnostic of oxidation of the c-heme, because the oxidized (up to 20–25%). This process (see Insets in Fig. 2) occurs reference spectrum is that of a fully reduced c-heme. between 0.1 and 10 s, with the optical changes assigned to the The first spectrum recorded after reduced NIR was mixed with d1-heme preceding those of the c-heme (with rate constants of 2 and Ϫ 10 ␮M nitrite (at 6 ms) still contains a significant fraction of the 0.3 s 1, respectively). Finally, at times between 10 s and 4 min the ϩ2 ϩ2 starting reduced species (Fig. 2A). Because only minor oxidation of mixture slowly drifts toward the c d1 NO species, and the the c-heme is observed (Յ15%), it is likely that the Michaelis c-heme returns to the fully reduced state. ϩ2 ϩ2 Ϫ complex (c d1 NO2 ) is one of the species populated at this At least four spectra thus can be assigned during the overall time time. At higher nitrite concentrations (150 ␮M, Fig. 2B) more course (but only two of them refer to pure species): (i) that of the ϩ2 ϩ2 oxidized c-heme is observed at 6 ms. The initial mixture evolves fully reduced c d1 derivative, present in the driving syringe; (ii) through a nitrite-independent process and populates other species. that of the pre-steady-state mixture, clearly evident in the first After absorbance changes in the spectral regions characteristic of spectrum after mixing (6 ms); (iii) that corresponding to a more complex mixture, containing a clearly detectable amount of the both the c- and d1-hemes, it is observed that more c-heme becomes ϩ3 ϩ2 mixed valence complex c d1 NO; and (iv) that of the final species ϩ2 ϩ2 c d1 NO (dead-end state). The time course at 460 nm (Insets of

Table 1. Enzymatic activities of wt and mutant cd1 NIR Fig. 2 A and B) is thus representative of two processes, namely evolution of the initial mixture to the steady state and formation of Nitrite reductase* Oxygen reductase† the dead-end species. An overall scheme showing the various Ϫ1 Ϫ1 NIR Km, ␮M Turnover number, s Km, ␮M Kcat, ␮M min possible intermediates, as well as information obtained by George et al. (20), is depicted in Fig. 3 and detailed in the figure legend. wt 6 8 2.0 (Ϯ0.5) 1.5 (Ϯ0.5) Ϯ Ϯ Additional information on this reaction has been obtained with H327A — 0.08 1.8 ( 0.8) 2.6 ( 0.5) the following experiments: (i) if the steady-state mixture obtained H369A — 0.08 7.5 (Ϯ2) 1.9 (Ϯ0.6) as described above is quickly prepared and then mixed with NO ␮ ϩ2 ϩ2 *pH 6.2 and 25°C. Error is Ϯ20%. (100 M) within 20 s, a species with the spectrum of c d1 NO † Ϫ1 pH 7.0 and 20°C. Km values were obtained with cytochrome c551 as a substrate. is obtained with an apparent rate constant of 3 s , whereas mixing

2234 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.041365298 Cutruzzola` et al. Downloaded by guest on September 27, 2021 In the case of mutant H369A, the first spectrum corresponds largely to reduced unliganded NIR, which then evolves in a multiphasic process to yield the reduced, NO-bound species, more clearly so at high nitrite concentration, i.e., 0.5 mM (Fig. 2F). The observed time course (Fig. 2F, Inset) can be fitted to two exponentials; the faster process has the same rate (k ϭ 0.3 sϪ1) at high and low nitrite, whereas the slower one is concen- ϭ Ϫ1 Ϫ Ϫ1 tration dependent (k 0.045 s at 0.5 mM NO2 and 0.004 s ␮ Ϫ at 10 MNO2 ). Despite the complexity of the time course (which frustrated attempts to describe quantitatively the kinet- ics), it seems obvious from examination of the difference spectra (Fig. 2) that, compared with wt NIR, H369A has a considerably Ϫ Ϫ reduced affinity for NO2 . The dead-end state is reached more Fig. 3. A proposed scheme for the reaction mechanism of cd1 NIR with NO2 . Ϸ When nitrite is mixed with the reduced enzyme (species 1), the formation of the rapidly than wt ( 3 min), and the reaction scarcely requires Michaelis complex (species 2) is followed by very rapid formation of NO, involving cycling; thus compared with wt, either the mixed-valence NO bond breaking and loss of a hydroxide ion; yet this process is assumed to be complex is reduced faster or dissociation of NO occurs more ϩ2 ϩ3 ϩ2 ϩ2 ϩ reversible. The resulting mixed-valence species 3 or 4 (c d1 NO or c d1 NO ) slowly, or both. The faster evolution to the dead-end species at may dissociate NO and be reduced to species 1 (via 6 and 7) to enter a new higher substrate concentration may simply reflect the increase of productive cycle; however, in vitro it seems to be progressively inhibited to a the catalytic rate with substrate concentration, given that in this dead-end state with NO bound to the ferrous d1-heme (species 8). Species 1–5 mutant all backward rate constants are probably higher than in equilibrate rapidly [see also George et al. (20)], accounting for a fraction of wt NIR. oxidized c-heme formed during the dead time of the stopped flow. The relative Reduction of the c-heme by ascorbate starting from the fully population of each species depends on experimental conditions, such as pH and concentration of substrate and reductant, and it may even differ in NIR from oxidized His mutants corresponds to a single kinetic process, Ϸ Ϫ1 ␮ different species. The Michaelis complex, formed rapidly even at low nitrite with a rate constant of 0.1 s at 75 M ascorbate, compared Ϫ1 concentrations (e.g., 100 ␮M), accounts for considerably less than 100% of the with heterogeneous kinetics with values of 0.1–0.01 s for the enzyme; therefore the bimolecular rate constant is fast, but the affinity is lower wt enzyme. When azurin was used as the reductant in the than previously suggested (24). Nevertheless, our kinetic data are consistent with presence of excess CO (21), reduction of the c-heme in the ϭ ␮ Ϫ the value of Km 6 MNO2 , which was independently determined (not shown). mutants occurs 2-fold more rapidly than for wt, whereas no ϭ Ϫ1 Species 6 builds up slowly (k 2s ) and incompletely under our experimental significant difference in the c-to-d1 electron transfer rate could conditions; the internal redox equilibrium between species 6 and 7 is assigned a be detected (W. Sun, M.A., A.B., F.C., and M.B., unpublished Ϫ1 rate constant of 0.3 s . Because it is well established that the electron accepting observations). site is the c-heme, only species 5 and 7 can be reduced with the use of ascorbate, azurin, or cytochrome c . In the scheme, there are two paths leading to cϩ2d ϩ2 551 1 X-Ray Structure of the H327A and H369A Mutants. In both mutants, NO, the dead-end species 8: either by reaction of the reduced enzyme with NO or Ϫ by reduction of species 5 by external reductants. In P. pantotrotrophus NiR, no 2Fo Fc electron density is visible at the place of the mutated Ϫ Ϫ species 5 forms completely and instantaneously at [NO2 ] ϭ 0.2–5.0 mM; there- His, whereas a strong negative density was observed in the Fo fc after it decays to species 4 at 38 sϪ1, as shown by George et al. (20); it is possible map, as expected (see Fig. 1 B and C). The whole N-terminal that a similar reequilibration also occurs in Pa-NIR, but if so, it is lost in the dead segment (residues 1–25) has no detectable electron density in either time. mutant, likely because of molecular flexibility. Consequently, Tyr-10 is not visible in the distal pocket of the d1-heme, contrary to what is seen in the oxidized wt protein (4, 5). For the H369A with degassed buffer has no effect (not shown). This experiment mutant, the other residues in the active site have conformations that shows that the initial mixture of species must transiently allow are almost unchanged compared with the wt or differ as different formation of the unliganded reduced derivative, either by cycling or data sets at this resolution usually do (rms deviation Х 0.5 Å). The by dissociation of the weakly bound substrate. (ii) If reduced NIR active site crevice of H327A is clearly more open (on average by 2 is mixed with NO and nitrite, only the NO complex is obtained Å), with the distances between the Fe and the C␤ of residues within the dead time of the instrument (2 ms), showing that the His-369, Ala-327, and Phe-425 increasing by about 3.8, 1.5, and 1.8 affinity and rate constants for combination with NO must be high. Å, as compared with the wt enzyme and the H369A mutant. (iii) Reduction of fully oxidized wt NIR by ascorbate populates a Both mutants H327A and H369A are dimers in solution, as mixed-valence intermediate and displays a complex time course: determined by gel filtration chromatography (data not shown). Ϫ1 the faster phase has a rate constant of 0.1–0.2 s , dependent on the Although a single monomer is contained in the crystal asymmetric concentration of ascorbate, and is followed by much slower pro- unit, a dimer identical to the wt enzyme can be reconstructed by cess(es) (not shown). This rate constant is compatible with the taking into account a 2-fold symmetry-related molecule (Fig. 4). hypothesis that irreversible inhibition of the enzyme in vitro may The crystallographic monomer is composed of the ␤-propeller occur by reduction of the mixed valence state with formation of domain carrying the d1-heme (residues 149–543) and of the c-heme ϩ2 ϩ2 c d1 NO in the presence of NO. domain (residues 26–115), linked by a 34-residue segment. Similar H369A and H327A Mutants. For the mutant H327A, the first to the wt enzyme (oxidized or reduced), the B factors of the c-heme spectrum after mixing depends on nitrite concentration (Fig. 2 C domains of both mutants are higher than those of the d -heme Ϫ ϭ ␮ 1 and D). At [NO2 ] 10 M (Fig. 2C) this spectrum corresponds domain, to such an extent that for the mutant H369A, segments of to a mixture containing a significant percentage of the fully reduced the polypeptide chain were difficult to follow. unliganded enzyme (see, for example, the band at 472 nm); at In both mutants, the d1-heme domain is superimposable on that Ϫ ϭ [NO2 ] 0.5 mM (Fig. 2D) the more populated component is not of the wt protein, whereas the c-heme domain’s orientation is one that can be obtained at equilibrium and recalls the spectrum of completely different (Fig. 4). The latter has been subjected to a the first intermediate observed in the experiment carried out on wt rigid-body gliding motion on top of the d1-heme domain surface, NIR. The final spectrum, corresponding to the reduced NO com- resulting in a rotation of about 60° around an axis parallel to the Ϸ Ϫ plex, is reached in 1 min, at least at the highest NO2 concen- 2-fold molecular axis and passing through Gln-115. As a result, the tration (0.5 mM). We conclude that substrate binding to the mutant c-heme domains have moved about 20 Å, on average, from the

H327A is somewhat similar to wt, but its conversion to the dead-end original position in the wt (Fig. 4). The distance between the c- and BIOCHEMISTRY species occurs faster. d1-heme iron (21 Å) is almost unchanged, whereas the edge-to-edge

Cutruzzola` et al. PNAS ͉ February 27, 2001 ͉ vol. 98 ͉ no. 5 ͉ 2235 Downloaded by guest on September 27, 2021 avoid inhibition. Structural and spectroscopic information on the enzyme from two different species is now available (3, 6, 8, 22–24), and very recently, the reaction of reduced P. pantotro- phus NIR with nitrite at very high concentration (e.g., 5 mM) and in the absence of excess reductant has been reported (20). Our study provides insight into the enzyme mechanism and allows us to propose the scheme shown in Fig. 3, which is an extension of that initially proposed by Averill (1). It implicitly assumes that functional interactions between the two monomers can be neglected, which may not necessarily be the case. Insight into the molecular mechanism of catalysis comes from the results on the two His mutants. Substitution of either of the two invariant His with Ala has a dramatic effect on nitrite reduction, but absolutely no effect on the oxygen reductase activity. In addition, it was somewhat surprising to discover that the two His are not equally important in the reaction with nitrite and to find out that His-369 is essential in controlling the affinity for this ligand. This disparity in importance is shown by the stopped-flow experiment carried out with the H369A mutant (see Fig. 2 E and F), in which no significant amounts of the Michaelis complex can be detected; Ϫ the reaction with NO2 proceeds from the reduced state (species 1, Fig. 3) to the dead-end product (species 8) without detectable intermediates. In the mutant H327A, on the other hand, a signif- icant fraction of the pre-steady-state mixture of species was de- tected, as shown in Fig. 2 C and D. It is known that the affinity of ferrous heme proteins for anions is usually very low; as an example, ferrous hemoglobin binds Ϸ cyanide with a Kd 1 M (25). On the other hand, in the case of NIR, the ferrous d1-heme displays high affinity for both nitrite and Ϸ Ϫ6 cyanide (Kd 10 M). The 3D structure of the cyanide derivative of reduced P. pantotrophus NIR (23) shows that the two His and the Tyr in the active site are within H-bonding distance of cyanide, suggesting that a positive electrostatic potential in the pocket is Fig. 4. Crystallographic structure of the two mutants of Pa-NiR. (A) Top view essential for stabilization of the bound anion. An unusually high of the mutant proteins superimposed on the wt enzyme. Color code for the positive charge density has also been found in the heme pocket of c-heme domains: dark green, H327A; light green, H369A; red, wt. It is evident the Escherichia coli sulfite reductase, an enzyme capable of binding that the c-heme domains glide away in a new configuration, which is almost both sulfite and nitrite with high affinity, catalyzing their reduction the same for the two mutants. Notice also that the 3D structure of the d1-heme to sulfide and ammonia, respectively (26). The unique role of domain (gray) is identical for the three proteins. (B) Side view of the three His-369 in stabilizing the enzyme–substrate complex, although structures (same color code); only one monomer is shown for the sake of unexpected, seems to be in agreement with the structural data clarity. shown in Fig. 1. In fact, although both His are fairly close to the bound nitrite, His-369 is a shorter distance from both oxygens of the substrate, thus accounting for a dominant effect on the stability of distance (11.4 Å in the wt) has increased by 2 and 4 Å for H327A the Michaelis complex, via the formation of (possibly) two H-bonds. and H369A, respectively. In the mutants, the two c-heme domains Over and above the visible loss of density due to substitution of in the dimer are closer to each other (3.8 Å), and the angle between His with Ala in the active site (Fig. 1 B and C), the most striking them is about 13°, perpendicular to the 2-fold molecular axis. The structural feature of both mutants is a large displacement of the c-heme domains, in their new positions, are involved in contacts whole domain containing the c-heme relative to the d1-heme, which with symmetry-related molecules: in H327A, the loop 92–95 inter- stays put (Fig. 4). An important issue is to understand whether and acts with the bottom of a symmetry-related d1-heme domain how this conformational change is related to the dramatic inhibition (Lys-431), the residues 61 and 70 interact with the Asn-502 of a of NIR activity reported above for the two mutants. The overall symmetry-related molecule, and the loop 71–85 interacts with the distance between the c- and d1-hemes and the surface of contact same loop of a symmetry-related molecule. In H369A, the segment between these two domains is similar in the His mutants compared 78–85 interacts with a symmetry-related loop (residues 522–524) of with wt; therefore the rate of intramolecular electron transfer the d1-heme domain. should not be drastically modified, a fact that has been experimen- The contact area between the c- and d1-heme domains in the tally verified (W. Sun, M.A., A.B., F.C., and M.B., unpublished 2 wt enzyme has a surface of about 350 Å . This area is almost observations). Furthermore, the large area of contact between the Ϸ 2 maintained in the mutant H369A) ( 300 Å ) and is increased to c- and d1-heme domains and the fact that the c-domain position is Ϸ 2 500 Å in the mutant H327A. Despite some similarities in the very similar for the two mutants (and therefore unlikely to be overall interdomain surface area, the high B values observed opportunistic) suggest that the structure is a rather stable one. with the H369A mutant may indicate a mobility of the c-heme These results illustrate the plasticity of the redox enzymes, a domain higher than those in the wt and H327A structures. phenomenon often postulated but seldom substantiated (27, 28). In our case the structural change shown in Fig. 4 may be used to gauge Discussion the decreased interactions of Tyr-10 with the two invariant His-327 Periplasmic cd1NIR catalyzes in denitrifying bacteria the con- and His-369 in the active site and the associated motion of the version of nitrite to NO. This reaction poses a problem because N-terminal arm away from the pocket. This structural change is the product NO is known to bind with very high affinity to accompanied by a relocation of the c-heme domain in a minimum reduced hemes and thus must be quickly released in the bulk to of lower free energy. A similar relocation of the c-heme domain

2236 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.041365298 Cutruzzola` et al. Downloaded by guest on September 27, 2021 may take place also in the mutant Y10F, which, despite being and should assist NO dissociation. This hypothesis is in agreement functionally and spectroscopically identical to wt (9), has been very with quantum mechanical calculations (29), indicating that inter- difficult to crystallize. In a nutshell, we suggest that the c-heme action with the active-site His can stabilize and orient nucleophilic domain relocation and the loss of favorable interactions of Tyr-10 ligands of the d1-heme, the presence of which imposes an unfavor- in the active site of the two His mutants may be due to reduction able orientation on NO and thereby facilitates its release. It is worth of the positive electrostatic potential in the pocket. Insofar as this noting that it has been recently shown that a buried carboxylate in hypothesis is correct, a structural interpretation of the inhibition of the active site of nitrophorins (a class of heme proteins with the NIR activity observed with either one of the two His mutants vasodilatory activity associated with NO release) promotes disso- may focus on the observation that they are trapped in the reduced ciation of NO from the ferric heme iron (30). Finally, the inter- NO-bound dead-end species faster than is wt NIR. According to the pretation proposed above is consistent with our finding that mu- scheme shown in Fig. 3, dissociation of NO from the mixed-valence tation of either one of the two invariant active-site His with Ala has ϩ2 ϩ3 species 3 (c d1 NO) should be faster than electron transfer to the no effect on the O2 reductase activity of NIR. In this case the d1-heme to yield a catalytically competent enzyme. Given that the substrate is uncharged, and its protonation may be assisted by the intramolecular electron transfer rate is unchanged in the His remaining His and bulk water, now accessible to the d1-heme distal mutants (see above), the increased probability of trapping the pocket, which is wide open. dead-end species may originate in a faster reduction of the mutants by ascorbate (as observed; see Results) or a decrease in the rate of We thank R. Dagai and L. Nicolini (Istituto Superiore di Sanita`, Rome, NO dissociation from the ferric d -heme of species 3, or both. We Italy) for fermentation of bacterial strains and V. Zamboni (Marseille, 1 France) for crystallizing the mutants. Grants from the Consiglio Nazionale thus propose that substitution of either one of the two invariant delle Ricerche of Italy (Target Project on Biotechnology) and the Min- active-site His with Ala and reduction of the positive potential in the istero dell’Universita`e della Ricerca Scientifica e Tecnologica of Italy pocket is associated with loss of the hydroxyl, which was found to (Progetto di Ricerca di Interesse Nazionale 1999, ‘‘Dinamica Strutturale di be coordinated with the ferric d1-heme iron in the wt enzyme (4) Emoproteine’’) are gratefully acknowledged.

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