Solution 1H NMR of the Active Site of Substrate-bound, Cyanide-inhibited Human Heme Oxygenase

COMPARISON TO THE CRYSTAL STRUCTURE OF THE WATER-LIGATED FORM*,

Gerd N. La MarDagger §, Anbanandam AsokanDagger , Bryan EspirituDagger , Deok Cheon YehDagger , Karine Auclair, and Paul R. Ortiz de Montellano

From the Dagger  University of California, Department of Chemistry, Davis, California 95616 and the  Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446

Received for publication, November 1, 2000, and in revised form, January 12, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The majority of the active site residues of cyanide-inhibited, substrate-bound human heme oxygenase have been assigned on the basis of two-dimensional NMR using the crystal structure of the water-ligated substrate complex as a guide (Schuller, D. J., Wilks, A., Ortiz de Montellano, P. R., and Poulos, T. L. (1999) Nat. Struct. Biol. 6, 860-867). The proximal helix and the N-terminal portion of the distal helix are found to be identical to those in the crystal except that the heme for the major isomer (~75-80%) in solution is rotated 180° about the alpha -gamma -meso axis relative to the unique orientation in the crystal. The central portion of the distal helix in solution is translated slightly over the heme toward the distal ligand, and a distal four-ring aromatic cluster has moved 1-2 Å closer to the heme, which allows for strong hydrogen bonds between the hydroxyls of Tyr-58 and Tyr-137. These latter interactions are proposed to stabilize the closed pocket conducive to the high stereospecificity of the alpha -meso ring opening. The determination of the magnetic axes, for which the major axis is controlled by the Fe-CN orientation, reveals a ~20° tilt of the distal ligand from the heme normal in the direction of the alpha -meso bridge, demonstrating that the close placement of the distal helix over the heme exerts control of stereospecificity by both blocking access to the beta , gamma , and delta -meso positions and tilting the axial ligand, a proposed peroxide, toward the alpha -meso position.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Heme oxygenase (HO)1 is a membrane-bound protein that carries out the NADPH-, O2-, and cytochrome P450 reductase-dependent regiospecific catabolism of heme to iron, alpha -biliverdin, and carbon monoxide (1). There are two well-established mammalian isoforms, the 288-residue inducible HO1 (2, 3) and the 316-residue constitutive HO2 (4), that share significant identity and have a common substrate, mechanisms, and products. The sequence of HO fails to exhibit significant identity to any previously structurally characterized enzyme, and hence, in the absence of a molecular model, the early research on the soluble enzyme focused on functional (5-13), mutational (5, 11), and spectroscopic (5, 14-18) investigations. The current understanding of HO is largely based on investigation of a recombinant, truncated 265-residue portion of HO1 from which the membrane-binding domain has been deleted, but which retains full activity (13). The mechanism of HO resembles that of cytochrome P450 in its ability to oxidize unactivated C-H bonds (19). However, in contrast to cytochrome P450 and the heme peroxidases, the action of HO does not pass through a ferryl intermediate (13, 20) but appears rather to involve a peroxo ligand capable of attacking the alpha -meso bridge.

Spectroscopic investigations agree on a normal iron-His (5-7, 14, 15) bond that more closely resembles that of Mb, with a neutral imidazole ligand, than that of the peroxidases, in which the axial ligand has significant imidazolate character (21). Mutational work identified His-25 as the axial ligand (5, 22). Whereas there is spectroscopic evidence for a distal titratable proton that interacts with exogenous ligands (7, 15) and hence potentially interacts with bound O2, it has not been possible to identify the distal base or its sequence origin. The structural basis for the remarkable stereoselectivity of HO, which exclusively excises the alpha -meso carbon, has been discussed in terms of both steric and electronic effects (8-10, 18, 23) but remains incompletely understood. Analysis of heme catabolism in Mb via the unrelated mechanism of coupled oxidation, which similarly leads primarily to alpha -biliverdin, has been proposed to result from differential steric access to the four meso positions (23). On the other hand, the influence of electronic withdrawing or electron-donating meso substitutions on the heme (9, 10) and the pattern of contact shifts of cyanide-inhibited, substrate-bound HO (18) suggest that electronic influences may also be operating.

Considerable progress has been made recently by solution of the crystal structure of a human HO-heme complex that reveals (24) a novel helical fold in which the heme is ligated near the surface by His-25. A schematic representation of active residues of interest here is shown in Fig. 1. The heme is wedged between two helices, with the distal helix exhibiting a significant kink that may be relevant to the entry of substrate and the exit of product. Prominent features of the active site, in contrast to the globins and peroxidases, are very close contacts between the heme and helical backbones in a fashion that appears to restrict access to all but the alpha -meso position (24). Hence a steric component to stereoselectivity has been clearly demonstrated. However, the structure identified no residues in the distal cavity that could serve as candidates for the titratable proton detected by UV-visible, resonance Raman (15), and EPR (17) spectroscopic analyses of the unligated HO-hemin complex. However, two observations suggest that the distal pocket may be very flexible and may be altered by relatively small perturbations in its environment. Thus, the unit cell contains two nonequivalent protein molecules (24) designated A and B that show a common proximal heme environment, but for which a portion of the distal helix (residues 139-147) on one side of the helix kink is 1-2 Å further from the heme in the B molecule than in the A molecule, and some side chain orientation differs significantly (i.e. Met-34). The two structures have been discussed as representing different degrees of cavity "closure" upon binding substrate (24). Moreover, numerous residues in the distal cavity of both molecules in the unit cell exhibited large thermal factors indicative of flexibility (24). Preliminary data on the rat HO-hemin complex reveal a structure very similar to that of human HO (hHO) (25).

1H NMR studies on hHO-hemin-CN prior to report of the crystal structure had located numerous aromatic residues and an Ala near the heme, showed that pyrrole C was exposed to solvent, and located a distal proton close enough to the iron to H-bond to the bound ligand (18). However, because of the large size of the enzyme, the spectral congestion due to the presence of significant equilibrium heme orientational disorder (16, 18), the absence of a homology model, and our inability to detect the crucial NH-Calpha H couplings required for sequence specific assignments, only the heme and axial His could be assigned. However, the unusual pattern of the dipolar shift for nonligated residues suggested that this complex exhibited a strong tilt from the heme normal to its magnetic axes (18), which can be correlated with the tilt of the Fe-CN unit from the heme normal (26-29). The recently determined crystal structure (24) now provides an invaluable guide to residue assignment that confirms the largely conserved solution heme cavity structure relative to that of the crystal but also brings into focus certain aspects of the structure that differ. These differences could result from the fact that the solution NMR has been carried out on the cyanide-ligated HO-hemin complex of the 265-residue truncated enzyme that is missing only the membrane binding tail (18), whereas the crystallography was carried out on a more extensively truncated 233-residue protein with water as ligand (24), or because different populations are observed in the solid state and in solution for an intrinsically flexible enzyme-substrate complex.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein Sample-- The 265-residue soluble portion of hHO was expressed and purified as described previously (30). Hemin was titrated into apo-hHO to a 1:1 stoichiometry in the presence of a 10-fold molar excess of KCN in a 90% H2O:10% 2H2O solution buffered at pH 8.0 with 100 mM phosphate. The final hHO-hemin-CN complex was ~2 mM. The solution was ultimately converted to 10% H2O:90% 2H2O in two steps of 70% 1H2O:30% 2H2O and 50% 1H2O:50% 2H2O using an Amicon ultrafiltration cell.

NMR Spectroscopy-- 1H NMR data were collected on a Bruker AVANCE 600 spectrometer operating at 600 MHz. Reference spectra were collected in both 1H2O and 2H2O over the temperature range 15 °C to 35 °C at a repetition rate of 1 s-1 using a standard one-pulse sequence with saturation of the water solvent signal. Chemical shifts are referenced to 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) through the water resonance calibrated at each temperature. Nonselective T1s were determined in both 1H2O and 2H2O at 20 °C , 25 °C, and 30 °C from the initial magnetization recovery of a standard inversion recovery pulse sequence. The distance of proton Hi from the iron, RHi, was estimated from the relation


<UP>R</UP><SUB><UP>H<SUB>i</SUB></UP></SUB>=<UP>R*<SUB>Fe</SUB></UP>[<UP>T<SUB>1</SUB>*/T<SUB>1i</SUB></UP>]<SUP><UP>1/6</UP></SUP><UP>,</UP> (Eq. 1)
using the heme for the alpha -meso-H for H* (R*Fe = 4.6Å and T1* = 50 ms) as reference (31, 32). NOESY (33) spectra (mixing time, 40 ms; 15 °C, 20 °C, 27 °C, 30 °C, and 35 °C) and Clean-TOCSY (34) spectra (27 °C and 30 °C, spin lock of 15 and 30 ms) using MLEV-17 were recorded over a bandwidth of 25 KHz (NOESY) and 14 KHz (TOCSY) with recycle times of 400 ms, 500 ms, and 1 s, using 512 t1 blocks of 128 and 250 scans each consisting of 2048 t2 points. Two-dimensional data sets were processed using Bruker XWIN software on a Silicon Graphics Indigo work station and consisted of 30° sine-squared-bell-apodization in both dimensions and zero-filling to 2048 × 2048 data points prior to Fourier transformation.

Magnetic Axes-- The location of the magnetic axes was determined by finding the Euler rotation angles, Gamma  (alpha , beta , and gamma ), that rotate the crystal structure-based, iron-centered reference coordinate system, x', y', and z', into the magnetic coordinate system, x, y, and z, where the paramagnetic susceptibility tensor, chi , is diagonal, i.e. (x, y, z) = (x', y', z') Gamma  (alpha , beta ,gamma ), where alpha , beta , and gamma  are the three Euler angles (26-29, 32, 35). Angle beta  dictates the tilt of the major magnetic axis, z, from the heme normal z' (Fig. 1B), angle alpha  reflects the direction of this tilt and is defined as the angle between the projection of the z axis on the heme plane and the x' axis (Fig. 1A), and kappa  ~ alpha  + bgamma is the angle between the projection of the x, y axes onto the heme plane and locates the rhombic axes (Fig. 1A). The magnetic axes were determined by a least square search for the minimum in the error function (26, 32, 35):
<UP>F/n</UP>=<LIM><OP>∑</OP><LL><UP>i=1</UP></LL><UL>n</UL></LIM>‖&dgr;<SUB><UP>dip</UP></SUB>(<UP>obs</UP>)−&dgr;<SUB><UP>dip</UP></SUB>(<UP>calc</UP>)‖<SUP>2</SUP> (Eq. 2)
where the calculated dipolar shift in the reference coordinate system, x', y', z', or R, theta ' Omega ', is given by:
&dgr;<SUB>dip</SUB>(<UP>calc</UP>)=(24&pgr;<UP>N</UP>)<SUP><UP>−1</UP></SUP>[<UP>2&Dgr;&khgr;<SUB>ax</SUB></UP>(<UP>3cos<SUP>2</SUP>&thgr;′</UP>−1)<UP>R<SUP>−3</SUP></UP>+ (Eq. 3)

3&Dgr;&khgr;<SUB><UP>rh</UP></SUB>(<UP>sin<SUP>2</SUP>&thgr;′cos2&OHgr;′</UP>)<UP>R<SUP>−3</SUP></UP>]<UP>&Ggr;</UP>(<UP>&agr;, &bgr;, &ggr;</UP>)
with Delta chi ax and Delta chi rh as the axial and rhombic anisotropies of the diagonal paramagnetic susceptibility tensor. The observed dipolar shift, delta dip(obs), is given by:
&dgr;<SUB><UP>dip</UP></SUB>(<UP>obs</UP>)<UP> = &dgr;<SUB>DSS</SUB></UP>(<UP>obs</UP>)<UP> − &dgr;<SUB>DSS</SUB></UP>(<UP>dia</UP>) (Eq. 4)
where delta DSS(obs) and delta DSS(dia) are the chemical shifts (in ppm), referenced to DSS, for the paramagnetic hHO-hemin-CN complex and an isostructural diamagnetic complex, respectively. In the absence of an experimental delta DSS(dia), it may be reliably estimated from the available molecular structure:
&dgr;<SUB><UP>DSS</UP></SUB>(<UP>dia</UP>)<UP> = &dgr;</UP><SUB><UP>tetr</UP></SUB>+&dgr;<SUB><UP>sec</UP></SUB>+&dgr;<SUB><UP>rc</UP></SUB> (Eq. 5)
where delta tetr, delta sec, and delta rc are the chemical shifts of an unfolded tetrapeptide relative to DSS (36) and the effect of secondary structure (37) and ring currents (38) on the shift, respectively.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Assembly of the hHO-Hemin-CN Complex-- Addition of hemin to apo-hHO in the presence of cyanide yields the 600 MHz 1H NMR spectra illustrated in Fig. 2. Immediately after assembly in 1H2O, three distinct species with methyl peaks m3, M3, and m* are observed, with m3 and M3 exhibiting comparable intensity (Fig. 2A). The new intermediate with peak m* has not been reported previously (18) and converts within a few days into the species with M3 (Fig. 2, A and B). Previous assignments by isotope labeling in rat HO-hemin-CN (16) and two-dimensional NMR on hHO-hemin-CN (18) had assigned M3 to the 3-CH3 peak of the complex with the heme in the orientation shown in Fig. 1A and m3 to the 3-CH3 of the complex with the heme rotated by 180° about the alpha -gamma -meso axis. Over a period of 3 weeks, equilibrium is reached where the species with m* is absent (Fig. 2C), and the M3:m3 ratio of intensities is ~3.5:1.0. Noteworthy observations are that, in 1H2O, the peaks for the alternate, minor (m3 and m28beta ) isomers are significantly broader than those of the major (M3,M28beta ) isomer (Fig. 2, A and B) and that upon exchange from 90% 1H2O to 10% 1H2O, the lines for this minor isomer become significantly narrower (Fig. 2, C-E). Comparison of the 1H2O and 2H2O NMR spectra in Fig. 2, C and E, reveals two strongly low-field shifted labile protons for the major component, labeled a and b; peak a exhibits full proton intensity for the major isomer, whereas peak b exhibits ~0.5 proton intensity (for the major isomer) due to ~50% saturation transfer from bulk water (as confirmed in 1:1 1H NMR spectra with and without H2O saturation) (18, 39) that is essentially independent of pH in the range 8.0-9.5, and temperatures in the range of 10 °C to 30 °C. The remainder of the study addresses the properties of the major isomer, and only brief reference will be made later as to the nature of the minor equilibrium isomer.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 1.   A, schematic representation of heme contact residues of interest in the heme pocket of substrate-bound human heme oxygenase, viewed from the proximal side, as found in the crystal structure (24) but with the heme rotated 180° about the alpha ,gamma -meso axis. The residues Thr-21, Val-24, Thr-23, Thr-26, Ala-28, and Glu-29 reside on the proximal helix, whereas residues Tyr-134, Thr-135, Leu-138, Gly-139, Ser-142, and Gly-143 reside on the distal helix. Proximal, distal, and peripheral residues are shown as rectangles, circles, and triangles, respectively. B, edge on view that illustrates the connection among 4 key aromatic residues in the distal pocket. The double-sided arrows indicate heme-residue and intra-residue dipolar connections detected by NMR in the cyanide-inhibited derivative. The axes x', y' in A and z' in B designate the reference coordinate system, as defined by the crystal structure. The magnetic axes x, y (in A) and z (in B) define the magnetic coordinate system in which chi  is diagonal. The Euler angles alpha  in A (the angle between the projection of the z axis on the x', y' plane and the x' axis), beta  in B (tilt of the major magnetic axis from the heme normal), and kappa  in A (= alpha  + gamma ) represent the direction of tilt, the magnitude of the tilt from the heme normal of the Fe-CN vector, and the location with rhombic magnetic axes in the heme plane, respectively (related to the orientation of the axial His imidazole plane, which is given by phi  in A).


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 2.   Resolved portions of the 600 MHz 1H NMR spectra of hHO-hemin-CN at the following time points: (A) immediately after assembly in 1H2O at 25 °C, revealing three species with low-field heme methyl peaks M3, m3, and m*, (B) 2 days later at 25 °C, showing a factor ~3 decrease in peak m* and a factor 2 decrease in peak m3 intensities, and (C) 3 weeks later in 1H2O at 30 °C, where peak m* has disappeared, and an equilibrium M3:m3 intensity ratio of 3.5:1.0 is reached. The effect of converting the sample solvent from 90% 1H2O:10% 2H2O to (D) 50% 1H2O:50% 2H2O at 30 °C and (E) 10% 1H2O:90% 2H2O is to decrease the intensity of labile proton peak a and to significantly narrow the minor compound peaks m3, m28beta , and h2beta s. The sample was maintained in 50 mM phosphate buffer at nominal pH ~8. The peaks are labeled Mi, Hi (methyl single proton), and mi,hi for the major and minor equilibrium forms, respectively, with i as either the heme position 1-8 (pyrrole, i.e. M3 = 3-CH3), alpha -delta (meso-H, i.e. Ha = alpha -meso-H), or the residue number and proton (M28A = Ala-28 Cbeta H3).

Assignment Protocols-- The large size and helical nature of the protein, paramagnetic relaxation, and the presence of dynamic molecular heterogeneity (see below) allow the detection of very few of the backbone NH-Calpha H TOCSY peaks required for standard sequence-specific assignments of the active site (40). However, because it is now known that the overwhelming majority of active site residues are on alpha -helices (24, 25), we use the expected (40) characteristic strong intra-residue Ni-alpha i, backbone inter-residue strong Ni-Ni+1, beta i-Ni+1, moderate alpha i-Ni+3, alpha i-beta i+3, and weak alpha i-Ni+1 NOESY cross-peaks to locate and assign numerous residues. The starting premise is that the solution structure is very similar to the crystal structure, and the differences between the two are more likely to be due to small to moderate readjustments of the positions of some residues than to large changes in the heme-residue and heme cavity intra-residue interactions. Hence, we use the crystal structure to guide assignments, in every case using the accessible TOCSY cross-peaks to confirm spin topology and paramagnetic relaxation to gauge proximity to the iron.

Previous 1H NMR in 2H2O at lower resolution had located all individual heme signals, although only a single chemical shift was detected for Hbeta s of the two propionates (18). Here we can resolve the individual Hbeta s and confirm all previous heme assignments. The only sequence-specific assignment offered previously was for the axial His-25 Calpha HCbeta H2 fragment (16, 18), which could be assigned because of the low-field contact shifts unique to this fragment in all low-spin ferric His-ligated heme proteins (32, 41). Assignments are most conveniently initiated for the proximal side that is essentially identical in the two molecules in the unit cell (24). Upon confirming a conserved solution structure for the proximal site, the magnetic axes are determined. The subsequently predicted dipolar shifts, together with relaxation data for the distal residues, and the crystal structure are then used to establish the degree and manner in which the distal site in solution resembles that for either of the two molecules in the unit cell. However, we have indicated previously (18) and definitively confirm herein that the heme of the 265-residue recombinant HO-hemin-CN in 75-80% of the molecules in solution is rotated by 180° about the alpha -gamma -meso axis with respect to the unique orientation found in the crystal structure of the more truncated 233-residue fragment (24) or the similarly truncated rat HO-hemin complex (25). Hence contacts predicted on the basis of the crystal structure are corrected for the rotated heme position, i.e. for residue Xright-arrow8-CH3 (5-CH3) reflects an expected dipolar connection between residue X and 5-CH3 in the crystal but is observed to the 8-CH3 in solution.

Scalar Connections-- TOCSY spectra (34) to support the residue assignments are provided as supplemental material (Figs. 1S and 2S). The previously detected, strongly upfield shifted CH3-CH fragment (labeled Ala S previously) (18) is here extended to a labile proton to uniquely identify an Ala, and another CH3-CH (previously labeled residue R) (18) is extended to another non-labile proton to identify a CH3-CH-CH fragment shown to result from Thr-135. A strongly low-field shifted, single proton is extended to include four additional protons for a Calpha HCbeta H2Cgamma H2 (previously residue J) (18) fragment later identified as a part of Lys-22, and a CH3-CH-OH fragment is observed that will be shown to represent a portion of Thr-21. A CH3CHCH fragment is detected with weak low-field shifts that will be uniquely traced to Thr-26. In the upfield portion of the spectra, the spin connectivity for a complete Leu, including its NH, is observed with strongly up-field shifted Cbeta Hs that can be shown to arise from Leu-138, and a five-spin system is observed with a moderately relaxed and strongly upfield shifted Cgamma H later shown to be due to Glu-29. A complete, weakly low-field shifted AlaX residue (including NH) was also detected.

Relaxation Properties-- Recovery of the magnetization after a 180° pulse led to estimates of T1s for resolved and partially resolved resonances in 2H2O and 1H2O. Heme resonances yielded the expected pattern (T1s) 3-CH3 (130 ms), 8-CH3 (170 ms), 2-Halpha (160 ms), 2Hbeta c(~200 ms), 4Halpha (160 ms), 6Halpha (150 ms), 7Halpha (150 ms), and alpha -meso-H (50 ms) that reflects their positions relative to the iron (32). The heme pocket residues characterized include labile proton b (50 ms), Phe-207 Cepsilon Hs (~80 ms), Ser-142 Cbeta 1H (85 ms), Ser-142 Cbeta 2H (50 ms), Glu-29 Cgamma 1H (~80 ms), Gly-143 Calpha H (~80 ms), and Ala-28 Cbeta H3 (200 ms). The paramagnetic contribution to the relaxation of heme substituents is small to negligible for all pyrrole substituents. However, the alpha -meso-H (R*Fe = ~4.6 Å) is dominated by paramagnetic relaxation and provides a convenient reference for using Equation 1 to estimate R*Fe for residue protons.

The Proximal Helix-- Convenient entry points to the proximal helix are the previously assigned His-25, Glu-29 with a backbone NH contact to the heme 2Halpha (3-CH3), and another, Thr-21, with a side chain OH contact with the heme 5-CH3(8-CH3), as well as the complete Ala-28 with expected strong contact with the pyrrole A/B junction. Indeed, the NH of Ala-28 (previously labeled Ala S (18)) is part of a series of strong Nj-Nj+i NOESY cross-peaks in a six-member helical fragment (40) Nj . . . . .-Nj+5 (shown by the arrows in Fig. 3E), with Ala-28 as j+4. In agreement, the Nj+5 (N29) exhibits the expected NOE to 3-CH3 (Fig. 3, A and F) and to the upfield Calpha HCbeta H2Cgamma H2 fragment (Fig. 4B), which, in turn, exhibits NOESY cross-peaks to 3-CH3 (Fig. 4K) and 4Halpha (Fig. 4I) that conclusively identify Glu-29. The Nj+1 (N25), as expected for His-25 NH, exhibits the NOESY cross-peak to the previously assigned (18) His-25 Calpha HCbeta H2 fragment (Fig. 3, C and E), and the TOCSY-detected Calpha HCbeta HCgamma H3 fragment exhibits the NEOSY cross-peak to Nj+2 (N26) characteristic of Thr-26 (Fig. 3E). The Calpha Hs of Val-24 and Gln-27 could not be located. However, N24, in common with N25, exhibits NOESY cross-peaks to two methyls (Fig. 3B) that the crystal structure uniquely assigns to the Val-24 Cgamma H3s. A strong NOESY cross-peak from the Calpha H of the low-field shifted Calpha HCbeta H2Cgamma H2 fragment (previously residue Q (18)) to the His-25 Cbeta Hs (Fig. 3, D and E) identifies the former as a portion of the side chain of Lys-22. The assignments to the proximal helix are confirmed by the detection of the expected (40) alpha i-Ni+3 and alpha i+beta i+3 connection for i = 25 (data not shown) and 26 (Fig. 3B) and alpha 22-beta 25 cross-peaks (Fig. 3E); the latter connections also provide stereospecific assignment of the His-25 and Glu-29 Cbeta Hs. Lastly, the labile proton of the TOCSY-detected CH3CH-OH fragment exhibits a NOESY cross-peak to 8-CH3 (Fig. 3E), as expected for the Thr-21 to 5-CH3 contact in the crystal structure (24). It was not possible to obtain any direct evidence for the side chain protons of either Asp-23 or Gln-27, largely because they are expected to be neither relaxed (RFe > 11Å) nor dipolar-shifted (see below). The observed characteristic dipolar contacts that provide the assignments of proximal helical residues are summarized in Fig. 5.


View larger version (74K):
[in this window]
[in a new window]
 
Fig. 3.   Portions of the 600 MHz NOESY spectrum (mixing time = 40 ms) of hHO-hemin-CN in 90% 1H2O:10% 2H2O at 27 °C illustrating the sequence of Nj-Nj+1 connections (via arrows) for the 6-residue proximal helical fragment Val-24-Glu-29 and the 4-residue distal helical fragment Leu-138-Asp-141 (E). Also shown are crucial beta i-Ni+1, alpha i-Ni+3, and alpha i-beta i+3 connections (A-D) that confirm that helical nature of the fragments. Also shown are the expected dipolar connections between the two helical labile protons and 8-CH3 (Leu-138, Gly-139, and Thr-21; E) and 3-CH3 (Glu-29; F). Both the NH of Glu-29 and Nepsilon H of Glu-38 that are degenerate at 27° exhibit NOEs to 3-CH3 in F; the distinct NOESY cross-peaks are readily resolved at lower temperature. The heme signals are labeled Mi, Hi (where i = 1-8 for pyrrole), and alpha -delta for meso substituents, and residue signals are labeled solely by the sequence number and the position of the proton in the side chain; the peptide NH is labeled solely by residue number.


View larger version (93K):
[in this window]
[in a new window]
 
Fig. 4.   Portions of the 600 MHz 1H NMR NOESY spectrum (mixing time = 40 ms) at 27 °C illustrating the side chain contacts between the heme and Glu-29 (B, C), Gln-38 (G, J), Thr-135 (B, E, I), Leu-138 (B, C, E, F, I), Tyr-134 (A, B, F, G), and Gly-139 (D, G, H). Both the NH of Glu-29 and Nepsilon H of Glu-38 that are degenerate at 27° exhibit NOEs to 3-CH3 in J; the distinct NOESY cross-peaks are readily resolved at lower temperature. Peaks are labeled as described in the Fig. 3 legend.


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 5.   Schematic summary of observed Ni-Ni+1, alpha i-Ni+1, beta i-Ni+1, alpha i-Ni+3, and/or alpha i-beta i+3 NOESY cross-peak patterns in support of the helical fragments (40) Val-24-Glu-29 and Leu-138-Gly-143 (the relative widths of the bars reflect the relative cross-peak intensities).

The confirmed axial His-25 Cbeta H and Calpha H have been shown (18) to exhibit NOESY cross-peaks to a TOCSY-detected residue AromB (18) (a third spin is detected at 600 MHz) aromatic ring that is also in contact with 8-CH3 (Fig. 3E) and 1-CH3 (data not shown). This ring also exhibits a NOESY cross-peak to the Ala-28 Calpha H (data not shown) and Cbeta H3 (Fig. 3A) and is uniquely identified as Phe-207 in the crystal structure with a 180° rotated heme. The T1 of ~80 ms for the two-proton peak at 5.9 ppm identifies it as the more relaxed Cepsilon Hs closer to the iron in the crystal structure. The Thr-21 Cbeta HCgamma H3 fragment identified above exhibits the expected significant intensity NOESY cross-peaks to the Phe-207 ring (Fig. 4H).

Determination of Magnetic Axes-- Using the assigned proximal side residue protons (listed in Table I) with significant dipolar shifts as constraints, the crystal coordinates of hHO-hemin-H2O (24), and the magnetic anisotropies of isoelectronic metMbCN (42), Delta chi ax = 2.48 × 10-8 m3/mol and Delta chi rh = -0.58 × 10-8 m3/mol, as input, the orientation of the magnetic axes was determined (26) by the three-parameter (alpha , beta , and gamma ) least square search for the minimum in the error function in Equation 2. The results yield alpha  = 234 ± 4, beta  = 20 ± 2, and kappa  = alpha  + gamma  = 25° ± 13° when using the crystal molecule A coordinates and alpha  = 238 ± 11, beta  = 19 ± 3, and kappa  = alpha  + gamma  = 11° ± 14° when using the crystal B molecular coordinates. Both result in acceptably low residual error functions and good correlation between delta dip(obs) and optimized delta dip(calc) in Table I, as shown in Fig. 6, A and B. The direction and magnitude of kappa  that defines the rhombic axes are qualitatively consistent with the expectation (43) based on the orientation of the axial His-25 imidazole orientation angle phi  ~40°. The difference in the angles for molecules A and B is well within the uncertainties of their determination, such that it can be concluded that the magnetic axes are essentially the same for molecules A and B. However, differences between the two molecules are readily evident for the distal side (see below). The two sets of magnetic axes agree that significant (>1 ppm) upfield delta dip are expected only for the distal Thr-135, Leu-138, Gly-139, and Ser-142, whereas significant (>1 ppm) low-field delta dip are expected only for distal Asp-140, Gly-143, Gly-144, and Leu-147. The detailed predicted delta dip values are listed in Table I. Differences in delta dip(calc) for the two molecules in the unit cell will be considered when the relevant residue is assigned.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Chemical shift data for hHO-hemin-CN


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 6.   Plot of delta dip(obs) versus delta dip(calc) for the optimized magnetic axes determined in Equations 1-3 from the data on the proximal side residues given in Table I, using the coordinates for (A) molecule A and (B) molecule B in the hHO-hemin-H2O crystal structure, each shown as filled circles. The predicted delta dip(calc) versus delta dip(obs) for the distal residues not used as input data are shown as filled triangles in C for molecule A and filled triangles in D for molecule B in the crystal structure. The straight line reflects a perfect fit.

Distal Helix-- Convenient entry points to the distal helix suggested by the crystal structure (24) are the expected strong contacts of two backbone NHs (Leu-138 and Gly-139) and the Calpha H of Thr-135, each with the 5-CH3(8-CH3). NOESY spectra reveal the Nk-Nk+1 cross-peaks characteristic (40) of a four-member helical fragment Nk-Nk+3, for which Nk and Nk+1 exhibit NOESY cross-peaks to 8-CH3 (Fig. 3E). Nk is part of the complete TOCSY-detected Leu and hence identifies the first two members of this fragment as Leu-138 and Gly-139. TOCSY failed to detect the Calpha Hs of Gly-139, but a NOESY cross-peak from N139 to a moderately relaxed (~80 ms) but very broad upfield shifted proton on the high-field shoulder of the diamagnetic envelope (Fig. 4H) provides a strong candidate for Calpha 1H. This assignment is confirmed upon assignment of Ser-142 below. The expected alpha 138N139 cross-peak (Fig. 4D) confirms the direction of the helical fragment. The side chain of Asp-140 (whose Calpha H should exhibit a significant low-field delta dip; see Table I) and Leu-141 (which does not exhibit delta dip) were not located. The Leu-138 side chain is expected to make extensive dipolar contact to 5-CH3 (8-CH3) and 6Halpha s (7Halpha s), and such contacts are observed between the moderately relaxed Cbeta Hs and 8-CH3 (Fig. 4I) and 7Halpha (Fig. 4H). The two Cbeta Hs are assigned stereospecifically based on the stronger relaxation of Cbeta H and the observed beta '138-N139 NOESY cross-peak. Whereas both Leu-141 and Leu-147 are expected to exhibit NOEs to 3-CH3 (2Halpha ), and numerous unassigned cross-peaks are observed (Fig. 4K), spectral congestion precluded the detection of the TOCSY cross-peak necessary to define the corresponding spin system. The detected backbone NOESY cross-peaks characteristic of the distal helix are summarized in Fig. 5B.

TOCSY spectra extend a previously located low-field shifted CH3-CH (residue R (18)) to a CH3-CH-CH fragment, of which the terminal single proton exhibits a very intense NOESY cross-peak to the 8-CH3 (Fig. 4I) and 1-CH3 (data not shown) as expected for the Thr-135 Calpha H to 5-CH3(8-CH3) in the crystal, thus identifying Thr-135 (its NH could not be detected). The assignment is confirmed by the alpha 135N138 cross-peak (seen weakly in Fig. 4D but more clearly at a lower temperature (data not shown)) expected for the distal helix (Fig. 5B). The previously located (18), two-spin aromatic ring in contact with both Leu-138 and 8-CH3 (Fig. 3E) is thus uniquely assigned to the Tyr-134 ring (residue G with AromB(Phe-207) part of aromatic cluster 4C (18)). A labile proton c, which is in contact with the Tyr-134 ring (Fig. 4F) and the 7Halpha and 7Hbeta s (Fig. 4A), is that expected for the Tyr-134 ring OH.

The strongly relaxed (T1 = ~50 ms, RFe = ~4.6 Å, T1 = ~85 ms, RFe = ~5.6 Å) and upfield shifted, TOCSY-connected (18) CH2 fragment (residue U (18)) exhibits NOESY (but not TOCSY) cross-peaks to an upfield shifted proton at 2.5 ppm (Fig. 7B), its likely Calpha H, and to a labile proton peak e at 8 ppm (Fig. 7A), its likely NH, and to the 6-propionate H6alpha s (see supplemental material (Figs. 1S and 2S)). The 2.5 ppm peak similarly exhibits an NOE to the labile proton peak e at 8 ppm (data not shown). The obvious distal location (see below) of this strongly relaxed and upfield shifted residue is consistent only with its attribution to Ser-142, with the Calpha H at 2.5 ppm. The nearly identical T1s (~50) for alpha -meso-H and Ser-142 Cbeta 2H argue for very similar RFe = ~4.6 Å, and this is consistent with the distance in either molecule A (4.7 Å) or molecule B (4.6 Å) (24). The longer T1 of ~80 ms for Ser-142 Cbeta 1H, moreover, is again consistent with larger RFe in both molecules A (~5.8 Å) and B (5.6 Å), and this differential relaxation assigns the Ser-142 Cbeta Hs stereospecifically. Weak NOESY cross-peaks are detected between the Ser-142 Cbeta Hs and the proposed Calpha H of Gly-139 (data not shown; see supplemental material (Figs. 1S and 2S)), as expected (Fig. 5B).


View larger version (63K):
[in this window]
[in a new window]
 
Fig. 7.   Portions of the 600 MHz 1H NOESY spectrum (mixing time = 40 ms at 30 °C) illustrating contacts involving Ser-142, Gly-143, the three labile protons (a, b, and d), and the cluster of four aromatic rings labeled PheA (Phe-167), PheD (Phe-166), AromC (Tyr-58), and AromE (Tyr-137). The individual protons of a ring are labeled arbitrarily (i.e. PheA with ring protons A1, A2, and A3).

The low-field labile proton b in Fig. 2 is too strongly relaxed (T1 = ~50 ms) to exhibit any TOCSY cross-peaks. It does, however exhibit numerous NOESY cross-peaks (Fig. 7D) to the 5-CH3 (weak), to the proposed Ser-142 labile proton e (moderate), to two protons of the TOCSY-detected PheA (peaks A1and A2), to NH of Asp-141, and to two relaxed, non-labile protons at 7.6 and 7.8 ppm, each of whose shift moves strongly to lower field as the temperature is lowered (data not shown). This clearly identifies labile proton b as arising from a distal residue. The NOE to the labile e proton in common with Ser-142 Cbeta Hs confirms the distal placement of the latter residue as well. The lower field (and possibly also the higher field) of these two non-labile protons exhibits NOESY cross-peaks to the 5-CH3 (Fig. 7C). The strong dipolar coupling among b and the two non-labile protons at 7.8 and 7.6 ppm and the proximity to the 5-CH3 and to the iron, together with the large low-field delta dip (predicted by either magnetic axes), dictate the origins of proton b as the Gly-143 NH. The similar T1 (50 ms) of b (Gly-143 NH), Cbeta 2H of Ser-143 (T1 = ~50 ms, RFe = ~4.6 Å), and alpha -meso-H (50 ms, RFe = ~4.6 Å) dictates a similar (4.7 Å) distance from the iron. The backbone inter-residue NOESY contacts, which support the assignment of a portion of the distal helix, are summarized schematically in Fig. 5.

Peripheral Residues-- Two other labile protons exhibit strong NOESY cross-peaks to the 3-CH3 (Fig. 4J) and to each other (Fig. 4G), but to no other labile protons. The strong cross-peak between them indicates that the NH2 group of Gln-38 is located near pyrrole A(B) in the crystal structure. The assignment to Gln-38 is confirmed by the detection of the expected NH2 NOEs to the Glu-29 Cbeta Hs (data not shown). The previously characterized Phe ring (PheJ) (18) in contact with the 2-vinyl (strong), 3-CH3 (weak), and Phe-28 is due to Phe-214, which is in close contact with the 3-CH3 in the crystal.

Predictions in delta dip for Distal Residues-- The results of predicting delta dip(calc) for the assigned distal residues, using the magnetic axes according to either molecule A or B, and the comparison with delta dip(obs) are illustrated in Fig. 6, C and D, respectively. For molecule A, all assigned distal protons are extremely well predicted, except for the NH and Calpha H of Gly-143 (Fig. 6C). In the case of molecule B, the protons for neither Ser-142 nor Gly-143 are well predicted (Fig. 6D). Hence the distal structure for the beginning of the helix, including residues through residue Gly-139, can be assumed to be the same in solution as it is in both molecules A and B in the unit cell. Conversely, both Ser-142 and Gly-143 appear to differ in position for at least one of the two molecules in its unit cell. The inclusion of distal residues delta dip(obs) for residues Tyr-134 to Gly-139 in the least square search to redetermine the magnetic axes leads to magnetic axes that are insignificantly different from those obtained using only proximal residue data (see supplemental material (Figs. 1S and 2S)).

The Distal Aromatic Cluster and Labile Proton a-- In the crystal structure (24), four aromatic rings interact strongly pairwise in the distal pocket some ~10-11 Å from the iron, in the pattern Phe-166 left-right-arrow Phe-167 left-right-arrow Tyr-58 left-right-arrow Tyr-137. The magnetic axes for either molecule A or B predict moderate (~1-2 ppm) low-field delta dip for the two Phe (and a large, upfield ring current shift for one) and small to negligible delta dip for the two Tyr, as observed (Table I). NMR had originally detected these four aromatic rings (cluster 4A (18)) and their interactions, PheAleft-right-arrow PheD and AromCleft-right-arrow AromE, with only the weakest NOESY cross-peak visible between PheA and AromC. The pattern of NOEs and shifts clearly dictates that the residues can be confidently assigned to Phe-166, Phe-167, Tyr-58, and Tyr-137 for rings D, A, C, and E, respectively. However, the interaction among the rings for at least the two Tyr in solution differs significantly from that in the crystal (24). First, because the Phe-166(D)left-right-arrow Phe-167(A) NOESY cross-peak intensities are consistent with the relative disposition of the two rings in the crystal structure, the much weaker Phe-167(A)-Tyr-58(C) NOESY cross-peak must result from a reorientation of the latter ring away from Phe-167, which brings it closer to Tyr-137(E). The altered nature of the Tyr-58(C) and Tyr-137(E) intensities is documented in Fig. 7C. The extreme low-field labile proton peak a exhibits a strong NOE to another labile proton, proton d, at 9.6 ppm and relatively strong NOEs to the rings of both Tyr-58(C) and Tyr-137(E) (Fig. 7E), as well as a moderate NOE to the Calpha H of Ser-142 (Fig. 7F). The strong NOESY cross-peak between the two labile protons, a and d, each with close contact to the rings of Tyr-58(C) and Tyr-137(E), strongly suggests that their origin is the interacting hydroxyls of the two Tyr, Tyr-58 and Tyr-137, that participate in strong hydrogen bonds.

Such an interaction between a pair of Tyr is not detected in the crystal structure (24). Qualitative modeling suggests that the interaction could take place between Tyr-58 and Tyr-137, if the former rotates chi 1 by 20-25°, and both rings reorient somewhat. We clearly detect weak to moderate NOESY cross-peaks between proton b (the Gly-143 NH) and the ring of Phe-167(A) (Fig. 7D), despite >= 7 Å separations indicated in the crystal structure (24). Because the Gly-143 NH is indicated to be at the crystallographic distance to the iron (RFe = ~4.6 Å), it is likely that the whole aromatic cluster has moved slightly closer to the iron. Such a movement is also suggested by the observation of Phe-167(A) and Tyr-58(C) ring NOEs (Fig. 7C) to the Calpha H of a complete AlaX (NH, Calpha H, and Cbeta H3 shifts of 7.45, 5.45, and 1.58 ppm, respectively) for which there are no obvious candidates in the crystal. Hence, more detailed modeling of the Tyr-58-Tyr-137 interaction was considered unproductive. The T1 of peak a is >250 ms, and it exhibits an essentially temperature-independent shift unaffected by paramagnetism and indicative of a strong hydrogen bond. The strength and importance of the H bond involving the two Tyr ring hydroxyls is manifested in two novel observations. First, peak a is split in 1H2O/2H2O solution mixtures, with the relative intensity parallel to the solvent isotope composition (as shown in Figs. 8, A-C), such that the strength of the H bond involving proton a depends on the isotope for proton d. Secondly, the NOESY cross-peaks for both split a peaks exhibit NOESY cross-peaks to Tyr-58 and Tyr-137 rings, but, as clearly shown in Fig. 8, D and E, both the Tyr-58 and TyrE ring protons have slightly smaller low-field shifts when hydrogen d is occupied by a 2H as compared with a 1H.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 8.   The effect of the solvent isotope composition of hHO-hemin-CN, 50 mM phosphate, pH 8, and 30 °C on the position and intensity of labile proton a in (A) 90% 1H2O:10% 2H2O, (B) 50% 1H2O:50% 2H2O, and (C) 10% 1H2O:90% 2H2O; note the splitting of the peak a in mixed solvent and the correlation between relative intensities of the split components and solvent isotope composition. Portions of the NOESY spectrum (mixing time = 40 ms) in 50% 1H2O:50% 2H2O and cross-peaks between the split components of peak a and ring Cepsilon Hs of (D) Tyr-58(C), (E) Tyr-137(E), and (F) labile proton d are shown. Note that the ring protons are slightly further to low-field when d is occupied by 1H than when d is occupied by 2H.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Structural Characterization-- Solution NMR has proven reasonably effective in identifying the majority of the residues in the substrate binding cavity, despite the fact that the crucial fingerprint peaks needed for de novo sequence-specific assignments (40) were very elusive. Whereas the spin type of numerous residues was correctly identified prior to the availability of the crystal structure, only a single residue could be sequence-specifically assigned (18), and one crucial distal residue was suggested to arise from a Tyr, based on the chemical shifts in the aromatic window, but is now known to arise from a strongly low-field dipolar shifted Gly-143. Hence the availability of the crystal structure of the water-ligated complex is an essential component of any quantitative NMR analyses. The combination of the crystal coordinates (24), together with the remarkably accurately predicted delta dip based on the magnetic axes generated from the assigned proximal helix (see Table I and Fig. 6), allowed the detection of the NOESY peaks to many of the expected distal residue protons with the appropriate paramagnetic relaxation and delta dip. In this respect, the magnetic axes and predicted delta dip were similar for the distal residues Tyr-134-Gly-139 defined by the alternate molecules A and B in the unit cell. The success of the assignments is a strong confirmation of the approach in which magnetic axes generated interactively with reasonably robust assignments allow a definition of the structure that could not have been achieved on the basis of scalar and dipolar correlations alone. It is reasonable to expect that using the delta dip values as constraints, a more quantitative description of the active site is attainable once NOESY rise curves have allowed quantitative estimates of internuclear distances.

The inefficiency in detecting TOCSY peaks is surprising, because the helical segments of both isoelectronic but bigger 44-kDa HRP-CN (44) and 65-kDa metHbCN (45) have yielded the necessary fingerprint cross-peaks. The difficulty with hHO-hemin-CN is likely due to line broadening from the dynamic averaging of more than one unique structure in solution (see "Dynamic Properties of the Pocket"). Dynamic heterogeneity, moreover, is the likely origin for our failure to detect the signal for the functionally important Gly-144 and Leu-147 (see below). Planned NMR studies with uniform 15N-hHO may provide these as well as other additional assignments.

Similarities in the Solution and Crystal Active Site Structures-- The pattern of intra-residue and heme-residue NOESY cross-peaks for the proximal helix residues Lys-22-His-25, Thr-26, Ala-28, Glu-29, Phe-207, and Thr-21 are those predicted by the essentially identical proximal structures of either molecule in the unit cell (24). The excellent definition of the magnetic axes (see Fig. 6, A and B) by proximal residues is additional support for conserved proximal side structure. The distal side residues of the helix, Tyr-134-Gly-139, in solution exhibit the heme-residue and inter-residue dipolar contacts predicted by the very similar structures for either the A or B molecule in the crystal (24). Moreover, the delta dip values for these residues are exceptionally well predicted by the magnetic axes (see Table I and Fig. 6), such that it is reasonable to conclude that the position of the beginning of the distal helix through at least Gly-139 in solution is conserved. Whereas the NHs of neither Tyr-134 nor Thr-135 could be located, the NHs of Leu-138 and Gly-139 are clearly detected for a few days after transferring the protein into 2H2O, indicating that this portion of the distal helix is both well formed and dynamically stable.

Differences in Solution and Crystal Active Site Structure-- The most obvious difference between the active site structure in solution of the 265-residue hHO-hemin-CN complex and the 233-residue hHO-hemin-H2O complex in the crystal (24) is that the heme orientation is unique in the crystal, whereas the solution reflects an equilibrium between two heme orientations differing by 180° rotation about the alpha -gamma -meso axis, with the ratio of isomers of ~3.5:1. Most importantly, the crystallographic heme orientation corresponds to the solution minor component. Unfortunately, the minor isomer in solution could not be characterized to the same detail as the major isomer due in part to its lower population but primarily because its signals are broadened severely by a dynamic equilibrium heterogeneity (see below). A 180° rotation of the heme about the alpha -gamma -meso axis in solution (46) relative to that in the crystal (47) has been documented for Chironomus thummi thummi cyano-methemoglobin. The formation of crystals with the unique heme orientation of the minor isomer in solution could result from a lower solubility of the minor isomers for both cases.

The two molecules in the unit cell differ primarily in the position of the portion of the distal helix starting with Ser-142, as the following two residues, Gly-143 and Gly-144, have been proposed to serve as a "hinge" in the broken distal helix that allows substrate entry and product release (24). In general, this position of the distal helix is closer to the heme plane in molecule A relative to that in molecule B but translated somewhat away from the iron toward the crystallographic pyrrole C (and hence toward pyrrole D in the solution heme orientation). The fact that the relaxation properties of Ser-142 are well predicted by either the A or B molecule, whereas the delta dip values are somewhat better predicted by molecule A than B (compare Fig. 6, C and D), indicate that the solution position of Ser-142 is closer to that in molecule A than that in molecule B. The Gly-143 NH exhibits relaxation (T1s = ~50 ms, RFe = ~4.6 Å) consistent with the expectation for molecule A (RFe = ~4.7 Å) but not molecule B, in which it is further (RFe = ~5.7 Å) from the iron, confirming the close approach of this position of the distal helix to the heme in the complex in solution.

However, the delta dip(obs) for the Gly-143 NH is much larger than that predicted by either molecule A or B (Fig. 6, C and D; Table I). The small delta dip(calc) is due to the position of Gly-143 NH near the magic angle of the magnetic axes where delta dipright-arrow0. The much larger delta dip(obs) for Gly-143 (~9 ppm), with conserved RFe of ~4.6 Å relative to the crystal structure, would result in the residue being located ~1-2 Å closer to the heme center, placing the NH within the strong, low-field part of the dipolar field reflected in delta dip(obs). Such a small translation of the distal helix would still result in a sterically hindered access to beta -, gamma -, and delta -meso positions, as found in the crystal. Quantitative modeling of this movement was not considered practical in the absence of detection and assignment of residue Asp-140, Leu-141, Gly-144, and Leu-147. The failure to detect these residues, despite their large low-field dipolar shifts (Table I), may not be a simple experimental failure. Rather, it is likely that these signals are extensively broadened by a dynamic molecular heterogeneity that reflects two interconverting distal structures. The close proximity of both Gly-139 and Gly-143 to the heme iron in solution, as found in the crystal (24), provides a rationalization for the loss of oxygenase activity for both and conversion to peroxidase activity upon mutating the latter residue (48).

Distal Aromatic Cluster-- The distal aromatic cluster of Phe-166, Phe-167, Tyr-58, and Tyr-137 is too far removed from the heme iron to directly participate in the catalytic action of the enzyme (24). However, it could provide important structural stabilization of the distal helix to facilitate the steric influence of this helix on the stereospecificity of the reaction. It is obvious that some rearrangements of aromatic side chains in the crystal structure are necessary to rationalize the NOESY pattern for the 4-residue cluster that also provides the remarkable H bond between the side chain hydroxyls of Tyr-58(C) and Tyr-137(E) in solution. The closer proximity of Phe-167 to Gly-143 (estimated to be 4-5 Å; predicted to be >=  7.5 Å) indicated by the moderate to weak NOE intensity in Fig. 7D, together with the observed strong hydrogen bonding interaction between Tyr-58 and the likely Tyr-137 hydroxyls, could result in the aromatic cluster moving closer to the heme.

The alternate molecules A and B in the crystal structure have been interpreted (24) as representing two different positions between the "open" pocket that admits substrate and releases product and a more closed pocket that enforces stereoselectivity on the reaction, with the molecule B representing the more open pocket, and molecule A representing the more closed pocket. The present NMR data suggest that in cyanide-ligated hHO-hemin in solution, the pocket is even more collapsed or closed than in molecule A and that both the aromatic inter-ring interactions and the H bonding interaction of Tyr-58(C) and Tyr-137(E) are important in stabilizing the restricted pocket that leads to high stereoselectivity in the ring cleavage reaction. Whether this difference in the arrangement of this aromatic cluster reflects the solution versus crystal environment, the difference in protein length between 265 and 233 residues, or the effect of ligation by an analog of O2 is not known.

Magnetic Axes and Distal Steric Tilt-- Sizable tilt of the major magnetic axis in both globins (26-28, 45, 46) and peroxidases (29, 32, 49) can be correlated with tilting/bending of the axial ligand from the heme normal. The tilt is smaller in globins and peroxidases than in hHO and in highly variable directions from the heme normal (26-29, 32, 45, 46, 49). The well-defined magnetic axes based on either molecule A or B coordinates for the proximal helix agree remarkably on the fact that the major magnetic axis is tilted by about ~20° from the heme normal in the general direction (alpha  = 230° ± 10°) of the gamma -meso position (alpha  = 225°; see Fig. 1A). However, because the z axis is oriented toward the proximal side, the Fe-CN unit on the distal side (aligned with -z), which controls the major magnetic axes, must be tilted by ~20° toward the alpha -meso position. The strong tilt of the Fe-CN unit toward the alpha -meso position had been suggested previously (18), based solely on the pattern of dipolar shifts around the heme periphery. A strong steric tilt of the Fe-O2 unit has similarly been proposed on the basis of resonance Raman spectroscopy (8), but the direction of the tilt could not be defined. Hence, a significant portion of the remarkable stereoselectivity in the ring opening of heme by hHO can be attributed to two distinct steric influences. On the one hand, the close placement of the distal helix to the heme face blocks access to the beta , gamma , and delta  positions, as shown in the crystal structures (24, 25). On the other hand, as documented herein, the bound ligand is sterically tilted toward the alpha -meso position to facilitate its attack on that position.

It is not possible at this time to determine whether the Gly-143 NH is close enough to the bound cyanide to form a stabilizing hydrogen bond. However, the proposed small lateral translation of the distal helix toward the heme center relative to that in the crystal structure would place Gly-143 NH closer to the axial ligand than predicted by the crystal structure. A potential electronic contribution to stereoselectivity, suggested previously (16, 18) by the larger spin density of the alpha -meso position as compared with the other three meso positions, cannot be further elaborated here, and discussion will be postponed until planned definitive 13C NMR studies are completed.

Dynamic Properties of the Pocket-- It is noted that the m3 (3-CH3), m28beta (Ala-28 Cbeta H3), and h2 (2-vinyl) peaks of the minor heme orientation isomer are much broader in 1H2O than those of the major isomer (compare Fig. 2, C and E) and become narrower upon transferring the complex to 2H2O. Even in 2H2O, the minor component peaks are significantly broader than those in the major component (i.e. compare h2beta s and H2beta s in Fig. 2E). The line broadening in 2H2O is more obvious at lower temperature (data not shown). Thus the minor isomer, which corresponds to the crystallographic heme orientation (24), is undergoing interconversion on the fast-to-intermediate NMR time scale between two structures (50) for which the hyperfine shifts differ significantly. The nature of the individual species is not known at this time but could represent interconversion between states with different degrees of closure of the distal pocket.

A similar dynamic heterogeneity of the heme pocket plagues the major isomer in solution. Thus several resonances, most notably the Gly-139 Calpha H at -0.7 ppm, exhibit a considerably larger linewidth, ~250 Hz, than expected based on the paramagnetic relaxation (T1 = ~80 ms) and are not detected in the NOESY map in 1H2O or in 2H2O below 30 °C. Similarly, the NOESY cross-peaks characteristic of the proposed Gly-143 Calpha Hs at 7.6 and 7.8 broaden faster at lower temperatures than other peaks. Perhaps, most obviously, all NOESY contacts involving the 4-vinyl group are selectively obliterated upon lowering the temperature. Therefore, this dynamic heterogeneity is the likely basis for the failure to detect the signal for Asp-140 and Leu-147, both of which are expected to exhibit large low-field delta dip values (differential for molecule A and B; see Table I), although it is not enough to result in resolved signals for either molecule. Large thermal ellipsoids indicative of conformational flexibility have been observed in the crystal structure for the distal helix past the Gly-Gly hinges (24). The NMR confirms that there are alternate structures in the distal helix but, to date, cannot provide information on the nature of the conformational alternate. However, only part of the distal helix may be flexible. The extremely slow exchange rates of the Leu-138 NH proton (seen after a week in 2H2O) and the relatively slow rates for Gly-139 (seen in 2H2O for many hours) indicate that the beginning of the distal helix is rigid.

Two serious resolution problems, the heme orientation isomerism and the equilibrium micro-heterogeneity that causes line broadening for each heme orientation, may be resolved using a symmetric heme and working at more elevated temperatures. Planned studies include uniform 15N labeling to facilitate location of the unassigned distal residue signals and investigation of the effect of heme modification and mutation on the influence on both the thermodynamics and dynamics of the structural heterogeneity. Optimal candidates would either speed up the interconversion to assign the signals for residues beyond Gly-143 or slow down the interconversion to allow probing of the individual structures.

    ACKNOWLEDGEMENTS

We are indebted to Dr. M. Webba da Silva for some experimental assistance and to Prof. T. L. Poulos for providing a set of refined coordinates prior to publication.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM26226 and GM62830 (to G. N. L.) and DK30297 (P. R. O. d. M.). The instruments used in this research were funded in part by National Institutes of Health Grants RR11973, RR04795, and RR08206 and National Science Foundation Grant 90-16484.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Figs. 1S and 2S.

§ To whom correspondence should be addressed: Dept. of Chemistry, University of California, Davis, CA 95616. Tel.: 530-752-0958; Fax: 530-752-8995; E-mail: lamar@indigo.ucdavis.edu.

Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M009974200

    ABBREVIATIONS

The abbreviations used are: hemin, iron(III) protoporphyrin IX; HO, heme oxygenase; hHO, human heme oxygenase; NOE, nuclear Overhauser effect; NOESY, two-dimensional nuclear Overhauser spectroscopy; TOCSY, two-dimensional total correlation spectroscopy; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate; Mb, myoglobin; HO-hemin-CN, cyanide-ligated hemin-bound heme oxygenase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Tenhunen, R., Marver, H. S., and Schmid, R. (1969) J. Biol. Chem. 244, 6388-6394[Abstract/Free Full Text]
2. Mueller, R. M., Taguchi, H., and Shibahara, S. (1987) J. Biol. Chem. 262, 6795-6802[Abstract/Free Full Text]
3. Yoshida, T., Biro, P., Cohen, T., Mueller, R. M., and Shibahara, S. (1988) Eur. J. Biochem. 171, 457-461[Abstract]
4. Rotenberg, M. O., and Maines, M. D. (1990) J. Biol. Chem. 265, 7501-7506[Abstract/Free Full Text]
5. Sun, J., Loehr, T. M., Wilks, A., and Ortiz de Montellano, P. R. (1994) Biochemistry 33, 13734-13740[Medline] [Order article via Infotrieve]
6. Takahashi, S., Wang, J., Rousseau, D. L., Ishikawa, K., Yoshida, T., Host, J. R., and Ikeda-Saito, M. (1994) J. Biol. Chem. 269, 1010-1014[Abstract/Free Full Text]
7. Takahashi, S., Wang, J. L., Rousseau, D. L., Ishikawa, K., Yoshida, T., Takeuchi, N., and Ikeda-Saito, M. (1994) Biochemistry 33, 5531-5538[Medline] [Order article via Infotrieve]
8. Takahashi, S., Ishikawa, K., Takeuchi, N., Ikeda-Saito, M., Yoshida, T., and Rousseau, D. L. (1995) J. Am. Chem. Soc. 117, 6002-6006
9. Torpey, J., and Ortiz de Montellano, P. R. (1996) J. Biol. Chem. 271, 26067-26073[Abstract/Free Full Text]
10. Torpey, J., and Ortiz de Montellano, P. R. (1997) J. Biol. Chem. 272, 22008-22014[Abstract/Free Full Text]
11. Mansfield Matera, K., Zhou, H., Migita, C. T., Hobert, S. E., Ishikawa, K., Katakura, K., Maeshima, H., Yoshida, T., and Ikeda-Saito, M. (1997) Biochemistry 36, 4909-4915[CrossRef][Medline] [Order article via Infotrieve]
12. Migita, C. T., Mansfield Matera, K., Ikeda-Saito, M., Olson, J. S., Fujii, H., Yoshimura, T., Zhou, H., and Yoshida, T. (1998) J. Biol. Chem. 273, 945-949[Abstract/Free Full Text]
13. Ortiz de Montellano, P. R. (1998) Acc. Chem. Res. 31, 543-549[CrossRef]
14. Yoshida, T., and Kikuchi, G. (1978) J. Biol. Chem. 253, 4224-4229[Medline] [Order article via Infotrieve]
15. Sun, J., Wilks, A., Ortiz de Montellano, P. R., and Loehr, T. M. (1993) Biochemistry 32, 14151-14157[Medline] [Order article via Infotrieve]
16. Hernández, G., Wilks, A., Paolesse, R., Smith, K. M., Ortiz de Montellano, P. R., and La Mar, G. N. (1994) Biochemistry 33, 6631-6641[Medline] [Order article via Infotrieve]
17. Fujii, H., Dou, Y., Zhou, H., Yoshida, T., and Ikeda-Saito, M. (1998) J. Am. Chem. Soc. 120, 8251-8252[CrossRef]
18. Gorst, C. M., Wilks, A., Yeh, D. C., Ortiz de Montellano, P. R., and La Mar, G. N. (1998) J. Am. Chem. Soc. 120, 8875-8884[CrossRef]
19. Ortiz de Montellano, P. R. (1995) in Cytochrome P450: Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P. R., ed), 2nd Ed. , pp. 245-304, Plenum Press, New York
20. Wilks, A., Torpey, J., and Ortiz de Montellano, P. R. (1994) J. Biol. Chem. 269, 29553-29556[Abstract/Free Full Text]
21. Dunford, B. H. (2000) Heme Peroxidases , Wiley & Sons, New York
22. Wilks, A., Black, S. M., Miller, W. L., and Ortiz de Montellano, P. R. (1995) Biochemistry 34, 4421-4427[Medline] [Order article via Infotrieve]
23. Brown, S. B., Chabot, A. A., Enderby, E. A., and Nort, A. C. T. (1981) Nature 289, 93-95[Medline] [Order article via Infotrieve]
24. Schuller, D. J., Wilks, A., Ortiz de Montellano, P. R., and Poulos, T. L. (1999) Nat. Struct. Biol. 6, 860-867[CrossRef][Medline] [Order article via Infotrieve]
25. Sugishima, M., Omata, Y., Kakuta, Y., Sakamoto, H., Noguchi, M., and Fukuyama, K. (2000) FEBS Lett. 471, 61-66[CrossRef][Medline] [Order article via Infotrieve]
26. Emerson, S. D., and La Mar, G. N. (1990) Biochemistry 29, 1556-1566[Medline] [Order article via Infotrieve]
27. Rajarathnam, K., Qin, J., La Mar, G. N., Chiu, M. L., and Sligar, S. G. (1994) Biochemistry 33, 5493-5501[Medline] [Order article via Infotrieve]
28. Qin, J., La Mar, G. N., Ascoli, F., and Brunori, M. (1993) J. Mol. Biol. 231, 1009-1023[CrossRef][Medline] [Order article via Infotrieve]
29. La Mar, G. N., Chen, Z., Vyas, K., and McPherson, A. D. (1995) J. Am. Chem. Soc. 117, 411-419
30. Wilks, A., and Demontellano, P. R. O. (1993) J. Biol. Chem. 268, 22357-22362[Abstract/Free Full Text]
31. La Mar, G. N., and de Ropp, J. S. (1993) in Biological Magnetic Resonance (Berliner, L. J. , and Reuber, J., eds), Vol. 12 , pp. 1-78, Plenum Press, New York
32. La Mar, G. N., Satterlee, J. D., and de Ropp, J. S. (1999) in The Porphyrin Handbook (Kadish, K. , Smith, K. , and Guilard, R., eds), Vol. 5 , pp. 185-298, Academic Press, San Diego
33. Jeener, J., Meier, B. H., Bachmann, P., and Ernst, R. R. (1979) J. Chem. Phys. 71, 4546-4553[CrossRef]
34. Griesinger, C., Otting, G., Wüthrich, K., and Ernst, R. R. (1988) J. Am. Chem. Soc. 65, 355-360
35. Williams, G., Clayden, N. J., Moore, G. R., and Williams, R. J. P. (1985) J. Mol. Biol. 183, 447-460[Medline] [Order article via Infotrieve]
36. Bundi, A., and Wüthrich, K. (1979) Biopolymers 18, 285-297
37. Wishart, D. S., Sykes, B. D., and Richard, F. M. (1991) J. Mol. Biol. 222, 311-333[Medline] [Order article via Infotrieve]
38. Cross, K. J., and Wright, P. E. (1985) J. Magn. Reson. 64, 240-231
39. Plateau, P., and Gueron, M. (1982) J. Am. Chem. Soc. 104, 7310-7311
40. Wüthrich, K. (1986) NMR of Proteins and Nucleic Acids , Wiley Interscience, New York
41. Bertini, I., Turano, P., and Villa, A. J. (1993) Chem. Rev. 93, 2833-2932
42. Nguyen, B. D., Xia, Z. C., Yeh, D. C., Vyas, K., Deaguero, H., and La Mar, G. N. (1999) J. Am. Chem. Soc. 121, 208-217[CrossRef]
43. Shokhirev, N. V., and Walker, F. A. (1998) J. Biol. Inorg. Chem. 3, 581-594[CrossRef]
44. Chen, Z., de Ropp, J. S., Hernández, G., and La Mar, G. N. (1994) J. Am. Chem. Soc. 116, 8772-8783
45. Kolczak, U., Han, C., Silvia, L. A., and La Mar, G. N. (1997) J. Am. Chem. Soc. 119, 12643-12654[CrossRef]
46. Peyton, D. H., La Mar, G. N., and Gersonde, K. (1988) Biochim. Biophys. Acta 954, 82-94[Medline] [Order article via Infotrieve]
47. Steigemann, W., and Weber, E. (1979) J. Mol. Biol. 127, 309-338[Medline] [Order article via Infotrieve]
48. Liu, Y., Koenigs Lightning, L., Huang, H.-w., Moënne-Loccoz, P., Schuller, D. J., Poulos, T. L., Loehr, T. M., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 34501-34507[Abstract/Free Full Text]
49. Banci, L., Bertini, I., Pierattelli, R., Tien, M., and Vila, A. J. (1995) J. Am. Chem. Soc. 117, 8659-8667
50. Sandström, J. (1982) Dynamic NMR Spectroscopy , Academic Press, New York


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.