Báo cáo khoa học: H NMR study of the molecular structure and magnetic properties of the active site for the cyanomet complex of O2-avid hemoglobin from the trematode Paramphistomum epiclitum pdf

Eur J Biochem 270, 2707–2720 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03638.x H NMR study of the molecular structure and magnetic properties of the active site for the cyanomet complex of O2-avid hemoglobin from the trematode Paramphistomum epiclitum Weihong Du1, Zhicheng Xia1, Sylvia Dewilde2, Luc Moens2 and Gerd N La Mar1 Department of Chemistry, University of California, Davis, CA, USA; 2Department of Biomedical Sciences, University of Antwerp, Wilrijk, Belgium The solution molecular and electronic structures of the active site in the extremely O2-avid hemoglobin from the trematode Paramphistomum epiclitum have been investigated by 1H NMR on the cyanomet form in order to elucidate the distal hydrogen-bonding to a ligated H-bond acceptor ligand Comparison of the strengths of dipolar interactions in solution with the alternate crystal structures of methemoglobin establish that the solution structure of wild-type Hb more closely resembles the crystal structure of the recombinant wild-type than the true wild-type met-hemoglobin The distal Tyr66(E7) is found oriented out of the heme pocket in solution as found in both crystal structures Analysis of dipolar contacts, dipolar shift and paramagnetic relaxation establishes that the Tyr32(B10) hydrogen proton adopts an orientation that allows it to make a strong H-bond to the bound cyanide The observation of a significant isotope effect on the heme methyl contact shifts confirms a strong contact between the Tyr32(B10) OH and the ligated cyanide The quantitative determination of the orientation and anisotropies of the paramagnetic susceptibility tensor reveal that the cyanide is tilted % 10° from the heme normal so as to avoid van der Waals overlap with the Tyr32(B10) Og The pattern of heme contact shifts with large low-field shifts for 7-CH3 and 18-CH3 is shown to arise not from the 180° rotation about the a-c-meso axis, but due to the % 45° rotation of the axial His imidazole ring, relative to that in mammalian globins Globins (hemoglobin, Hb, and myoglobin, Mb) are ferrous heme-containing, O2-binding proteins found widespread in nature [1,2] They exhibit an extraordinary range of ligation rates and affinities, as well as autoxidation rates (conversion to the nonfunctional ferric hemin) in spite of a highly conserved folding topology (the Mb fold) The majority of globins, which consists of % 150 residues, are arranged in a compact globule consisting of eight (A–H) helices, with the heme wedged between the E and F helices A completely conserved His F8 (eighth residue on helix F) provides the only covalent bond to the protein, although a conserved aromatic ring (CD1) (first residue and the loop between helices C and D) provides considerable stabilization by p-stacking on the heme [1–3] Some recently discovered ÔtruncatedÕ (% 100–120 residues) globins from bacteria exhibit the general Mb fold but retain only four of the helices, leaving a largely conserved active site with respect to more conventional globins [4,5], and one has an unprecedented Tyr (CD1) [3] The modulation of the extreme range of ligation rates in monomeric globins appears to be controlled primarily by limited sets of residues on the distal (opposite side to the proximal His F8) side of the heme, which determine the distal pocket polarity, provide stabilizing H-bonds to ligands and/or sterically interfere with ligand binding [6] Among the extensively studied monomeric mammalian Mbs, the key residues have been identified at positions E11 and E7 (generally His, but occasionally Gln [7]), where the latter provides the crucial H-bond to stabilize O2 Val E11 makes van der Waals contact with the ligand and, in certain cases, may sterically destabilize ligands [6,7] The B10 residue is generally a Leu, except in elephant Mb [8,9], where a Phe at B10 (and a Gln at E7) results in an Mb with a relatively reduced autoxidazibility but conserved O2 affinity relative to other mammalian Mbs The globins from invertebrates exhibit much more variability in both the nature of the distal residues that provide the stabilizing H-bond to the O2 and their positions in the globin [10–17] Thus the sea hare Aplysia limacina possesses a Val E7, but H-bond stabilization of O2 is provided by Arg E10 [18] The monomeric Hb from Glycera dibranchiata possesses a Leu E7, and the absence of an alternate H-bond donor leads to rapid O2 off-rates [19] Some of the most unusual Hbs characterized to date are those from nematodes and trematodes (mammalian parasites) such as the nematode Ascaris suum (As) [11,12] and the trematode Paramphistomum epiclitum (Pe) [14,15], which exhibit extraordinarily Correspondence to G N La Mar, Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA Fax: + 530 752 8995, Tel.: + 530 752 0958, E-mail: lamar@indigo.ucdavis.edu Abbreviations: Hb, hemoglobin; Mb, myoglobin; WT, wild type; rWT, recombinant WT; metHbCN, cyanide ligated ferric hemoglobin; NOE, nuclear Overhauser effect; DSS, 2,2-dimethyl-2-silapentane5-sulfonate; WEFT, water-eliminated Fourier transform; Pe, Paramphistomum epiclitum Note: a website is available at http://www.chem.ucdavis.edu/faculty/ (Received 20 December 2002, revised 24 March 2003, accepted 28 April 2003) Keywords: hemoglobin; trematode; H-bonding; dipolar shift; NMR Ó FEBS 2003 2708 W Du et al (Eur J Biochem 270) high O2 affinity via an exceptionally slow off-rate Their offrates are too slow to allow these globins to participate in aerobic respiration, and their exact physiological role is openly debated [14,15,20] The nematode/trematode globins share the property of a Tyr at position B10, which in conjunction with or without the E7 H-bond donor, provides an extremely strong H-bond to bound O2 [11,12,16] It must be noted, however, that a Tyr B10 occurs in other globins such as native Lucina pectinata (Lp) Hbs [21], with ÔordinaryÕ O2 affinity, where the B-helix is too far from the heme to allow a strong H-bond A Tyr B10 has been incorporated into a mammalian Mb variant, where its relatively minor influence on O2 affinity was attributed to the B-helix being too close to the heme to allow a robust H-bond to O2 [22] The extremely O2-avid monomeric Pe Hb is unique in that it possesses Tyr at both the B10 and E7 positions [14] Solution H NMR on Pe WT HbO2 [23] had found two labile protons in the vicinity of the bound O2, both arising from Tyr, implying that both residues are oriented into the heme pocket However, mutagenesis on Pe Hb [16] has shown that substituting Tyr E7 does not, while substituting Tyr B10 does, strongly reduce O2 affinity via a faster O2 off-rate These studies firmly establish that the O2 avidity does not require an H-bond by Tyr E7 However, these results not alone establish whether the Tyr E7 is oriented into or out of the heme pocket While crystals of Pe HbO2 have not been prepared to date, the oxidized Pe wild type (WT) and recombinant wild-type (rWT) metHb have crystallized in two different forms [16,17], and the detailed structures provide important information on the novel Hb The Tyr66(E7) ring is oriented out of the heme pocket in both forms with the Tyr32(B10) in one of the structures [16,17] serving as a H-bond acceptor to a ligated water molecule The two structures of WT metHb and rWT metHbH2O, exhibit significant differences in the interaction of the FG loop with the heme and in the position of the B-helix, with ˚ Tyr32(B10) closer to the iron by 1–2 A in WT than rWT metHb, such that the ligated water is lost [16,17] The sizable structural accommodation to crystal forms for Pe metHb is unprecedented and reflects a surprising ÔplasticityÕ whose functional relevance is unknown The differences in the two structures could be rationalized by interactions between two molecules in the unit cell in one, but not the other, crystal form [16,17] Spectral congestion precludes more definitive 1H NMR structural studies of diamagnetic Pe WT HbO2 at present [23], and the molecule does not crystallize Hence, we have embarked on a 1H NMR study of the paramagnetic Pe WT metHbCN form whose ligand, like O2, is an (albeit weaker) H-bond acceptor [24–27], and whose tilting from the heme normal reflects steric repulsive and/or H-bonding attractive interactions in the distal pocket [24,28–30] The large hyperfine shifts, moreover, provide highly enhanced resolution to the active site residue protons that greatly facilitates their assignment, and whose hyperfine shifts contain a wealth of information on the detailed molecular structure not readily obtained in an analogous diamagnetic complex [30,31] At the same time, the paramagnetic-induced relaxation is sufficiently weak so as not to interfere with effective conventional 2D NMR experiments [32] that will confirm whether the active site structure is closer to one or the other crystal forms, or distinct from both The hyperfine shifts of the heme are sensitive to the presence of an H-bond to ligated cyanide [24–26,33], and hence provide direct information on distal H-bonding interactions Lastly, a relatively robust interpretive basis of the hyperfine shift of the heme and active site residue for low-spin ferric globins in terms of distal ligand tilt [28–30] and axial His orientation [26,34–38] has been established on the basis of characterizing a variety of hemoproteins with different properties The Pe metHb provides a novel His F8 [16] orientation that will allow further testing of this procedure Experimental procedures Protein samples The monomeric wild-type (WT) hemoglobin, labeled WT Hb, from the trematode Paramphistomum epiclitum (Pe) and Isoparorchis hypselbagi (Ih) were isolated and purified as described previously [14] The cyanomet complexes were prepared by adding approximately five molar equivalents of KCN to the air-oxidized Hb The final concentration of Pe metHbCN complex was % mM and that of Ih metHbCN was 0.2 mM The 1H2O solution was subsequently converted to 2H2O solution using an Amicon ultrafiltration cell Solution pH was adjusted with NaO1H (NaO2H) or 1HCl (2HCl) solution NMR spectroscopy H NMR data were collected on a Brucker AVANCE 600 spectrometer, operating at 600 MHz for protein samples in both 1H2O and 2H2O over the temperature range 15–35 °C, at a repetition rate of s)1 with presaturation of the solvent signal Water-eliminated Fourier transform (WEFT) [39] spectra were recorded to detect broad, strongly relaxed proton signals Chemical shifts were referenced to 2,2-dimethyl-2silapentane-5-sulfonate (DSS) through the water resonance Non-selective T1s, with ± 15% uncertainty, were determined for the resolved strongly relaxed protons at 25 °C from the initial magnetization recovery of a standard inversionrecovery pulse sequence The distance of proton H (with T1) from the iron, RFeH, was estimated from the relation: 1=6 à RFeÀH ẳ R FeH ẵT1 =T1i 1ị where R*Fe-H is the distance for a reference proton with T1* ˚ Using both the heme 18-CH3 for H* (R*Fe ¼ 5.88 A, ˚ T1* ¼ 140 ms) and F8 NdH (R*Fe ¼ 5.01 A, T1* ¼ 37 ms) as reference The : [40] and steady-state NOE spectra were recorded at 30 °C as described previously in detail [41] NOESY [42] and TOCSY [43] spectra were collected (512t1 blocks, 2048t2 points) at 25 °C, 30 °C and 35 °C in order to identify scalar and dipolar connectivities among heme and amino acid residues Spectral widths of 13 kHz and mixing times of 80 ms for NOESY and 50 ms for TOCSY were used Scans (192) were collected for each block with a repetition time of 1.0 s)1 Two-dimensional data sets were processed by XWINNMR software on a Silicon Graphics Indigo workstation Both NOESY and TOCSY spectra were processed by a 30° shifted sine bell squared apodization, and zero-filled to 2048 · 2048 points prior to Fourier transformation Ó FEBS 2003 Active site structure of trematode cyanomet Hb (Eur J Biochem 270) 2709 Magnetic axes determination The observed dipolar shifts are given by: Experimental dipolar shifts for the structurally conserved residues and backbone protons were used as input to search for the Euler rotation angles a, b and c These transform the molecular pseudosymmetry coordinates x¢, y¢ and z¢ (Fig 1) obtained from crystal coordinates [16,17] into the magnetic axes x, y and z, by minimizing the error function according to the following equation [2830,44]: X F=n ẳ ẵjddip (obs) ddip (calc)Fa; b; cịj 2ị where ddip calcị ẳ 12plNo ÞÀ1 ½2Dvax ð3cos2 h0 À 1ÞRÀ3 0 À3 þ 3ðDvrh sin h cos 2X ÞR Š ð3Þ ddip obsị ẳ dDSS obsị dDSS diaị 4ị where ddip(obs) and dDSS(dia) are the chemical shifts (in p.p.m.) referenced to DSS, for the paramagnetic Pe metHbCN complex and an isostructural diamagnetic complex, respectively Limited dDSS(obs) are available from Pe HbO2 [23]; for other residues dDSS(dia) may be reliably estimated from the available molecular structure [8,28,29] as follows: dDSS diaị ẳ dtetr ỵ dsec ỵ drc 5ị where dtetr, dsec and drc are the chemical shifts of an unfolded tetrapeptide relative to DSS [45], the effect of secondary Fig Schematic representation of the heme pocket of Pe Hb as found in the crystal structure of Pe metHbH2O [16] and as confirmed herein by solution 1H NMR of Pe metMbCN Proximal and distal residues are shown as rectangles and circles, respectively, and arrows connecting heme substituents and residues, and between residues, reflect NMR observed, and crystallographically expected, contacts The reference, x¢, y¢, z¢, as well as the magnetic coordinate systems, x, y, z, are shown, where b represents the tilt of the major magnetic axis, z, away from the heme normal (z¢ axis), a is the angle between the projection of the tilt of the z axis on the x¢, y¢ plane (defined direction of tilt of z and the x¢ axis), and j % a + c defines the location of the rhombic (x, y) axes The orientation of the axial His imidazole plane relative to the heme is given by / Ó FEBS 2003 2710 W Du et al (Eur J Biochem 270) structure [46] and the heme-induced ring current shift [47], respectively Structural modeling Protons were added to the crystal coordinates of recombinant WT Pe metHbH2O [16] and WT metHb [17] using the program INSIGHT-II (Accelrys) This provided unique coordinates for all protons of interest except the Tyr32(B10) hydroxyl proton, as its position was determined from the 1H NMR spectral parameters Results The 1H NMR spectra of Pe WT metHbCN in 1H2O and H2O are illustrated in Fig 2A,B A WEFT spectrum [39] designed to emphasize strongly relaxed signals is shown in Fig 2C Comparison of the traces in Fig 2A,B reveals the presence of two strongly relaxed labile protons at 32.5 p.p.m (T1 % 10 ms) and 17.5 p.p.m (T1 % 35 ms), as well as a weakly relaxed one at 11.4 p.p.m and several inconsequentially relaxed peaks in the 11–9 p.p.m window The 2H2O WEFT trace in Fig 2C locates a broad Fig 1H NMR spectra (600 MHz) of Pe WT metHbCN at 30 °C, pH » 7.0 (A) Relaxed (repetition rate s)1) reference trace in 1H2O; (B) relaxed, reference trace in 2H2O; (C) WEFT spectrum (relaxation delay 30 ms, repetition rate 10 s)1) in 2H2O which allows detection of strongly relaxed, broad signs at p.p.m.; and (D) steady-state NOE difference trace at 35 °C upon saturating the Tyr32(B10) OH signal (vertical arrow) Heme resonances are labeled as shown in Fig 1, and residues are labeled by position numbers and protons Ó FEBS 2003 Active site structure of trematode cyanomet Hb (Eur J Biochem 270) 2711 Table 1H NMR spectral parameters for the heme and His98(F8) signals in Pe metHbCN Chemical shifts in p.p.m are referenced to DSS in 1H2O 100 mM phosphate, pH 6.8, 30 °C Non-selective T1, in ms, in square brackets for resolved resonances Protons Heme Heme His98(F8) dDSS(obs) [T1] 2-CH3 7-CH3 12-CH3 18-CH3 3-Ha 3-Hbs 8-Ha 8-Hbs 13-Ha 13-Hbs 17-Has 17-Hbs 5-H 10-H 15-H 20-H NH CaH CbH CbH NdH 6.18 23.64 [88] 7.97 14.10 [143] 12.40 [182] )4.88 [253], )4.23 [235] 8.83 )1.22 [186], 0.29 14.32 [110], 5.44 )3.34 [152], )2.63 [165] 13.29 [92], 4.86 )3.80 [130], )169 [155] )1.02 [52] 8.41 )0.09 6.23 12.81 [129] 8.05 8.67 10.59 [84] 17.51 [37] (% 300 Hz) and very strongly relaxed (T1 < ms) peak on the low-field shoulder of the diamagnetic envelope Heme pocket residue assignments were pursued by backbone connectivities as standard in a diamagnetic protein [48], with the remainder of the target residues assigned by detection of NOESY residue–heme and interresidue cross-peaks solely by the standard Mb fold, and the observation of relaxation effects and/or sufficient TOCSY cross-peaks to identify the side chain uniquely The identification of hyperfine shifted and relaxed resonances was greatly facilitated by variable temperature studies to define unique scalar/dipolar connectivities as described previously [49] Assignments deduced herein are given by the heme-labeling scheme shown in Fig and the residue numbers and proton The chemical shifts for the heme and axial His98(F8) are listed in Table 1, and those for assigned residues with significant dipolar shifts are listed in Table T1 values for predominantly paramagnetically influenced protons are given in parentheses Heme assignments TOCSY spectra (not shown) identify four (two three-spin and two four-spin) hyperfine shifted and relaxed spin systems with dipolar contacts (not shown) to four strongly temperature-dependent methyl peaks [two resolved (Curie behavior) and two nonresolved methyl peaks (antiCurie behavior)], that uniquely identify the pyrrole substituents [30] Dipolar contacts to adjacent meso-Hs (5-H, 10-H, 15-H, 20-H), with their unique low-field intercept at T)1 ¼ 0, locate the four meso-Hs (as listed in Table 1) Sequence-specific assignments The detection of the Ni–Ni+1, ai–Ni+1, bi–Ni+1, ai–Ni+3 and/or ai–bi+3 NOESY connection diagnostic of helical fragments [48] with limited (but sufficient) TOCSY-identified side chains leads to the identification of the six segments labeled I–VI (Fig 3) Fragment I is represented by Zi-AMXi+1-Glyi+2-Zi+3-AMXi+4-AMXi+5-, where Z is >4 spin side chain, and AMXi+1 and AMXi+4 exhibit significant low-field dipolar shifts The relaxed, low-field labile proton at % 17 p.p.m exhibits a NOE to the AMXi+5 and its peptide NH that is unique for the proximal His98(F8), and AMXi+1 is in dipolar contact with a twospin aromatic ring, as expected for Tyr94(F4); this identifies I as Gln93–His98 of the proximal F-helix(F3–F8) Further backbone (nonhelical) dipolar connections (Fig 3) allow the adjacent assignments of Thr99–Val103, the residues that constitute the FG corner (FG1–FG5), with expected dipolar contacts to pyrroles B and C (Fig 1) and residues in the C and G helices (see below) Fragment II is represented by AMXi-Alai+1-Zi+2-Thri+3-Leui+4-Zi+5-Zi+6-Alai+7, which the sequence uniquely identifies as the expected E-helix (E7–E16) segment Tyr66–Val75, with the ThrE10 and LeuE11 side chains exhibiting weak-to-moderate upfield shifts The detection of an inconsequentially shifted, two-spin aromatic ring in contact with AMXi (Fig 4E) confirms Tyr66(E7) Key dipolar contacts that define the orientation of the Tyr66(E7) ring include those to the 13-propionate Hbs (Fig 4B,C) and to the Phe46(CD1) ring, and the NHs of Arg48(CD3) and Leu49(CD4) (Fig 4G) It was not possible to detect a labile proton in dipolar contact with the Tyr66(E7) CeHs signal that would identify the side chain hydroxyl proton The observed heme-residue contacts are characteristic of the general Mb fold (Fig 1) Fragment III is detected as Glyi-Zi+1-Xi+2-Ilei+3AMXi+4-Thri+5-Zi+6-AMXi+7-AMXi+8 (Fig 3) where contacts of three-spin aromatic rings to AMXi+4 and AMXi+8 and a two-spin aromatic ring to AMXi+7 uniquely identify Gly111–Phe119 on the G-helix (G8– G16) An additional AMX spin-system connected to a hyperfine shifted aromatic ring identifies a Phe, and its backbone exhibits the ai-3-Ni cross peak to AMXi in III (Gly111); this identifies it as Phe108(G5) The side chains exhibit the expected NOESY cross peaks to the pyrrole A/B junction and to the E-helix as depicted in Fig Fragment IV, Vali-AMXi+1-Xi+2-Zi+3-Zi+4-Alai+5-Alai+6Zi+7-Zi+8 (Fig 3), is unambiguously identified as Val139– Ile147 on the H-helix (H15–H23) The dipolar contacts of Phe140(H16) and Met143(H19) to pyrrole A, as well as Ile147(H23) to the axial His98(F8), and the interresidue contacts to the G-helix (Fig 1) confirm their locations in a standard H-helix The helical fragment V is represented by Zi-AMXi+1Zi+2-Zi+3-AMXi+4 (Fig 3), where dipolar contacts of a two-spin aromatic ring to AMXi+1 (and to the 7-CH3 and 8-vinyl; Fig 1) identifies the Gln41–His45 fragment on the C-helix (C3–C7), with the expected moderate dipolar shifts, and which is in contact with the pyrrole B/C junction (Fig 1) Backbone NOESY connections allow the extension of sequential assignment of fragment V to include AMXi+5Seri+6 that must arise from Phe46(CD1) and Ser47(CD2) Ó FEBS 2003 2712 W Du et al (Eur J Biochem 270) Table 1H NMR spectral parameters for strongly dipolar shifted active site residues in Pe metHbCN Observed chemical shifts, dDSS(obs), in p.p.m., are referenced to DSS in 1H2O, 100 mM phosphate, pH 6.8 at 30 °C Diamagnetic chemical shifts, dDSS(dia), calculated via Eqn (5) using the WT Pe metHb H2O crystal coordinates [16] na, Not assigned Residue Proton dDSS(obs) dDSS(dia) Tyr32(B10) NH CaH CbH¢ CbH CdHs CeHs OH NH CaH CbHs CdHs CeHs CfH NH CaH CbH CdHs CeHs NH CaH CbH CdH NH CaH CbH CdHs CeHs CfH NH CaH CbH CbH¢ CdHs CeHs OH NH CaH CbH3 NH CaH CbH CcH3 NH CaH CbH CbH¢ CcH CdH3 CdH3¢ NH CaH CbH3 NH CaH CbH 8.50 5.04 3.90 3.60 8.50 11.79 32.51 9.60 5.01 3.43, 3.32 7.61 7.78 7.34 8.21 3.78 3.66 6.73 6.35 7.62 4.14 1.95 5.89 7.33 3.01 2.15 6.15 8.88 11.42 7.08 4.85 6.27 3.33 6.55 5.45 na 9.32 5.19 2.37 8.17 3.44 3.04 0.15 9.80 na 3.33 6.08 2.8 2.63 2.09 7.10 3.84 )0.03 8.62 6.44 3.87 7.41 3.27 2.44 2.32 5.87 5.48 8.12 8.07 4.08 3.12, 6.99 6.08 5.81 8.20 4.01 3.78, 7.04 6.85 8.25 4.45 3.21, 7.50 8.10 4.74 2.75, 7.09 6.04 6.44 7.70 3.51 1.86 1.64 7.42 6.75 8.50 7.78 3.87 1.06 7.79 4.03 4.45 1.05 8.49 3.29 0.62 0.91 0.56 )0.81 )1.10 7.81 4.60 1.70 7.93 3.94 3.03 Phe36(B14) Tyr42(C4) His45(C7) Phe46(CD1) Tyr66(E7) Ala67(E8) Thr69(E10) Leu70(E11) Ala73(E14) Tyr94(F4) Table (Continued) Residue Gly95(F5) Lys96(F6) Asp97(F7) 2.80 His98(F8) 3.36 2.98 2.67 Thr99(FG1) Val103(FG5) Phe108(G5) Gly111(G8) Phe115(G12) Phe140(H16) Met143(H19) Ile147(H23) Proton dDSS(obs) dDSS(dia) CbH¢ CdHs CeHs NH CaH CaH¢ NH CaH CbH CcHs CdHs CeHs NH CaH CbH CbH¢ NH CaH CbH CbH¢ NdH NH CaH CbH CcH3 NH CaH CbH CcH3 CcH3¢ NH CaH CbH CbH CdHs CfH NH CaH CaH¢ NH CbH CbH¢ CdHs CeHs CfH NH CaH CbH CdHs CeHs CfH NH CaH CbHs CcH CeH3 CaH CbH CcHs CcH¢ CdH3 3.25 7.40 6.57 10.47 5.71 6.80 9.8 5.78 2.90, 2.79 2.13 2.30 2.47 10.07 5.80 3.51 6.54 12.81 8.05 8.67 10.59 17.51 9.35 5.23 5.00 2.12 6.81 3.50 0.79 0.54 )0.05 8.08 1.34 2.43 1.75 4.22 3.76 6.65 1.58 2.92 8.50 3.01, 2.82 2.82 6.52 6.82 8.96 8.08 3.16 2.16 6.19 4.95 4.38 7.49 4.49 2.28 1.93 0.48 4.38 2.30 2.78 1.22 0.54 2.76 7.71 7.16 7.66 3.11 2.26 7.45 3.47 1.62, 1.49 1.09 1.47 2.73 7.50 4.08 2.57 1.50 6.66 2.71 0.91 0.67 6.42 7.45 3.55 3.91 0.68 8.06 4.08 2.67 )0.09 1.21 8.14 4.57 2.87 2.30 5.63 6.09 8.29 3.94 3.50 8.32 3.69 3.31 7.10 6.96 5.82 8.21 4.31 3.01, 3.31 7.39 7.46 7.90 7.89 3.64 0.98, 1.34 1.71 1.27 3.15 1.29 0.18 )0.15 )2.44 Ó FEBS 2003 Active site structure of trematode cyanomet Hb (Eur J Biochem 270) 2713 Comparison to the alternate crystal structures Fig Schematic representation of the sequential NOESY cross peak pattern for the six characterized helical fragments I–VI that identify key sections of the F, E, G, H, C and B helices, respectively Dipolar contact to a strongly relaxed and moderately dipolar shifted aromatic spin-system that is in contact with pyrrole C (Fig 4B,C) confirm both the assignment as Phe(CD1) and the orientation of the heme, as depicted in Fig and as found in the crystal structure The CfH of Phe46(CD1) exhibits the strong relaxation (T1 % 20 ms) characteristic for this residue The remaining helical segment VI, Zi-Thri+1-Glyi+2Zi+3 -Glyi+4-Alai+5-AMX i+6-AMX i+7-Ala i+8-Zi+9AMXi+10-Thri+11-Alai+12 (Fig 3), is unique to residues Glu26–Ala38 on the B-helix (B4–B16) The dipolar contact of a weakly shifted, three-spin aromatic ring to AMXi+10, and that of a strongly hyperfine shifted two-spin aromatic ring to AMXi+6 confirm the assignments of Phe36(B14) and the key Tyr32(B10) These side chains not exhibit NOESY cross peaks to the heme (as expected), but exhibit the expected contacts to helix E (Tyr32(B10) to Leu70(E11), Fig 4D; Ala67(E8), Fig 4F; and Tyr66(E7), Fig 4G, as depicted in Fig A strong NOE to the assigned Tyr32(B10 CeHs signal, upon saturating the extreme low-field, strongly relaxed (T1 % 10 ms) labile proton signal (Fig 2D), locates the residue side chain hydroxyl proton The pattern of NOESY cross peaks of the Tyr66(E7) ring to the heme (Fig 4A–C), Tyr32(B10) (Fig 4F), and in particular, to the Phe46(CD1) backbone (Fig 4E), as summarized in Fig 1, unequivocally establish that the Tyr66(E7) ring is oriented out of the heme pocket, exactly as found in both Pe metHb crystal structures [16,17] The position is further supported by the calculated and observed small ddip (and hence, negligible temperaturedependence to its shifts) for the crystallographic orientation of the Tyr66(E7) ring (see below) While its hydroxyl proton could not be located by its characteristic strong NOE to the definitively assigned CeHs, most likely due to its lability, there is no orientation of the OH group that ˚ can bring it close enough (>5 A) to interact with the bound cyanide The interresidue and residue-heme NOESY cross peak pattern that led to the schematic representation of the Pe metHbCN heme cavity structure in Fig is equally consistent with qualitative expectations of either of the two Pe metHb crystal structures [16,17] It is only upon quantitative consideration of cross peak intensities that such detailed structural distinctions can be made The two X-ray structures, one of WT and the other of rWT metHb, exhibit differences that include important portions of the protein that we have characterized above Thus parts of the FG corner move away from the heme and the B-helix moves closer to the heme in the WT metHb than in the rWT metHbH2O crystal structure, with the result that the ligated water is lost in the latter complex [16,17] Prior to determining the magnetic axes, which will allow us to elaborate the tilt of the ligated cyanide and characterize the H–bonding interaction of Tyr32(B10) with its ligand, it is necessary to establish which crystal structure better represents the solution structure This distinction can be made on the basis of three NMR observations, the NOESY cross peak intensities between proton i and j (/ rij)6), the paramagnetism-induced relaxation, TÀ1 / RÀ6 , of proton i, and the magnetic axes themselves 1i FeÀi; (see next section) Inspection of the two sets of crystal coordinates identifies a series of proton pairs whose separations differ significantly between the two structures [16,17] and which we have been able to identify unambiguously The contacts involve the FG corner and the position of helix B relative to the heme and E-helix backbone Table lists the alternate rij for eight sets of proton pairs in the two structures, as well as the ˚ observed NOESY cross peak intensity (s, < 2.5 A; m, 2.5– ˚ 4.6; w > 4.0 A) In each case, the distance in bold is the one in better agreement with the experiment, and each of the four distances dictate that the solution structure of WT metHbCN is consistent only with the crystal structure of rWT metHbH2O [16] (except for a labile proton on Tyr32(B10), see below) The alternate structures predict characteristic relaxation time differences for several proton sets in the alternate crystal structure, i.e Tyr32(B10), Thr99(FG1), Val103(FG5), but in only one case is the key resonance resolved so that its T1 can be quantitated Thus the movement of the B-helix towards the heme in the WT metHb relative to that in the rWT metHbH2O crystal Ó FEBS 2003 2714 W Du et al (Eur J Biochem 270) Fig Portions of the 600 MHz 1H NOESY spectra (mixing time 80 ms) of Pe metHbCN in H2O 100 mM in phosphate, pH 7.0 at 35 °C Dipolar contacts are illustrated, involving key distal residues Phe46(CD1) and helical ring cross peaks for Tyr32(B10) (F), Phe36(B14) (G), Phe46(CD1) (E), Tyr66(E7) (G) and Leu70(E11) (E), and to NHs of heme-residue contacts Tyr66(E7) and Phe46(CD1) to have propionates (A, B, C) and interresidue contacts from Tyr66(E7) to Phe46(CD1) and NHs of Arg48(CD3) Leu49(CD4) (G), and Tyr32(B10) (D) contacts to Ala67(E8) (F) The cross peak between Tyr32(B10) CeHs and Tyr66 (E7) CdHs is observed only at a lower contour level structure leads to the reduction of the RFe-i for the two Tyr ˚ B10 CeHs of 5.8 and 7.9 A in rWT metHbH2O (with expected T1 % 20–30% shorter than that for a heme ˚ methyl), to 4.5 and 5.8 A in WT metHb such that a T1 is expected closer to that of a meso-H (T1 % 50 ms) The observed T1 (Tyr32(B10) (CeH) % 100 ms, is consistent with the former, but not the latter distances, such that the relaxation effects similarly confirm a WT metHbCN solution structure similar to the rWT metHbH2O, but not the WT metHb crystal structure Magnetic axes The orientation of the magnetic axes was determined by using the ddip(obs) via Eqns (4) and (5) for Pe metHbCN The anisotropies at 30 °C, which have been shown to be highly conserved in a wide variety of cyanomet globins [8,26,28–30], are Dvax ¼ 2:48  10À8 m3ÁmsÀ1 and Dvrh ¼ À0:58  10À8 m3ÁmsÀ1 , as reported for sperm whale metMbCN The coordinates that determine R, h¢ and W¢ in Eqn (3) were taken alternatively from the Pe rWT Ó FEBS 2003 Active site structure of trematode cyanomet Hb (Eur J Biochem 270) 2715 Table Comparison of predicted and observed NOESY cross peak intensity for the two crystal structures of Pe metHb Inter-proton ˚ separation rij (A) Pe rWT metMbH2O crystal structure [16], Pe WT metHb crystal structure [17] Observed NOESY cross peak intensities, ˚ ˚ s (strong, rij < 2.5 A), m (moderate, 2.5 < rij < 4.0 A), weak (weak, ˚ 4.0 < rij < 5.0 A) Distances in bold are in agreement with the NMR observations ˚ rij (A) rWT metHbH2O F-helix/FG-corner NH(FG1)-CaH(F8) CaH(FG1)-CaH(H23) CaH(FG1)-CcH(H23) CbH(FG1)-CaH(F6) B-helix CaH2(B10)-CaH(E8) CeH2(B10)-CbH3(E8) CbH2(B10)-CbH1(E7) CeH2(B10)-CbH2(E11) WT metHb NOE 3.49 2.55 3.47 3.80 2.45 5.92 6.22 7.02 m mỈs)1 m m 2.22 4.33/4.86 2.91 3.14 3.11 5.33 2.52 2.38 s m m m metHbH2O [16] (case I) or the WT metHb [17] (case II) crystal structures In order to utilize the information in ddip for distinguishing between the two crystal structures, the experimental shifts and crystal coordinates initially used to determine the magnetic axes were only for those protons where the residue exhibited the same position in the alternate crystal structures The results lead to equally welldetermined orientations of a ¼ 203 ± 10, b ¼ 9° ± 1, j ¼ 50 ± 10 and residual F/n ¼ 0.14 p.p.m.2 for case I, and a ¼ 206 ± 10, b ¼ 10 ± 1, j ¼ 40 ± 10° and residual F/n ¼ 0.20 p.p.m.2 for case II The plot of ddip(obs) vs the ddip(calc) ( ,j) for each set of magnetic axes are given in Fig 5A (case I) and 5B (case II), and each represents a good fit The differences in b not reflect differences in tilt  Fig Plot of the ddip(obs) vs ddip(calc) for the magnetic axes of Pe WT metHbCN as based on the crystal coordinates of rWT metHbH2O; and WT metHb (A) rWT metHbH2O; and (B) WT metHb, using as input only the ddip(obs) for protons whose positions are the same in the two crystal structures [16,17], with Dvax ẳ 2.48 à 10)8 m3ặmol)1 and Dvrh ¼ )0.58 · 10)8 m3Ỉ mol)1 as reported for sperm whale metMbCN [29] The solid markers represent the input data for the structurally conserved protons, while open markers are for those protons whose positions differ significantly in the two crystal structures of the axis so much as a small difference in the reference coordinate system x¢, y¢ and z¢ in the two structures (due to different nonplanarity of the heme) The ddip(obs) and ddip(calc) for those protons whose coordinates differed significantly in the two structures are shown as s and h For the most part, in particular Thr99(FG1) and Val103(FG5), residues with different geometries exhibit reasonable fits for both cases in Fig 5, in part because ddip is small, but also because their position is not very sensitive to dipolar shift However, it is noted that the Tyr32(B10) CeHs exhibit a very reasonable fit for case I (Fig 5A), but an unacceptable fit for case II (Fig 5B) Hence the magnetic axes completely concur with the results of both NOESY intensity analysis and paramagnetic relaxation effects in finding the Pe metHbCN active site solution structure to coincide with the crystal structure of rWT metHbH2O [16] but not WT metHb [17] Redetermination of the magnetic axes orientation (a, b, c), from a large variety of available input data using only the pertinent Pe rWT metH2O crystal structure [16] led to a ¼ 202 ± 10°, b ¼ ± 1° and j ¼ 52 ± 10° for a three-parameter search using the sperm whale metMbCN anisotropies [29], and yielded minimally changed orientation, a ¼ 202 ± 10, b ¼ ± and j ¼ 51 ± 10 for the five-parameter search that yielded Dvax ẳ 2:36 ặ 0:04 108 m3 mol1 and Dvrh ẳ 0:59 ặ 0:06 10À8 m3 ÁmolÀ1 which are within the uncertainties of the respective determinations (not shown) The tilt of the major magnetic axes is correlated with Fe-CN tilt [8,28,30] (with the negative z axis), and indicates that the cyanide is tilted % 10° in the direction of the 5-H position The rhombic axes are defined by j % 50° in Fig The difference in the overall shift dispersion pattern of Pe metHbCN relative to, for example, any of the mammalian metMbCN where both the FG corner and PheCD1 residues exhibit large upfield and downfield shifts, respectively, is due to the smaller tilt, b 2716 W Du et al (Eur J Biochem 270) Ó FEBS 2003 Structural simulation of Tyr32(B10) The good correlation between ddip(obs) and ddip(calc) for the Tyr32(B10) ring in the magnetic axes based on the rWT metHbH2O crystal structure [16] indicates that the ring (and hence B-helix) occupies the same position as in the crystal This conclusion is supported by the relaxation properties of the CeHs signal (see above) The hydroxyl proton position is not directly determined in the crystal structure, but can be inferred by the position of other Hbond acceptor/donors in the immediate vicinity The proposal that the Tyr32(B10) hydroxyl proton acted as a donor to the carbonyl of Tyr66(E7) in the rWT ˚ metHbH2O crystal structure [16] places it % 5.9 A from the iron with an angle of % 17° with the Tyr32(B10) Ce-Cf-O-H plane; we define this angle w ¼ In the rWT metHbH2O crystal structure [16], the heme ligand (water molecule) is an H-bond donor, while in metHbCN, it (cyanide) is an H-bond acceptor, so that a significantly different OH orientation can be expected A plot of the effect of the angle, w, between the Tyr32(B10) Cf-OH and ring planes, on the three distinctive variables that depend critically on the orientation of the OH group is illustrated in Fig The shaded areas correspond to the observed values of ddip(calc) (Fig 6A), distance to the ˚ iron, RFe ¼ 4.0–4.5 A (Fig 6B), as indicated by T1 % 10 ms, and Tyr32(B10) OH to Tyr66(E7) CdH distance, rij of Tyr66(E7), Fig 6C, as indicated by a weak-to-moderate NOESY cross peak intensity Inspection of Fig reveals a single orientation, w ¼ )140 ± 20°, that essentially quantitatively and simultaneously accounts for the three observations The effect on the Tyr32(B10) O-H.N(cyanide) angle on Y is illustrated in Fig 6D The position of the Tyr32(B10) Og relative to the cyanide ligand tilted by % 10° in the direction of the 10-H position is illustrated in Fig and reveals a van der Waals contact between Tyr32(B10) and the cyanide Discussion Active site structure The combination of NOESY cross peak intensities, paramagnetic relaxation and the magnetic axes data provide compelling evidence that WT Pe metHbCN much more closely resembles the structure found in the crystal structure of rWT Pe metHbH2O [16] than of WT Pe metHb [17] Because the WT Pe metHbCN active site structure is essentially the same as rWT metHbH2O, and different from WT metHb, the present data support the interpretation that the structural differences between rWT and WT Pe metHb in crystals result from the extensive interaction between the two molecules in the unit cell for WT metHb, rather than from significant structural differences between isolated WT and rWT Pe Hb molecules [17] The distal Tyr66(E7) ring was found oriented out of the heme pocket in both rWT metHbH2O and WT metHb [16,17] Our NMR data on Pe metHbCN confirm that Tyr66(E7) is similarly oriented away from the heme iron in a position essentially the same as in the crystal structure with its OgH much too far removed Fig Plots of ddip(calc) derived from optimized magnetic axes, the distance to iron via paramagnetic relaxation RFe, distance to Tyr66(E7) CdH(via NOESY cross peak intensity) and /O-H-N angle as a function of the CfO-H to aromatic plane dihedral angle, w, for the Tyr32(B10) hydroxyl group, with the ring position as defined in the rWT metHbH2O crystal structure [16] and confirmed for the WT metMbCN solution structure described here The shaded portions represent the observed values (and their uncertainties) of the three variables Note that a simultaneous fit for all three variables occur only for w % )140 ± 20° ˚ (>6 A) from the cyanide to provide a H-bond The failure to resolve the OgH signal for Tyr66(E7) can be attributed to its expected rapid exchange with solvent While cyanide is a H-bond acceptor and a weak mimic of O2, it does not induce a rearrangement of the Tyr66(E7) ring into the heme pocket relative to the high-spin, metHb complexes H NMR data on Pe WT HbO2 had shown that there are two interacting labile protons from two Tyr in the distal pocket capable of interacting with the bound O2 [23] Moreover, NOESY cross peaks between the Tyr66(E7) ring and the terminus of Leu70(E11) indicated that the Tyr66(E7) ring is oriented into the heme pocket There appears to be no obvious rationalization for these contradictory results It has been demonstrated that Tyr66(E7) can be substituted without significantly affecting the extreme O2 ligation dynamics/thermodynamics [16] However, these results not alone determine that Tyr66(E7) is not oriented into the heme pocket, they only demonstrate that any interaction of the Tyr66(E7) ring with O2 does not incrementally increase the H-bond stabilization of bound O2 A crystal structure or solution NMR structure of Pe HbO2 is clearly important Ó FEBS 2003 Active site structure of trematode cyanomet Hb (Eur J Biochem 270) 2717 Distal hydrogen bonding Magnetic axes It is noteworthy that for Pe WT metHbCN, only a single labile proton (Tyr32(B10) (OgH) is found sufficiently near the cyanide ligand to participate in H-bonding to the ligated cyanide The van der Waals surfaces for the Tyr32(B10) Og and the 10° tilted cyanide ligand are shown in Fig and establish that the Tyr Og and cyanide N are in van der Waals contact, with a (Tyr32(B10))Og-N(cyanide) separ˚ ation of 2.6 A It appears, moreover, that the 10° tilt of the cyanide results from a steric interaction between the Tyr32(B10) Og and the nitrogen, because a zero tilt angle ˚ would lead to an % 0.2–0.3 A overlap of the two van der Waals surfaces A moderate-to-strong H-bond [50] can occur when the donor/acceptor heteroatoms are separated ˚ by % 2.5–3.0 A, and the O-H.N bond angle is close to 180° Figure 6D shows that the unique orientation of the Tyr32(B10) OgH that optimally accounts for the NOESY, relaxation and dipolar shift data, i.e w ¼ )140° ± 20, is precisely what would lead to the strongest H-bond, with an O-H.N angle of % 170° Experimental verification of a moderate-to-strong single H-bond to bound cyanide is obtained from the effect of solvent isotope (1H/2H) composition [24,26,27,41,51] on the heme electronic structure Thus both the 18-CH3 and 7-CH3 exhibit two separate resonances in 1H2O/2H2O mixtures, whose relative intensities directly reflect the solvent isotope composition The splitting of the two methyls in 50% 1H2O/ 50% 2H2O is illustrated in the insets to Fig 2A, where the larger heme methyl contact shifts result from a single 2H rather than the 1H in a single H-bond to a heme ligand This single H-bond is clearly that of the Tyr32(B10) OH described above The magnetic axes could be determined to relatively high accuracy on the basis of the rWT metHbH2O crystal structure [16] The optimized anisotropies of Dvax ¼ 2.38 · 10)8 m3ặmol)1 and Dvrh ẳ )0.56 à 10)8 m3ặmol)1, are within the uncertainties of those reported for sperm whale metMbCN, [29] 2.48 0.03 à 10)8 m3ặmol)1, Dvrh ẳ )0.58 ± 0.04 · 10)8 m3Ỉmol)1, and numerous of its point mutants [26,30], and confirm the strong conservation of the magnetic anisotropies of low-spin hemin with His/cyanide ligation The 10° tilt of the major magnetic or z-axis is shown to be consistent with the % 10° tilt of the Fe-CN vector from the heme normal to avoid van der waals overlap with the Tyr32(B10) Og The location of the rhombic axes, j % 50 ± 10°, is in agreement with the expectations [30,35] of the counter-rotation principle which dictates that the rhombic axes, j % 50°, rotate relative to an N-Fe-N vector in opposite direction, but with the same magnitude, as the axial His imidazole orientation relative to the N-Fe-N vector, here given by / ¼ 52° (Fig 1) It has been proposed that the pattern of the meso-H hyperfine shift is determined largely by dipolar shifts due to the rhombic anisotropy [30,36,37] The D(obs) ¼ 1/2[dhf (5-H) ) dhf(10-H) + dhf(15-H) ) dhf(20-H)] ¼ 8.2 p.p.m., while D(calc) ¼ 1/2[ddip(5-H) ) ddip(10-H) + ddip(15-H) ) ddip(20-H)] ¼ ± p.p.m., which confirms the prediction Lastly, a pattern of heme hyperfine shift with the two largest low-field shifts for 7-CH3 and 18-CH3 is generally interpreted as indicating that the heme orientation is rotated % 180° about the a-c-meso axis relative to mammalian globins, as observed in Chironomus Hbs [26] This interpretation has as its premise that the axial His orientation is conserved relative to globins for which both 1H NMR and crystallographic characterization has been carried out The normal His F8 imidazole plane orientation for mammalian globins is close to / % in Fig [52] For the present Pe Hb, the heme orientation is, in fact, identical to that in mammalian globin (Phe CD1 near the 12-CH3) but its His F8 imidazole is rotated % 45° clockwise relative to the mammalian globin, and it is the His F8 rotation, not the heme rotation, that leads to the low-field 7-CH3 and 18-CH3 signals Hence it is clear that heme methyl assignments not yield information on heme orientation unless the axial His orientation is known Dynamic properties Fig Model of the distal ligand environment as determined herein which shows the van der Waals contact between the Tyr32(B10) side chain and the » 10° tilt of the ligated cyanide It is noted that the tilt of the CN– is necessary to avoid steric interaction with the Tyr32(B10) Og The remarkably slow autoxidation rate for Pe Hb has been suggested [16] to result from a greater dynamic stability of Pe Hb relative to other more autoxidizable globins A reliable indicator of the dynamic stability of distal heme pockets in globins is the rate of reorientation of the aromatic rings in the heme pocket [37,53] The rate of reorientation can be estimated by the excess line broadening at lowtemperature of the averaged CeH peaks if the chemical shift differences are known [37,53] Two such rings of interest are Tyr32(B10) and Phe46(CD1) The TyrB10 ring of the nematode Ascaris metHbCN with relatively ÔnormalÕ autoxidation rate exhibits [37] % 450 Hz excess line broadening at 15 °C that could be attributed to slow reorientation of the ring The difference in the TyrB10 CeH chemical shifts, as Ó FEBS 2003 2718 W Du et al (Eur J Biochem 270) determined from the magnetic axes [37], was found to be 6.0 p.p.m at 600 MHz For the present Pe metHbCN, the Tyr32(B10) CeHs signal exhibits 102 times faster in Pe metHbCN than As metHbCN The Phe46(CD1) averaged CeHs peak in Pe metHbCN is narrow, in contrast to some % 50 Hz excess linewidth due to ring reoriented observed for Phe(CD1) in sperm whale [53] and elephant [54] metMbCN Again, very similar chemical shift differences for the two CeH are predicted by the magnetic axes for sperm whale and Pe metHbCN, indicating that the PheCD1 ring in Pe metHbCN reorients >10 times faster than in the mammalian metMbCN complexes [53,54] Hence the heme pocket in Pe Hb does not appear to be more dynamically stable than those of other nematodes/trematodes or mammalian globins with ÔordinaryÕ autoxidation rates One possibility that cannot be discounted is that the Pe Hb possesses a limited flexibility that involves the position of the B-helix, as already witnessed by the facility with which the position of the B-helix and FG corner accommodates perturbations such as interprotein contacts [17] The limited ÔflexibilityÕ may be required to allow the facile reorientation of the Tyr66(E7) ring from ÔoutsideÕ the heme pocket in oxidized Pe globins to ÔinsideÕ the pocket in Pe HbO2 complexes, as observed [26] in the 1H NMR data of Pe HbO2 The static structure of neither WT nor rWT metMb would allow the TyrE7 ring reorientation without significant distortion of the heme pocket Comparison to other nematode/trematode Hbs The low-field portion of the 1H NMR spectra of Pe metHb-CN in Fig 8A is compared with those for Ih metHbCN and Dicrocoelium dendriticum (Dd) metHbCN [55] In each case, the saturation of the strongly relaxed low-field labile protons (not shown) results in NOEs to a two-proton signal [55] (Fig 2D; not shown for Ih metHbCN) indicative of a hyperfine shifted Tyr The similar relaxation of the labile proton (T1 % 10 ms) in all three complexes [55] indicates that the Tyr is similarly oriented with respect to the iron and positioned to provide the H-bond to the bound cyanide, and by implication, to the bound O2 in the reduced form in each of the three globins The extensive sequence homology for Pe and Ih Hbs [14] argues for very similar heme pocket structures For Dd Hb, for which a complete sequence has never been reported [56], earlier NMR studies [55] had proposed a distal Tyr at position E7 as the source of the H-bond to ligand on the basis of a partial sequence, which had indicated a Tyr on the distal E-helix The similarity in the 1H NMR spectra of the three globin complexes in Fig dictates that the distal Tyr in Dd metHbCN that results in the H-bond to the ligand is Tyr(B10), rather than TyrE7 Acknowledgements The authors are indebted to Dr Yuyang Wu for obtaining preliminary H NMR spectra Dr S Dewilde is a postdoctoral researcher of the F.W.O The results were supported by grants from the National Institutes of Health, HL16087 (G.N.L.) and the Fund for Scientific Research Flanders (F.W.O.), G0314.00 N (L.M.) 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S1 Portions of the 500 MHz TOCSY spectrum (mixing time 30 ms, repetition rate s)1) in 1H2O, 100 mM in phosphate, pH 7.2 at 25 °C, showing the heme proton spin systems Figure S2 Portions of the 500 MHz NOESY spectrum (mixing time 60 ms, repetition rate s)1) in 2H2O, 100 mM in phosphate, pH 7.1 at 30 °C, illustrating heme proton dipolar contacts Figure S3 Portion of the 600 MHz NOESY spectrum (mixing time 80 ms, repetition rate s)1) in 1H2O, 100 mM in phosphate, pH 6.8 at 35 °C, illustrating the E- and F-helix Ni–Ni+1 connections (solid lines and arrows), the F-helix – Ni+1 connections (dashed lines), and the dipolar contacts between Tyr32(B10), Tyr66(E7) and Phe46(CD1)  ... CbHs CdHs CeHs CfH NH CaH CbH CdHs CeHs NH CaH CbH CdH NH CaH CbH CdHs CeHs CfH NH CaH CbH CbH¢ CdHs CeHs OH NH CaH CbH3 NH CaH CbH CcH3 NH CaH CbH CbH¢ CcH CdH3 CdH3¢ NH CaH CbH3 NH CaH CbH... NH CaH CbH CbH¢ NdH NH CaH CbH CcH3 NH CaH CbH CcH3 CcH3¢ NH CaH CbH CbH CdHs CfH NH CaH CaH¢ NH CbH CbH¢ CdHs CeHs CfH NH CaH CbH CdHs CeHs CfH NH CaH CbHs CcH CeH3 CaH CbH CcHs CcH¢ CdH3 3.25... of WT and the other of rWT metHb, exhibit differences that include important portions of the protein that we have characterized above Thus parts of the FG corner move away from the heme and the