J. Mol.
Biol.
(1991) 217, 409412
X-ray Crystal Structure of the Ferric Sperm Whale Myoglobin : Imidazole Complex at 2-OA Resolution Claudia
Lionettil,
Maria
Grazia Guanziroli’, Francesco and Martin0 BolognesilT
Frigerio’,
Paolo Ascenzi’
1Dipartimento di Genetica e Microbiologia, Sezione di Cristallograjia Centro Interuniversitario per lo Studio della Struttura e dei Rapporti tra Struttura e Funzione in Macromolecule Biologiche Informazionali Universita’ di Pavia, Via Torquato Taramelli 16, I-27100 Pavia, Italy 2CNR,
Centro di Biologia
Molecolare, Dipartimento di Scienze Biochimiche di Roma “La Sapienxa” Aldo Moro 5, I-00185 Roma, Italy
Universita’
Piazzale
(Received 26 June 1990; accepted 11 October 1990) The X-ray crystal structure of the ferric sperm whale (Physeter catodon) myoglobin: imidazole complex has been refined at 2.0 A resolution, to a final R-factor of 148%. The overall conformation of the protein is little affected by binding of the ligand. Imidazole is co-ordinated to the heme iron at the distal site, and forces distinguishable local changes in the surrounding protein residues. His64(E7) swings out of the distal pocket and becomes substantially exposed to the solvent; nevertheless, it stabilizes the exogenous ligand by hydrogen bonding. The side-chains of residues Arg45(CD3) and Asp60(E3) are also affected by imidazole association.
Sperm whale (Physeter catodon) myoglobin (Mb$) is generally considered to be the prototype of monomeric heme proteins. In particular the modulating role of amino acid residues at the distal site of the heme pocket on ligand binding has been extensively studied by kinetic, thermodynamic and structuralmolecular viewpoints (Brunori et al., 1986, 1989; Kuriyan et al., 1986; Olson et al., 1988; Perutz, 1989). Binding of imidazole to sperm whale Mb crystals has been analyzed in solution and in the crystalline state; in addition, a preliminary account of the unrefined crystal structure has been given (Bolognesi et al., 1982). In the present study we report on the crystallographic analysis and refinement of the ferric sperm whale Mb :imidazole complex at 2.0 A resolution (1 B = @l nm).
Ferric sperm whale Mb was purified from commercial hemoprotein preparations (type II, from Sigma Chemical Company, St Louis, MO, U.S.A.; lot no. M-0380) as described (Antonini & Brunori, 1971). All chemicals (from Merck AG, Darmstadt, F.R.G.) were of analytical grade, and used without further purification. Crystals of the complex were prepared by soaking the native ferric sperm whale Mb crystals in solutions containing saturating levels of imidazole, at pH 7-O (Bolognesi et al., 1982). The crystals of the sperm whale Mb : imidazole complex are isomorphous with those of the native ferric hemoprotein (Takano, 1977). A total of 9169 unique reflections (with 1> 1.0 o(1)) were collected on the same crystal on an Enraf-Nonius CAD4 conventional four-circle diffractometer, equipped with a helium-flushed extension arm (368 mm). Data were corrected for crystal decay (which reached a maximum of 145%) and X-ray absorption by the crystal/capillary system (North et al., 1968). Initial structure factors, FC> and phases were calculated using the atomic co-ordinates from the crystallographic refinement of sperm whale Mb : ethylisocyanide (Johnson et al., 1989), from which the ligand and His64(E7) sidechain had been omitted. At this stage the crystallographic R-factor at 2.0 A resolution was 21.1%. A Sim-weighted difference Fourier map (Sim, 1959)
t Author to whom all correspondence should be addressed. $ Abbreviations used: Mb, myoglobin. Amino acid residues have been identified by their 3-letter codes, followed by the sequence number (wherever appropriate), and by their topological position within the 8 helices of the globin fold. For the numbering of the heme-group atoms, the scheme of Takano (1977) has been adopted. Water molecules located on the surface of the protein and at the intra- and inter-molecular contacts 160.
have been numbered
sequentially,
starting
from
409 WZ-2836/91/030409~4
$03.00/O
0 1991 Academic Press Limited
410
C. Lionetti et al.
was calculated with 2Fo-Pc coefficients, and calculated phases (F, indicates the observed structure factors for the sperm whale Mb : imidazole complex). Inspection of this electron density map indicated prominent difference features around the distal site, compatible with: (1) imidazole co-ordination to heme iron; (2) a new conformation for His64(E7) side-chain, which points towards the outer part of the heme crevice; and (3) other side-chain movements, mostly among the distal residues. Restrained crystallographic refinement of the structure was achieved by means of the TNT program package (Tronrud et al., 1987); electron density maps were inspected on an Evans & Sutherland PS330 graphics display, using the program FRODO (Jones, 1978). Four main refinement cycles were run: each consisted of about 25 steepest descent/conjugate gradients refinement steps, and of thorough inspection of F,- Fc and 2F,- Fc difference Fourier maps, with model update. In addition, individual atomic temperature factors were refined. For the 8822 reflections in the 10.0 to 2-O a resolution range (94.6% of all possible reflections), the crystallographic R-factor is 148 ye. Root-mean-square deviations from ideal values of
stereochemical parameters are 0019 -4, for bond lengths, and 3.24” for bond angles. The model includes 119 solvent molecules and one sulfate ion. The atomic co-ordinates and structure factors of the ferric sperm whale Mb : imidazole complex have been deposited with the Brookhaven Protein Data Brookhaven Chemistry Department, Bank, National Laborat,ory, Upton NY 11973, U.S.A., from which copies are available (Bernstein et al., 1977). Figure
1 is a stereoscopic view of the distal site of the sperm whale Mb :imidazole complex, as seen from the solvent side. Imidazole is co-ordinated to the heme iron, with a Fe-NE1 distance of 2-17 A. The ligand adopts an orientation that is 16” tilted with respect to a plane normal to the heme; moreover, the imidazole ring plane forms an angle cp of -41” with the pyrrole N2-N4 direction. Considering
the orientation
of the proximal
histi-
dine with respect to the same direction ($6”). bound imidazole and His93(F8) planes form an angle of 47”, close to the value of 50” reported for the sperm whale Mb : phenylhydrazine complex (Binge et al., 1984). The v, angle found for the imidazole ligand is very close to that observed in the
(a)
60
(b)
Figure 1. (a) Stereoscopic view of the distal site of the sperm whale myoglobin : imidazoie complex, as observed from the solvent side of the heme crevice. (b) The same region of the complex, in a comparable orientation, with the difference electron density observed before crystallographic refinement. His64(E?) side-chain was omitt,ed from the phase calculation. W indicates water molecule 164 in (a) and 0164 in (b).
Communications Mb : ethylisocyanide complex (Johnson et al., 1989). In the sperm whale Mb : CO complex one of the two refined CO orientations has cp= -62” (Kuriyan et al., 1986). The orientation of imidazole is dictated by the co-ordination bond to the heme iron, and by a hydrogen bond (3.03 A) between the ligand NE2 atom and ND1 of His64(E7), which has rotated about 80” around the C-@bond, as compared to aquo-ferric sperm whale Mb (Takano, 1977). The x1, x2 torsional angles defining the His64(E7) side-chain conformation are respectively 263” and 266”, indicating that substantial rotation around the C@-Cy bond has also occurred. Upon binding of imidazole, the iron atom moves essentially into the heme plane; the proximal bond to the His93(F8) NE2 atom is 2.04 A, the average Fe-pyrrole nitrogen distance is 1.94 A. Crystallographic refinement of the occupancies of the ligand, and of the side-chains of residues Arg45(CD3), Asp60(E3) and His64(E7), indicates that in the crystal the distal site is fully occupied by imidazole, and no alternative side-chain conformations are present for these residues. The pyrrole III (upper in Fig. 1) propionate 3601 atom is hydrogen-bonded to the Arg45(CD3) NE atom (288 A), and via an ordered water molecule (W 164)) to the Arg45(CD3) peptide nitrogen. Residue Arg45(CD3) adopts a conformation different from that observed in aquo-ferric sperm whale Mb (Takano, 1977), and is bonded to the carboxylate of ASPGO( mainly through atom 45 NEHl (45 NEHl . .600Dl, 287 A; 45 NEHl .600D2, 3.24 8). The conformation of the Arg45(CD3) . . . Asp60(E3) ion pair is practically coincident with that observed in the sperm whale Mb : ethylisocyanide complex (Johnson et al., 1989). In the lower part of the distal site binding of imidazole has little, if any, effect on the side-chain of residue Val68(Ell): x1, for the side-chain of Val68(Ell), is 163”. This is in agreement with what is observed in the sperm whale Mb : ethylisocyanide complex (Johnson et al., 1989), and corrects our previous observation about this residue (Bolognesi et al., 1982). A rotation of approximately 100” of Val68(Ell) isopropyl substituent is reported, on the other hand, for the Mb : phenylhydrazine complex (Ringe et al., 1984). The propionate substituent of the heme pyrrole ring IV interacts with two proximal residues: the 4701 atom is hydrogen-bonded (2.97 A) to the NE2 atom of residue His97(FG2), whereas 4702 is at 2.82 A from OG of Ser92(F7). In the sperm whale Mb : imidazole complex a sulfate ion is located between the side-chains of residues Ser58(El) and Asp60(E3), in a similar way to what has been observed by Johnson et al. (1989). LyslG(A14) and Lys34(B15) of a symmetry-related molecule provide charge compensation for the bound anion in the crystal lattice. Absence of a sulfate anion from the distal region, on the other hand, has been observed upon binding of the phenyl group in the reaction of sperm whale Mb with phenylhydrazine (Ringe et al., 1984) and in the structure of sperm whale Mb : CO complex (Phillips & Schoenborn, 1981; Kuriyan et al., 1986).
411
Concerning crystal contacts, it should be noted that the side-chain conformation adopted by residue Arg45(CD3) can be partly affected by an interaction with the symmetry-related residue His113(H14), mediated by an ordered water molecule (W176): bridging the two side-chains. As in the case of sperm whale Mb : ethylisocyanide and Mb : phenylhydrazine complexes, the distal site open conformation of the imidazole adduct indicates that the distal histidine residue can effectively play a role in stabilizing and/or regulating access of the ligand to the heme iron. Upon removal of imidazole from the complex structure, the “open door” described above for residue conformation His64(E7) allows accessibility (Lee & Richards, 1971) of a 1.8 A radius probe (simulating the oxygen molecule) to the distal site. Thus, swinging of the His64(E7) side-chain towards the protein surface creates an aperture large enough for the gaseous ligand to enter the distal site (Nobbs, 1966; Ringe et al., 1984; Johnson et al., 1989). In the case of imidazole, the bulky ligand keeps the E7 door in an open conformation; small diatomic ligands allow subsequent closure of the E7 door and formation of a stabilizing ligand . . His64(E7) hydrogen bond (Phillips, 1980; Phillips & Schoenborn, 1981; Perutz, 1989). These effects were first detailed by comparison of the X-ray crystal structures of the ferric “aquomet” and cyanide derivatives of Chironomus thummi thummi monomeric erythrocruorin. Indeed, in the aquomet form, which lacks the iron-bound water, the distal His is kept in an open door conformation mostly because of the size of residue IleEl 1. Upon ligand association a strong cyanide N . . HisE7 NE2 hydrogen bond forces the distal residue side-chain in the “closed door” geometry (Steigemann & Weber, 1979). It is interesting to notice that, despite the large size of imidazole as a ligand, this stabilizing ligand : His(E7) interaction is conserved in the sperm whale Mb : imidazole complex. The energy barrier for the opening of a direct channel to the heme iron atom through relaxation of the distal site residues, particularly His64(E7), has been estimated to be approximately 5 kcal/mol (1 cal= 4.184 J: Case & Karplus, 1979; Kottalam & Case, 1988). Moreover, empirical energy surfaces of ligand : protein interactions calculated with the ring of the distal histidine in an open conformation (x1 and x2 of His64 set of 280” and 275”, respectively) show an energetically open path from the protein surface to the heme iron, fairly broad in the E7 region (Kuriyan et al., 1986). As described above, the His64(E7) side-chain conformation in the sperm whale imidazole : Mb structure is characterized by the x1 and x2 values of 263” and 266”, respectively. In the sperm whale Mb : ethylisocyanide complex, the aperture of the distal site “door” is more pronounced (x1 and x2, 288” and 244”) due to a hydrogen bond of the His64 ND1 atom to the carbonyl oxygen atom of residue Asp6O(E3). Finally, the results of this study, and in particular the swinging movement of residue His64(E7), are in
412
C. Lionetti
accordance with the structural perturbations observed upon binding of imidazole to ferric horse hemoglobin (Bell et al., 1981), the crystals of which, however, do not tolerate full ligand saturation. We thank Dr George N. Phillips, Jr, for providing the refined co-ordinates of sperm the whale Mb : ethylisocyanide complex prior to publication, and Professor Maurizio Brunori for helpful discussion. This study has been supported by the Italian National Research Council target oriented project Biotecnologie e Biostrumentazione, and by the special project Peptidi Bioatt,ivi. References Antonini, E. & Brunori, M. (1971). Hemoglobin and Myoglobin in Their Reactions with Ligandz, pp. 6-9, Elsevier/North-Holland Publishing Co., Amsterdam. Bell, J. A., Korszun, Z. R. & Moffat, K. (1981). Structure of Imidazole Methemoglobin. J. ivoZ. Biol. 147. 325335. Bernstein, F. C., Koetzle, T. F.. Williams, G. J. B., Meyer, E. F., Jr, Brice M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T. & Tasumi, M. (1977). The Prot,ein Data Bank: A Computer-based Archival File for Macromolecule Struct,ures. J. Mol. Biol. 112, 535-542. Bolognesi, M.: Gannillo, E., Ascenzi, P., Giacometti, G. 31.: Merli, A. & Brunori, M. (1982). Reactivity of Ferric Aplysia and Sperm Whale Myoglobins towards Imidazole. X-ray and Binding Study. J. Mol. Biol. 158, 305-315. Brunori, M., Ascenzi, P. & Coletta, M. (1986). Control of Heme Reactivity in Hemes and Hemoproteins. In Proceedings of the Interrztional Congress on Xupermolecules: Biological and Chemical Aspects, pp. 55-73, Rome, 9 November 1984, Accademia Nazionale dei Lincei, Roma. Brunori, M., Coletta, M., Ascenzi, P. & Bolognesi, M. (1989). Kinetic Control of Ligand Binding Processes in Hemoproteins. J. Mol. Liquids, 42, 175-193. Case, D. A. & Karplus, M. (1979). Dynamics of Ligand Binding to Heme Proteins, J. Mol. Biol. 132, 343-368. Johnson, K. A., Olson, J. S. &, Phillips, G. N., Jr (1989). The Structure of Myoglobin-ethylisocyanide: Histidine as a Swinging Door for Ligand Entry. J. Mol. Biol. 207: 459-463. Jones, T. A. (1978). A Graphics ,Modei Building and Refinement System for Macromolecules. J. AppZ. Crystallogr. 11, 268-272.
Edited
et al.
Kottalam, J. & Case, D. A. (1988). Dynamics of Ligand Escape from the Heme Pocket of Mgoglobin. J. Amer. U&em. Sot. 110: 7690-7697. Kuriyan, J., Wilz: S., Karplus, M. & Petsko, G. A. (1986). X-ray Struct.ure and Refinement of Carbon-monoxy (Fe II)-Myoglobin at 1.5 a Resolution. J. &loZ. Riol. 192, 133-154. Lee, R. K. & Richards, F. M. (1971). The Interpretat,ion of Protein Structures: Estimation of Static Accessibility. J. Mol. Biol. 55. 379-400. Nobbs, C. L. (1966). Structure and ligand binding of deoxymyoglobin. In Hem,es and Hemoproteins (Chance, B.; Estabrook, R. W. 8: Yonetani. T.. eds). pp. 143-148. Academic Press. New York and London. North, .4. C. T., Philips, D. C. & Matthews, F. S. (1968). A Semi-Empirical Method of Absorption Correct.ion. Acta Crystallogr. sect. A. 24, 351-359. Olson., J. S., Matthews, A. J., Rohlfs, R. J., Springer. B. A., Egeberg, K. D., Sligar, S. G.: Thame. 5.; Renaud, J.-P. & Nagai, K. (1988). The Role of the Distal Histidine in Myoglobin and Haemoglobin. .Xatw-e (London), 336, 265-266. Perutz, M. F. (lg89). Myoglobin and Haemoglobin: Role of Distal Residues in Reactions with Haem Ligands. Trends Biochem. Sci. 14, 42-44. Phillips, S. E. V. (1980). Structure and Refinement of Oxymyoglobin at 1.6 a Resolution. J. Mol. BioE. 142, 531-554. Philtips, S. E. V. & Schoenborn, B. P. (1981). Neutron Diffraction Reveals Oxygen-Histidine Hydrogen Bond in Oxymyoglobin. Nature (London]: 292, 81-82. Ringe, D., Petsko, G. A., Kerr, D. E. & Ortiz de Montellano, P. R. (1984). Reaction of Myoglobin with Phenylhydrazine: A Molecular Doorstop. Biochemistry, 23, 2-4. of Phase Angles for Sim: G. A. (1959). The Distribution Structures Containing Heavy Atoms. II. A Modification of the Piormal Heavy-atom Method for Non-eentrosymmetrical Structures. Acta Crystallqr. 12, 813-815. Steigemann, W. $ Weber, E. (1979). Structure of Erythrocruorin in Different Ligand States Refined at 1.4 ,& Resolution. J. Mol. BioE. 127, 309-338. Takano, T. (1977). Structure of Myoglobin Refined at 2.0 a Resolution. Crystallographic Refinement, of Metmyoglobin from Sperm Whale. J. Mol. Biol. 110. 537-568. Tronrud, D. E., Ten Eyck, 1~. F. & MvIatt~hewq K. ‘8. (1987). An Efficient, General-purpose Least-squares Refinement Program for Macromolecular Struct,ures. Acta Crystallogr. sect. A, 43, 489-501,
by R. Huber
Biol.
(1991) 217, 409412
X-ray Crystal Structure of the Ferric Sperm Whale Myoglobin : Imidazole Complex at 2-OA Resolution Claudia
Lionettil,
Maria
Grazia Guanziroli’, Francesco and Martin0 BolognesilT
Frigerio’,
Paolo Ascenzi’
1Dipartimento di Genetica e Microbiologia, Sezione di Cristallograjia Centro Interuniversitario per lo Studio della Struttura e dei Rapporti tra Struttura e Funzione in Macromolecule Biologiche Informazionali Universita’ di Pavia, Via Torquato Taramelli 16, I-27100 Pavia, Italy 2CNR,
Centro di Biologia
Molecolare, Dipartimento di Scienze Biochimiche di Roma “La Sapienxa” Aldo Moro 5, I-00185 Roma, Italy
Universita’
Piazzale
(Received 26 June 1990; accepted 11 October 1990) The X-ray crystal structure of the ferric sperm whale (Physeter catodon) myoglobin: imidazole complex has been refined at 2.0 A resolution, to a final R-factor of 148%. The overall conformation of the protein is little affected by binding of the ligand. Imidazole is co-ordinated to the heme iron at the distal site, and forces distinguishable local changes in the surrounding protein residues. His64(E7) swings out of the distal pocket and becomes substantially exposed to the solvent; nevertheless, it stabilizes the exogenous ligand by hydrogen bonding. The side-chains of residues Arg45(CD3) and Asp60(E3) are also affected by imidazole association.
Sperm whale (Physeter catodon) myoglobin (Mb$) is generally considered to be the prototype of monomeric heme proteins. In particular the modulating role of amino acid residues at the distal site of the heme pocket on ligand binding has been extensively studied by kinetic, thermodynamic and structuralmolecular viewpoints (Brunori et al., 1986, 1989; Kuriyan et al., 1986; Olson et al., 1988; Perutz, 1989). Binding of imidazole to sperm whale Mb crystals has been analyzed in solution and in the crystalline state; in addition, a preliminary account of the unrefined crystal structure has been given (Bolognesi et al., 1982). In the present study we report on the crystallographic analysis and refinement of the ferric sperm whale Mb :imidazole complex at 2.0 A resolution (1 B = @l nm).
Ferric sperm whale Mb was purified from commercial hemoprotein preparations (type II, from Sigma Chemical Company, St Louis, MO, U.S.A.; lot no. M-0380) as described (Antonini & Brunori, 1971). All chemicals (from Merck AG, Darmstadt, F.R.G.) were of analytical grade, and used without further purification. Crystals of the complex were prepared by soaking the native ferric sperm whale Mb crystals in solutions containing saturating levels of imidazole, at pH 7-O (Bolognesi et al., 1982). The crystals of the sperm whale Mb : imidazole complex are isomorphous with those of the native ferric hemoprotein (Takano, 1977). A total of 9169 unique reflections (with 1> 1.0 o(1)) were collected on the same crystal on an Enraf-Nonius CAD4 conventional four-circle diffractometer, equipped with a helium-flushed extension arm (368 mm). Data were corrected for crystal decay (which reached a maximum of 145%) and X-ray absorption by the crystal/capillary system (North et al., 1968). Initial structure factors, FC> and phases were calculated using the atomic co-ordinates from the crystallographic refinement of sperm whale Mb : ethylisocyanide (Johnson et al., 1989), from which the ligand and His64(E7) sidechain had been omitted. At this stage the crystallographic R-factor at 2.0 A resolution was 21.1%. A Sim-weighted difference Fourier map (Sim, 1959)
t Author to whom all correspondence should be addressed. $ Abbreviations used: Mb, myoglobin. Amino acid residues have been identified by their 3-letter codes, followed by the sequence number (wherever appropriate), and by their topological position within the 8 helices of the globin fold. For the numbering of the heme-group atoms, the scheme of Takano (1977) has been adopted. Water molecules located on the surface of the protein and at the intra- and inter-molecular contacts 160.
have been numbered
sequentially,
starting
from
409 WZ-2836/91/030409~4
$03.00/O
0 1991 Academic Press Limited
410
C. Lionetti et al.
was calculated with 2Fo-Pc coefficients, and calculated phases (F, indicates the observed structure factors for the sperm whale Mb : imidazole complex). Inspection of this electron density map indicated prominent difference features around the distal site, compatible with: (1) imidazole co-ordination to heme iron; (2) a new conformation for His64(E7) side-chain, which points towards the outer part of the heme crevice; and (3) other side-chain movements, mostly among the distal residues. Restrained crystallographic refinement of the structure was achieved by means of the TNT program package (Tronrud et al., 1987); electron density maps were inspected on an Evans & Sutherland PS330 graphics display, using the program FRODO (Jones, 1978). Four main refinement cycles were run: each consisted of about 25 steepest descent/conjugate gradients refinement steps, and of thorough inspection of F,- Fc and 2F,- Fc difference Fourier maps, with model update. In addition, individual atomic temperature factors were refined. For the 8822 reflections in the 10.0 to 2-O a resolution range (94.6% of all possible reflections), the crystallographic R-factor is 148 ye. Root-mean-square deviations from ideal values of
stereochemical parameters are 0019 -4, for bond lengths, and 3.24” for bond angles. The model includes 119 solvent molecules and one sulfate ion. The atomic co-ordinates and structure factors of the ferric sperm whale Mb : imidazole complex have been deposited with the Brookhaven Protein Data Brookhaven Chemistry Department, Bank, National Laborat,ory, Upton NY 11973, U.S.A., from which copies are available (Bernstein et al., 1977). Figure
1 is a stereoscopic view of the distal site of the sperm whale Mb :imidazole complex, as seen from the solvent side. Imidazole is co-ordinated to the heme iron, with a Fe-NE1 distance of 2-17 A. The ligand adopts an orientation that is 16” tilted with respect to a plane normal to the heme; moreover, the imidazole ring plane forms an angle cp of -41” with the pyrrole N2-N4 direction. Considering
the orientation
of the proximal
histi-
dine with respect to the same direction ($6”). bound imidazole and His93(F8) planes form an angle of 47”, close to the value of 50” reported for the sperm whale Mb : phenylhydrazine complex (Binge et al., 1984). The v, angle found for the imidazole ligand is very close to that observed in the
(a)
60
(b)
Figure 1. (a) Stereoscopic view of the distal site of the sperm whale myoglobin : imidazoie complex, as observed from the solvent side of the heme crevice. (b) The same region of the complex, in a comparable orientation, with the difference electron density observed before crystallographic refinement. His64(E?) side-chain was omitt,ed from the phase calculation. W indicates water molecule 164 in (a) and 0164 in (b).
Communications Mb : ethylisocyanide complex (Johnson et al., 1989). In the sperm whale Mb : CO complex one of the two refined CO orientations has cp= -62” (Kuriyan et al., 1986). The orientation of imidazole is dictated by the co-ordination bond to the heme iron, and by a hydrogen bond (3.03 A) between the ligand NE2 atom and ND1 of His64(E7), which has rotated about 80” around the C-@bond, as compared to aquo-ferric sperm whale Mb (Takano, 1977). The x1, x2 torsional angles defining the His64(E7) side-chain conformation are respectively 263” and 266”, indicating that substantial rotation around the C@-Cy bond has also occurred. Upon binding of imidazole, the iron atom moves essentially into the heme plane; the proximal bond to the His93(F8) NE2 atom is 2.04 A, the average Fe-pyrrole nitrogen distance is 1.94 A. Crystallographic refinement of the occupancies of the ligand, and of the side-chains of residues Arg45(CD3), Asp60(E3) and His64(E7), indicates that in the crystal the distal site is fully occupied by imidazole, and no alternative side-chain conformations are present for these residues. The pyrrole III (upper in Fig. 1) propionate 3601 atom is hydrogen-bonded to the Arg45(CD3) NE atom (288 A), and via an ordered water molecule (W 164)) to the Arg45(CD3) peptide nitrogen. Residue Arg45(CD3) adopts a conformation different from that observed in aquo-ferric sperm whale Mb (Takano, 1977), and is bonded to the carboxylate of ASPGO( mainly through atom 45 NEHl (45 NEHl . .600Dl, 287 A; 45 NEHl .600D2, 3.24 8). The conformation of the Arg45(CD3) . . . Asp60(E3) ion pair is practically coincident with that observed in the sperm whale Mb : ethylisocyanide complex (Johnson et al., 1989). In the lower part of the distal site binding of imidazole has little, if any, effect on the side-chain of residue Val68(Ell): x1, for the side-chain of Val68(Ell), is 163”. This is in agreement with what is observed in the sperm whale Mb : ethylisocyanide complex (Johnson et al., 1989), and corrects our previous observation about this residue (Bolognesi et al., 1982). A rotation of approximately 100” of Val68(Ell) isopropyl substituent is reported, on the other hand, for the Mb : phenylhydrazine complex (Ringe et al., 1984). The propionate substituent of the heme pyrrole ring IV interacts with two proximal residues: the 4701 atom is hydrogen-bonded (2.97 A) to the NE2 atom of residue His97(FG2), whereas 4702 is at 2.82 A from OG of Ser92(F7). In the sperm whale Mb : imidazole complex a sulfate ion is located between the side-chains of residues Ser58(El) and Asp60(E3), in a similar way to what has been observed by Johnson et al. (1989). LyslG(A14) and Lys34(B15) of a symmetry-related molecule provide charge compensation for the bound anion in the crystal lattice. Absence of a sulfate anion from the distal region, on the other hand, has been observed upon binding of the phenyl group in the reaction of sperm whale Mb with phenylhydrazine (Ringe et al., 1984) and in the structure of sperm whale Mb : CO complex (Phillips & Schoenborn, 1981; Kuriyan et al., 1986).
411
Concerning crystal contacts, it should be noted that the side-chain conformation adopted by residue Arg45(CD3) can be partly affected by an interaction with the symmetry-related residue His113(H14), mediated by an ordered water molecule (W176): bridging the two side-chains. As in the case of sperm whale Mb : ethylisocyanide and Mb : phenylhydrazine complexes, the distal site open conformation of the imidazole adduct indicates that the distal histidine residue can effectively play a role in stabilizing and/or regulating access of the ligand to the heme iron. Upon removal of imidazole from the complex structure, the “open door” described above for residue conformation His64(E7) allows accessibility (Lee & Richards, 1971) of a 1.8 A radius probe (simulating the oxygen molecule) to the distal site. Thus, swinging of the His64(E7) side-chain towards the protein surface creates an aperture large enough for the gaseous ligand to enter the distal site (Nobbs, 1966; Ringe et al., 1984; Johnson et al., 1989). In the case of imidazole, the bulky ligand keeps the E7 door in an open conformation; small diatomic ligands allow subsequent closure of the E7 door and formation of a stabilizing ligand . . His64(E7) hydrogen bond (Phillips, 1980; Phillips & Schoenborn, 1981; Perutz, 1989). These effects were first detailed by comparison of the X-ray crystal structures of the ferric “aquomet” and cyanide derivatives of Chironomus thummi thummi monomeric erythrocruorin. Indeed, in the aquomet form, which lacks the iron-bound water, the distal His is kept in an open door conformation mostly because of the size of residue IleEl 1. Upon ligand association a strong cyanide N . . HisE7 NE2 hydrogen bond forces the distal residue side-chain in the “closed door” geometry (Steigemann & Weber, 1979). It is interesting to notice that, despite the large size of imidazole as a ligand, this stabilizing ligand : His(E7) interaction is conserved in the sperm whale Mb : imidazole complex. The energy barrier for the opening of a direct channel to the heme iron atom through relaxation of the distal site residues, particularly His64(E7), has been estimated to be approximately 5 kcal/mol (1 cal= 4.184 J: Case & Karplus, 1979; Kottalam & Case, 1988). Moreover, empirical energy surfaces of ligand : protein interactions calculated with the ring of the distal histidine in an open conformation (x1 and x2 of His64 set of 280” and 275”, respectively) show an energetically open path from the protein surface to the heme iron, fairly broad in the E7 region (Kuriyan et al., 1986). As described above, the His64(E7) side-chain conformation in the sperm whale imidazole : Mb structure is characterized by the x1 and x2 values of 263” and 266”, respectively. In the sperm whale Mb : ethylisocyanide complex, the aperture of the distal site “door” is more pronounced (x1 and x2, 288” and 244”) due to a hydrogen bond of the His64 ND1 atom to the carbonyl oxygen atom of residue Asp6O(E3). Finally, the results of this study, and in particular the swinging movement of residue His64(E7), are in
412
C. Lionetti
accordance with the structural perturbations observed upon binding of imidazole to ferric horse hemoglobin (Bell et al., 1981), the crystals of which, however, do not tolerate full ligand saturation. We thank Dr George N. Phillips, Jr, for providing the refined co-ordinates of sperm the whale Mb : ethylisocyanide complex prior to publication, and Professor Maurizio Brunori for helpful discussion. This study has been supported by the Italian National Research Council target oriented project Biotecnologie e Biostrumentazione, and by the special project Peptidi Bioatt,ivi. References Antonini, E. & Brunori, M. (1971). Hemoglobin and Myoglobin in Their Reactions with Ligandz, pp. 6-9, Elsevier/North-Holland Publishing Co., Amsterdam. Bell, J. A., Korszun, Z. R. & Moffat, K. (1981). Structure of Imidazole Methemoglobin. J. ivoZ. Biol. 147. 325335. Bernstein, F. C., Koetzle, T. F.. Williams, G. J. B., Meyer, E. F., Jr, Brice M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T. & Tasumi, M. (1977). The Prot,ein Data Bank: A Computer-based Archival File for Macromolecule Struct,ures. J. Mol. Biol. 112, 535-542. Bolognesi, M.: Gannillo, E., Ascenzi, P., Giacometti, G. 31.: Merli, A. & Brunori, M. (1982). Reactivity of Ferric Aplysia and Sperm Whale Myoglobins towards Imidazole. X-ray and Binding Study. J. Mol. Biol. 158, 305-315. Brunori, M., Ascenzi, P. & Coletta, M. (1986). Control of Heme Reactivity in Hemes and Hemoproteins. In Proceedings of the Interrztional Congress on Xupermolecules: Biological and Chemical Aspects, pp. 55-73, Rome, 9 November 1984, Accademia Nazionale dei Lincei, Roma. Brunori, M., Coletta, M., Ascenzi, P. & Bolognesi, M. (1989). Kinetic Control of Ligand Binding Processes in Hemoproteins. J. Mol. Liquids, 42, 175-193. Case, D. A. & Karplus, M. (1979). Dynamics of Ligand Binding to Heme Proteins, J. Mol. Biol. 132, 343-368. Johnson, K. A., Olson, J. S. &, Phillips, G. N., Jr (1989). The Structure of Myoglobin-ethylisocyanide: Histidine as a Swinging Door for Ligand Entry. J. Mol. Biol. 207: 459-463. Jones, T. A. (1978). A Graphics ,Modei Building and Refinement System for Macromolecules. J. AppZ. Crystallogr. 11, 268-272.
Edited
et al.
Kottalam, J. & Case, D. A. (1988). Dynamics of Ligand Escape from the Heme Pocket of Mgoglobin. J. Amer. U&em. Sot. 110: 7690-7697. Kuriyan, J., Wilz: S., Karplus, M. & Petsko, G. A. (1986). X-ray Struct.ure and Refinement of Carbon-monoxy (Fe II)-Myoglobin at 1.5 a Resolution. J. &loZ. Riol. 192, 133-154. Lee, R. K. & Richards, F. M. (1971). The Interpretat,ion of Protein Structures: Estimation of Static Accessibility. J. Mol. Biol. 55. 379-400. Nobbs, C. L. (1966). Structure and ligand binding of deoxymyoglobin. In Hem,es and Hemoproteins (Chance, B.; Estabrook, R. W. 8: Yonetani. T.. eds). pp. 143-148. Academic Press. New York and London. North, .4. C. T., Philips, D. C. & Matthews, F. S. (1968). A Semi-Empirical Method of Absorption Correct.ion. Acta Crystallogr. sect. A. 24, 351-359. Olson., J. S., Matthews, A. J., Rohlfs, R. J., Springer. B. A., Egeberg, K. D., Sligar, S. G.: Thame. 5.; Renaud, J.-P. & Nagai, K. (1988). The Role of the Distal Histidine in Myoglobin and Haemoglobin. .Xatw-e (London), 336, 265-266. Perutz, M. F. (lg89). Myoglobin and Haemoglobin: Role of Distal Residues in Reactions with Haem Ligands. Trends Biochem. Sci. 14, 42-44. Phillips, S. E. V. (1980). Structure and Refinement of Oxymyoglobin at 1.6 a Resolution. J. Mol. BioE. 142, 531-554. Philtips, S. E. V. & Schoenborn, B. P. (1981). Neutron Diffraction Reveals Oxygen-Histidine Hydrogen Bond in Oxymyoglobin. Nature (London]: 292, 81-82. Ringe, D., Petsko, G. A., Kerr, D. E. & Ortiz de Montellano, P. R. (1984). Reaction of Myoglobin with Phenylhydrazine: A Molecular Doorstop. Biochemistry, 23, 2-4. of Phase Angles for Sim: G. A. (1959). The Distribution Structures Containing Heavy Atoms. II. A Modification of the Piormal Heavy-atom Method for Non-eentrosymmetrical Structures. Acta Crystallqr. 12, 813-815. Steigemann, W. $ Weber, E. (1979). Structure of Erythrocruorin in Different Ligand States Refined at 1.4 ,& Resolution. J. Mol. BioE. 127, 309-338. Takano, T. (1977). Structure of Myoglobin Refined at 2.0 a Resolution. Crystallographic Refinement, of Metmyoglobin from Sperm Whale. J. Mol. Biol. 110. 537-568. Tronrud, D. E., Ten Eyck, 1~. F. & MvIatt~hewq K. ‘8. (1987). An Efficient, General-purpose Least-squares Refinement Program for Macromolecular Struct,ures. Acta Crystallogr. sect. A, 43, 489-501,
by R. Huber
