The effect of anions on azide binding to myoglobin: an unusual functional modulation

Biochimica et Biophysica Acta 1594 (2002) 341^352 www.bba-direct.com

The e¡ect of anions on azide binding to myoglobin: an unusual functional modulation M. Cristina De Rosa, Claudia Bertonati, Bruno Giardina *, Enrico Di Stasio, Andrea Brancaccio Institute of Chemistry and Clinical Chemistry, and C.N.R. Centre of Receptor Chemistry, Catholic University of Rome, Largo F. Vito 1, 00168 Rome, Italy Received 24 July 2001; received in revised form 30 October 2001; accepted 29 November 2001

Abstract The effect of increasing concentrations of several anions on the azide (N3 3 ) binding properties of sperm whale and horse ferric myoglobin has been studied. Surprisingly, a number of anions may act as heterotropic effectors, decreasing the affinity 3 3 3 3 23 of myoglobins for N3 3 , in the following order: ClO4 = I s Br s Cl and SO4 , which mirrors the increase in their charge 3 3 density. The largest effects were measured using ClO4 and I , which produce a 4-fold and 8-fold reduction of the N3 3 binding affinity in horse and sperm whale myoglobins, respectively. A dissociation equilibrium constant (Kd ) ranging from 150 to 250 3 mM was estimated for ClO3 4 and I binding to myoglobins. In order to analyse the molecular mechanism producing the 3 reduction of the N3 binding affinity to ferric myoglobin, the potential anionic binding sites within ferric myoglobin were investigated by a molecular modelling study using the program Grid. Analysis of the theoretical results suggests two particularly favourable binding sites: the first, next to the distal side of the haem, whose occupancy might alter the electrostatic potential surrounding the bound N3 3 ; the second, involving residues of helices B and G which are far from the haem iron atom, thus implying a long range effect on the bound N3 3 . Based on the evidence that no significant 3 conformational changes are found in the three-dimensional structures of N3 3 -free and N3 -bound myoglobin and on previous 3 results on N3 binding to ferric myoglobin mutants in CD3 positions, we favour the first hypothesis, suggesting that the functional heterotropic modulation of monomeric myoglobin is mainly depending on a decrease of the positive charge density induced by the binding of anions to the haem distal side. ß 2002 Elsevier Science B.V. All rights reserved. Keywords: Myoglobin; Ligand a¤nity; Heterotropic e¡ector; Computer modeling; Binding site

1. Introduction Recent progress in the characterization of ion^protein interactions for several proteins, such as serine proteases, ¢brinogen, and DNA binding proteins has suggested the existence of a broad range of previ-

* Corresponding author. Fax: +39-6-305-3598. E-mail address: [email protected] (B. Giardina).

ously unidenti¢ed ion-dependent allosteric systems [1,2]. Myoglobin (Mb) is a monomeric haem protein which represents a paradigmatic case for protein molecules which do not display any signi¢cant functional modulation either by homotropic or heterotropic e¡ectors [3]. There are three physiologically relevant ligation states for myoglobin: deoxymyoglobin, in which the haem iron atom is in the ferrous state and has an unoccupied sixth coordination site;

0167-4838 / 02 / $ ^ see front matter ß 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 0 1 ) 0 0 3 2 7 - 2

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liganded myoglobin, such as oxymyoglobin and carbomonoxymyoglobin, in which the iron is in the ferrous state with a ligand covalently bound to the sixth coordination site; and aquometmyoglobin, in which the iron is in the ferric state and has a water molecule co-ordinated to the haem [4^6]. The residues which surround the ligand, forming the so-called distal pocket, and in particular the distal histidine (E7) were demonstrated to be important in ¢ne-tuning ligand a¤nity and reactivity in Mb [7^9]. On the other side of the haem, the proximal F8 histidine binds to the haem iron atom, thus linking the haem to the globin. Conformational changes occurring in Mbs upon ligand binding are very small, compared with the corresponding tertiary structural change of each subunit of haemoglobin [10,11]. The relation between conformational motions and function in Mb has been investigated [12^14] and more recently Giardina et al. [15] have reported the e¡ect of lactate on the functional properties of ferrous Mb. The possibility of modulating ligand a¤nity in Mb prompted us to study the e¡ect of increasing concentrations of several anions on the azide (N3 3 ) binding properties of sperm whale and horse ferric myoglobin (met-Mb). In the ferric state, Mb can bind a 3 3 3 variety of anions such as N3 3 , CN , SCN and F [3,16,17] and a great deal of experimental work has been carried out on the reaction between CN3 , N3 3 and Mb [18^21]. In particular the N3 anion, 3 although not physiological, has been widely used to prepare stable low spin Fe(III) haem protein derivatives, whose properties resemble those of the complex between Fe(II) and oxygen, giving useful information about the general topic of protein^ligand interactions [22,23]. Herein we show that anions may modulate the a¤nity of N3 3 in met-Mb and that the protein could discriminate between monovalent ions according to their charge density. For a better understanding of the molecular interactions involved in the anionic binding to Mb, a molecular modelling study using the program Grid [24] has been carried out. Several computational approaches can be used to locate potential binding sites in a macromolecule [25^27]. The Grid program has been applied to various biological systems [28^30] and shown to be a very powerful method to identify

ligand binding sites. Grid calculations predicting the binding of a series of modi¢ed haems to sperm whale apo-Mb have also been reported [31,32]. Anionic binding sites predicted by the program Grid were checked by comparison with X-ray crystallographic structures of met-Mb and ligand^met-Mb complexes [16,23,33]. Our ¢ndings suggest that binding of anions to the haem distal site is responsible for a modulation of ligand binding in Mb and strengthen the concept that heterotropic modulation could be applied even to monomeric proteins. 2. Materials and methods 2.1. Functional studies Ferric horse heart (HH) and sperm whale (SW) myoglobins were purchased from Sigma (St. Louis, MO, USA) and used without further puri¢cation. All the reagents used were of analytical grade and purchased from Sigma or Fluka (Buchs, Switzerland). All the tested anions were used as sodium salts. The a¤nity binding constants for N3 3 were spectrophotometrically determined using a Cary 3 (USA) dual beam apparatus in 0.1 M sodium phosphate bu¡er at pH 6.5, 20³C as described elsewhere [34,18]. Brie£y, spectra were registered between 350 and 450 nm as a function of N3 3 concentration. Using OD values at 420 nm binding isotherms were evaluated and ¢tted using a single class of equivalent binding sites equation: Y = x/(Kd +x) where Y is fractional saturation (ODi /ODtot ), x is the concentration of ligand and Kd the equilibrium dissociation constant. A similar hyperbolic equation has been used to ¢t the data reported in Fig. 2, in order to evaluate the apparent Kd values for anion binding to Mbs. Control experiments were also carried out in 0.1 M 2-[N-morpholino]ethanesulphonic acid (MES) at pH 6.5 and 20³C in the absence of chloride ions. The N3 3 3 binding a¤nity in the presence of ClO3 4 and I was also measured at constant ionic strength using di¡er3 ent amounts of SO23 4 (for both Mbs) and Cl (only for HH Mb) in order to evaluate the e¡ect of nonspeci¢c electrostatic e¡ects [35]. For example, when we have tested 50 mM and 400 mM I3 , we have carried out experiments in the presence of 250 mM

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Table 1 List of probes used for the computations Probes F3 Cl3 I3 O: : O1 DRY

Fluoride anion Chloride anion Iodide anion Carboxy oxygen atom Aliphatic hydroxyl group Hydrophobic probe

VDWR

NEFF

ALPH

Q

1.36 1.81 2.16 1.60 1.65 1.65

9 13 31 6 7 7

0.80 2.90 6.30 2.14 1.20 1.20

31.0 31.0 31.0 30.45 30.1 0.0

The van der Waals radius (VDWR), number of e¡ective surrounding electrons (NEFF), polarizability (ALPH) and charge (Q) are shown for each probe.

23 and 133 mM SO23 4 , respectively. The proper SO4 amounts added were calculated according to the equation I = (1/2)4mi z2i , where I is the ionic strength, m is the molality and z is the charge of the ion. In Fig. 2, the Kd values, for N3 3 binding to Mbs, are those measured at constant ionic strength, whereas in Table 2 are reported those measured not at constant ionic strength. However, the apparent Kd values estimated for anion binding to Mbs (W200 mM for I3 ClO3 4 both to HH Mb and SW Mb, see Fig. 2) are very slightly a¡ected by the e¡ect of ionic strength (less than 10%, data not shown).

2.2. Molecular modelling The program Grid is particularly suitable for the study of proteins and was used to investigate the anionic binding sites in Mb. Grid calculations were performed on three di¡erent crystallographic structures of sperm whale met-Mb. The structures were obtained from the Protein Data Bank (PDB id: 4mbn [4,5,36]; 1a6k [6]; 1bz6 [33]) and appropriate parameters for Grid force ¢eld were assigned to each atom of the proteins. In the program, a three-dimensional grid surrounds the protein and the interaction energy between a probe, which represents a speci¢c chemical group, and each atom of the protein was then calculated for each grid point. The positions of all the core atoms are ¢xed but £exible side chain atoms of the protein can move towards the probe when there is attraction and away from it when there is repulsion. This approach gives a large volume of good attractive interactions between the probe and the macromolecule, since the £exible side chains always move in order to ¢nd the most favourable po-

sitions for interaction with the probe at each particular grid point [32]. The probes are characterized by their steric, electrostatic, and hydrogen bonding properties, and by their hybridization. The probes used for this molecular modelling study were selected from those provided by the program in order to represent monovalent anions with increasing ionic radius (Table 1). The carboxy oxygen (O: :), hydroxyl (O1) and hydrophobic (DRY) probes which all together represent the structure of lactate were also included in the list of the probes. The method of mirror charges [24] is used by Grid to compute the e¡ective dielectric, in this case to simulate the water environment surrounding the macromolecule. Grid results for halides are displayed as contour maps using InsightII (MSI, San Diego, CA, USA) showing regions of Mb where the anion would make favourable interactions. Dealing with a bigger ligand such as lactate, the results obtained by Grid are used as input data for the program Group [24] which, by ¢tting the Grid maps generated for O: :, O1, and DRY probes, positions the atoms of the ligand at favourable places on the protein. 3. Results 3.1. Functional studies The e¡ect of a number of anions, displaying signi¢cant charge density di¡erences, was tested on the binding a¤nity of HH and SW Mbs for N3 3 . The 3 3 , NO ions under analysis were: I3 , ClO3 4 3 , Br , 3 23 3 23 Cl , SO4 and H2 PO4 /HPO4 . In Table 2 is re-

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Table 2 Equilibrium dissociation constant (WM) for N3 3 binding to met-Mbs in the presence of di¡erent anions at 20³C, pH 6.5 Ion

HH Mb [anion] = 100 mM

SW Mb [anion] = 100 mM

HH Mb [anion] = 800 mM

SW Mb [anion] = 800 mM

23 H2 PO3 4 /HPO4 MES SO23 4 Cl3 Br3 NO3 3 ClO3 4 I3 Lactatea Lactateb

30 þ 4 28 þ 4 33 þ 3 32 þ 3 36 þ 3 47 þ 5 69 þ 6 72 þ 6 30 þ 3 30 þ 3

20 þ 3 19 þ 3 24 þ 3 27 þ 4 50 þ 5 42 þ 4 90 þ 8 82 þ 8 24 þ 3 25 þ 3

60 þ 6 ^ 58 þ 5 55 þ 5 80 þ 8 93 þ 9 127 þ 11 120 þ 9 ^ ^

43 þ 5 ^ 48 þ 5 84 þ 8 114 þ 11 124 þ 9 160 þ 10 168 þ 12 ^ ^

Every Kd value is derived from at least three independent experiments; standard deviations are reported. It should be noted that in the presence of 800 mM F3 , the dissociation constant for N3 3 was estimated higher than 2000 WM (the binding is clearly inhibited, see Fig. 1). Data refer to experiments carried out at the indicated anion concentration, without considering the non-speci¢c e¡ects related to the ionic strength (see Section 2 for details). a Experiment performed in 0.1 M sodium phosphate. b Experiment performed in 0.1 M MES.

ported the value of the dissociation equilibrium constant (Kd ) for the N3 3 binding both to SW and HH Mbs, measured at 100 mM and 800 mM anionic concentration. All the experiments were carried out in 0.1 M sodium phosphate at pH 6.5, T = 20³C. In Fig. 1, a representative set of N3 3 binding isotherms to Mbs is reported. 3 Strongly chaotrope ions, such as ClO3 4 and I , 3 produce an W4-fold increase of the Kd for N3 for HH Mb and an W8-fold increase for SW Mb at a concentration of 800 mM (see Table 2). A similar but

reduced e¡ect on SW Mb is also evident for NO3 3, 3 23 Br3 and Cl3 , whereas SO23 and H PO /HPO had 2 4 4 4 no e¡ect, considering as entirely non-speci¢c the effect due to ionic strength, which accounts for a 2-fold decrease of the a¤nity (see Table 2 and Section 4). The strength of the observed e¡ect follows the Jones^Dole scale that correlates with the ion charge density [37,38]. Fig. 2 shows the variation of Kd for the Mb^N3 3 3 3 reaction as a function of ClO3 and SO23 4 , I , Cl 4 concentration for HH Mb (Fig. 2a) and for SW Mb

Fig. 1. Binding isotherms referring to N3 3 binding to HH Mb (a) and SW Mb (b). Experimental conditions were: 0.1 M phosphate 3 a ( ), 800 mM SO23 (E), no salt (O) and 800 mM F3 bu¡er, pH 6.5, at 20³C, in the presence of 800 mM ClO3 4 (F), 800 mM Cl 4 3 (7). Mb concentration was always W2 WM, and data are reported in terms of free N3 concentration. The continuous and dotted lines refer to hyperbolic ¢ts (see Section 2). F3 competes for N3 3 binding to the haem iron atom of Mbs as already described by others [3], and it is reported as a control (only for HH Mb).

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3 3 Fig. 2. Dissociation constant (Kd ) for the Mb^N3 (O), Cl3 (a) and SO23 (E) concentration 3 reaction as a function of ClO4 (F), I 4 and Cl3 is non-speci¢c and is likely for HH Mb (a) and SW Mb (b). In the case of HH Mb, the slight e¡ect measured with SO23 4 shows a non-speci¢c e¡ect since Cl3 has an interdue to the increase of the ionic strength of the solution. For SW Mb only SO23 4 3 3 mediate e¡ect with respect to ClO3 and I . Continuous lines, for ClO , and dotted lines, for I3 , refer to hyperbolic ¢tting procedures 4 4 which all give a Kd of W200 mM. Experimental conditions were: 0.1 M phosphate bu¡er, pH 6.5, at 20³C. All the Kd values reported were measured at constant ionic strength (800 mM), except those referring to SO3 4 (see Section 2 for details).

Fig. 3. Absorbance spectra between 350 and 450 nm, SW Mb (a) and HH Mb (c), and between 450 and 700 nm, SW Mb (b) and HH Mb (d). Aquomet-Mb spectrum (continuous line), aquomet-Mb in the presence of 800 mM F3 (continuous line with b) and 3 3 2 mM azide (continuous line with E). Data are not shown for spectra collected in the presence of 800 mM I3 , ClO3 4 , NO3 , Br , 3 23 3 Cl , SO4 and PO4 to avoid overcrowding, since minimal di¡erences were detected with the aquomet-Mb spectrum. All the spectra were recorded at 20³C and pH 6.5; met-Mb concentration, for both SW Mb and HH Mb, was W2 WM for 350^450 nm spectra, and W10 WM for 450^700 nm spectra.

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(Fig. 2b). The slight e¡ect measured with SO23 for 4 3 both Mbs and with Cl for HH Mb is non-speci¢c and likely due to the increase of the ionic strength of the solution, as already discussed for SO23 4 by others 3 [39]. On the contrary, ClO3 and I are signi¢cantly 4 active on both Mbs, and their e¡ect is fully saturable with a binding a¤nity corresponding to a dissociation constant of W200 mM. It should be noted that 3 the data reported in Fig. 2, referring to ClO3 4 and I for both Mbs and to Cl3 only for SW Mb, were measured at constant ionic strength (800 mM), using decreasing amounts of an inert anion, such as SO23 4 , as the concentration of the anion under analysis 3 3 (ClO3 4 , I or Cl ) increases. It is intriguing that the anion lactate, previously

reported as a physiological heterotropic e¡ector of the Mb oxy^deoxy transition [15], did not produce any e¡ect (either in 0.1 M sodium phosphate or 0.1 M MES at pH 6.5) on the met-Mbs under analysis up to a concentration of 100 mM, which corresponds to its solubility limit (see Table 2), thereby suggesting the existence of structural and functional di¡erences between these two derivatives. The absorption spectra between 350 and 700 nm of both met-Mbs, collected in the presence of di¡erent anions, clearly show that the e¡ects measured were not due to a direct binding of the anions at the 6th coordination position of the haem iron atom, thus competing for the binding of N3 3 and reducing the 3 3 apparent Kd for N3 3 . As shown in Fig. 3, ClO4 , I ,

Fig. 4. Grid contour map at 320 kcal/mol for the F3 probe. The met-myoglobin structure by Takano (PDB code: 4mbn) is shown colour-coded by atom type (C, green; O, red; N, blue; Fe, magenta) and the contour map is white. Crystallographic sulphate ion (only the sulphur atom is reported in the structure) is shown, yellow coloured, low on the right. The predicted region of binding for F3 superimposes onto the crystallographically determined F3 in the Mb^£uoride complex. The contour maps are displayed using InsightII.

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M.C. De Rosa et al. / Biochimica et Biophysica Acta 1594 (2002) 341^352 3 23 Br3 , NO3 3 , Cl , SO4 at 800 mM did not change the spectroscopic properties of met-Mbs, indicating the lack of direct binding to the haem iron. As a control in Fig. 3 is also reported the e¡ect of F3 (800 mM) and N3 3 (2 mM), that instead change the maxima, shape and extinction coe¤cients of both met-Mbs via direct binding to the haem iron (see Table 2) [3]. An additional experiment was carried out to demonstrate that the e¡ect of the anions on Mbs was fully reversible. Namely, after an overnight incubation at high anionic concentration (800 mM), the met-Mbs solution was dialysed in a bu¡er containing no anions and afterwards both the spectroscopic properties and the a¤nity for N3 3 were completely restored.

3.2. Molecular modelling Energetically favourable binding sites within SW met-Mb were predicted for the ions provided by the program Grid, representing monovalent anions with increasing ionic radius (mostly halides, whereas no ClO3 4 probe was available, see Table 1). Grid calculations over three di¡erent structures of metMb show that the global interactions of the selected probes are similar with all Mbs; in particular, when the £exibility option of the program is selected and the amino acid side chains can move from their initial position, the low energy regions are the same for the three structures. The values of the most negative interaction energies for £uoride and iodide anions with the three met-Mb structures are reported in Table 3. In all cases Grid calculations on met-Mb using £uoride ion (F3 ) as a probe resulted in one minimum î for the 4mbn structure by which is close (2.15 A Takano) to the iron atom of the haem. This position is near the crystallographically determined £uoride position in the Aplysia limacina Mb^£uoride complex î from the iron atom (PDB ID: 3mba which is 2.23 A [16]). In Fig. 4 a contour map for F3 at 320 kcal/ mol is shown for the 4mbn structure. Di¡erent results, which can be ascribed to the decrease of charge density of the anion (see Table 1), are obtained when the interaction with larger halides is simulated. A Grid contour map at 316.0 kcal/mol was calculated from the 4mbn met-Mb structure for the iodide ion (I3 ) probe (Fig. 5A). The map shows one area of

347

Table 3 The most negative interaction energies (kcal/mol) for F3 and I3 probes with three di¡erent sperm whale met-Mb structures met-Mb structures (PDB code)

F3

I3

4mbn 1a6k 1bz6

322.61 321.14 322.88

317.03 318.50 317.19

The energies have been calculated using the Grid program [24].

interaction which is due to a few amino acids of the haem distal side (Phe CD1, Arg CD3 and His E7). The strongest energy of interaction between the I3 probe and the protein corresponds to the interaction with Phe CD1. The contour map for I3 at 311.0 kcal/mol (Fig. 5B) presents another main area of interaction located between the helices B and G of met-Mb, which is due to Ile B9, Arg B12, Leu B13, Phe G7 and Ala G11. This region was elected to be a potential anionic site in met-Mb since it is coincident with the binding site predicted by program Grid for the lactate molecule that has been found to modulate functional properties of ferrous Mb [15,40]. The binding mode of lactate, superimposed on the contours generated by I3 , is shown for 4mbn in Fig. 5B. It can be observed that the carboxyl group of lactate orientates, with respect to the protein, close to one crystallographic sulphate ion found by Kachalova et al. [33] in met-Mb. Both the predicted lactate and the crystallographic sulphate are hydrogen bonded to Arg B12 (Fig. 5B). The chloride ion probe (Cl3 ) shows a similar behaviour to I3 but its interaction with Phe CD1 is much weaker. 4. Discussion N3 3 , although not physiological, is a widely used ligand to analyse the functional and structural properties of haem proteins [3,17,19^23]. The binding of N3 3 to horse heart and sperm whale myoglobins can be modulated by a number of anions which, decreasing the a¤nity of N3 3 for Mb, may act as heterotropic e¡ectors. Several anions were studied and their di¡erent e¡ect on the Mb-N3 3 a¤nity constant is consistent with the anion properties as described by Collins [38]. Individual ions may be classi¢ed as

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Fig. 5. The predicted interaction between the I3 probe and met-Mb. The met-myoglobin structure by Takano (PDB code: 4mbn) is shown colour-coded by atom type (C, green; O, red; N, blue; Fe, magenta). (A) Grid contour map at 316 kcal/mol (white) displays the interaction between I3 and the distal side of the haem. The strong interaction between the anion and Phe CD1 can be observed. Crystallographic sulphate ion (only the sulphur atom is reported in the 4mbn structure) is shown, yellow coloured, low on the right. (B) Grid contour map at 311 kcal/mol (white) superimposed onto the lactate molecule in the orientation predicted by the program and onto the sulphate ion of the met-Mb structure by Kachalova et al. [33] (PDB code: 1bz6). The hydrogen bonds formed by lactate with Arg B12 are displayed.

chaotropes or kosmotropes by the Jones^Dole viscosity B coe¤cient [37] which correlates with the charge density and the strength of the interaction with H2 O molecules in solution. and H2 PO3 Kosmotrope anions such as SO23 4 4 bind water molecules tightly and therefore are little reactive with proteins. On the other hand, chaotrope 3 ions, such as ClO3 4 and I , are weakly hydrated and can easily interact with proteins by the removal of water molecules [41]. The e¡ects herein reported on the anionic-induced modulation of met-Mbs seem to re£ect the tendency of chaotrope ions to interact more favourably with protein moieties. Accordingly, we have measured the

most drastic e¡ects on the N3 3 a¤nity for Mbs using strong chaotrope anions such as I3 and ClO3 4 . Our results suggest that all the anions are capable of interacting with Mb, but with di¡erent a¤nities. In fact, the estimated dissociation constant (Kd ) of SW and HH Mbs for ClO3 4 ranges from 150 to 250 mM, in agreement with Kd values found also for other protein systems [42,35]. Accordingly, milder chaotropes, such as Br3 and Cl3 , show a weaker binding a¤nity that can be estimated in the molar range, and SO3 4 shows a slight e¡ect that can be totally ascribed to a non-speci¢c e¡ect [39]. It is noteworthy that the experiments were also carried out at constant ionic strength (I = 800 mM) using decreas-

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Fig. 5. (Continued).

ing concentrations of divalent SO23 4 with both Mbs, or monovalent Cl3 only with HH Mb due to the e¡ect that this anion has on N3 3 binding to SW Mb (see Fig. 2). Therefore, all the observed e¡ects can be fully ascribed to changes in the chemical potential of the ions, and not to changes in the ionic strength and/or in the Na‡ concentration, and cannot be accounted for by a non-speci¢c Debye^Huckel screening e¡ect. As a matter of fact, we may exclude that the e¡ect measured on the binding a¤nity for N3 3 to Mbs is dependent on non-speci¢c factors since (i) the e¡ect follows the dimension of the anions in solution; 3 (ii) the e¡ect of ClO3 shows a saturation 4 and I and is not competitive as that of £uoride, which binds directly to the haem iron [3]; and (iii) the anions do not induce any spectroscopic change on met-Mbs (see Fig. 3). It is remarkable that the induced change of a¤nity

for N3 3 is more evident in SW than in HH Mb. Despite a high degree of primary sequence homology found between sperm whale and horse heart myoglobin (88%), a few amino acid substitutions at the haem distal side (SW Arg CD3CHH Lys; SW Thr E10CHH Val) may account for the di¡erent functional behaviour displayed by the two proteins [43]. The molecular mechanism producing the reduction of N3 3 a¤nity to Mb can be interpreted on the basis of the molecular modelling results. Fluoride anion, which is representative of a series of ligands such as CN3 , N3 3 and imidazole, displays high a¤nity for ferric myoglobin, forms stable derivatives [3] and is predicted to bind iron atom (Fig. 4). The superimposition of the calculated binding site for F3 with the crystallographic F3 of the A. limacina Mb^£uoride complex [16] is a con¢rmation of the reliability of the Grid program in modelling ligand^protein interactions.

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According to molecular modelling, the observed anionic-induced changes of N3 3 binding in Mbs can be brought about by (i) the binding of chaotrope anions to amino acids next to the iron atom therefore altering the interaction between N3 3 and neighbouring amino acids, or (ii) the binding of chaotrope anions to amino acids far from the iron atom, thereby mediating a long range e¡ect on the interaction with N3 3 , which would imply a conformational rearrangement of the protein. The ¢rst hypothesis is strongly supported by our computational results, considering that the binding region due to the amino acids of the haem distal side corresponds to the global minimum of the Grid calculation for I3 (Fig. 5A). Also Fourier transforming infrared spectroscopy (FTIR) studies on the interaction of N3 3 with myoglobin mutants [43] suggest this mechanism. In fact, it was shown that the introduction of a negative charge in the distal side of the haem (Lys CD3CGlu) destabilizes the anionic N3 3 ligand, whereas the positively charged arginine (Val E10CArg) stabilizes azide binding. It was also observed that the major factor governing the stretching frequency Xco of CO bound to a number of Mbs is the electrostatic potential surrounding the bound ligand and not the steric hindrance within the haem pocket [44]. Therefore, we propose the existence of a direct interaction relating the decrease of the electrostatic potential at the haem distal side, which is due to the 3 binding of ClO3 to distal residues, with the 4 or I 3 a¤nity for N3 . This mechanism is consistent with the observation that the structure of the N3 3 ^SW Mb complex (PDB ID:1swm [45]), as well as of all the other N3 3 ^Mb complexes deposited in the Protein Data Bank [46], lacks the SO23 4 which is bound to the distal histidine in the SW aquomet structure (PDB ID: 4mbn [4,5]). It is worthwhile that Takano [4,5] correlates the absence of this sulphate ion in deoxy-Mb, with respect to the met form, to the loss of a net positive charge on the haem, and that more recently, Old¢eld [47] observed that the presence or absence of this sulphate ion is dependent not only on the charge of the iron atom and on the molecular packing in the crystal, but also on the conformation and charge of surrounding side chains. Nevertheless, despite the presence of a crystallo-

graphic sulphate ion at the haem pocket entrance of met-Mb, this anion was shown to produce a limited e¡ect on the Kd for N3 3 (see Table 2) [39]. Concerning this issue, it is important to note that the crystal growth is performed in highly concentrated ammonium sulphate solutions ( s 2.5 M), whereas anionic concentrations in our experiments range from 100 to 800 mM. Furthermore, as evident from Fig. 5A, the crystallographic sulphate ion is co-ordinated to His E7, Thr E10 and Arg CD3, whereas the iodide anion strongly interacts with Phe CD1. The second predicted binding site for I3 , located between helices B and G of Mb, may be considered negligible in modulating ligand a¤nity to Mb since a functional e¡ect due to its occupancy would imply a long range modulation of the a¤nity for N3 3 , which would not be easy to explain in the absence of a signi¢cant conformational change in Mb upon ligand binding. In order to better investigate the plausible binding sites of anions in met-Mb molecular dynamics simulations of the selected met-Mb^I3 complexes are going to be performed. In conclusion, our ¢ndings suggest that chaotrope anions, which have been formerly shown to in£uence the functional properties of multimeric proteins [48,49], may act as e¡ectors even on a monomeric protein like myoglobin. The mechanism herein proposed may be interpreted as an unusual heterotropic modulation and seems to support the recent idea that myoglobin may indeed act as an allosteric system with respect to its enzymatic function in concentrating and orienting diatomic molecules such as NO, CO and others [50]. Acknowledgements We gratefully acknowledge Maurizio Brunori (Rome) and John S. Olson (Houston) for their constant stimulating interest in our work. The ¢nancial support of MURST is greatly acknowledged.

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