Solution 1H NMR Investigation of the Active Site Molecular and Electronic Structures of Substrate-bound, Cyanide-inhibited HmuO, a Bacterial Heme Oxygenase from Corynebacterium diphtheriae*,

Yiming LiDagger , Ray T. SyvitskiDagger , Grace C. Chu§, Masao Ikeda-Saito**, and Gerd N. La MarDagger ||

From the Dagger  Department of Chemistry, University of California, Davis, California 95616 and the  Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970 and ** Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahura, Aoba-ku, Sendai 980-8577, Japan

Received for publication, November 4, 2002, and in revised form, December 9, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The molecular structure and dynamic properties of the active site environment of HmuO, a heme oxygenase (HO) from the pathogenic bacterium Corynebacterium diphtheriae, have been investigated by 1H NMR spectroscopy using the human HO (hHO) complex as a homology model. It is demonstrated that not only the spatial contacts among residues and between residues and heme, but the magnetic axes that can be related to the direction and magnitude of the steric tilt of the FeCN unit are strongly conserved in the two HO complexes. The results indicate that very similar contributions of steric blockage of several meso positions and steric tilt of the attacking ligand are operative. A distal H-bond network that involves numerous very strong H-bonds and immobilized water molecules is identified in HmuO that is analogous to that previously identified in hHO (Li, Y., Syvitski, R. T., Auclair, K., Wilks, A., Ortiz de Montellano, P. R., and La Mar, G. N. (2002) J. Biol. Chem. 277, 33018-33031). The NMR results are completely consistent with the very recent crystal structure of the HmuO·substrate complex. The H-bond network/ordered water molecules are proposed to orient the distal water molecule near the catalytically key Asp136 (Asp140 in hHO) that stabilizes the hydroperoxy intermediate. The dynamic stability of this H-bond network in HmuO is significantly greater than in hHO and may account for the slower catalytic rate in bacterial HO compared with mammalian HO.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Heme oxygenase (HO)1 is an alpha -helical enzyme that carries out the highly stereoselective conversion of hemin to alpha -biliverdin, iron, and CO, excising CO from exclusively the alpha -meso position (1). In contrast to the better understood heme peroxidases and cytochromes P450, which pass through the common ferryl intermediate, the reactive form of HO is a ferric hydroperoxy intermediate (2-4). In mammals, the ~300-residue membrane-bound enzyme occurs as an inducible HO-1, whose primary roles are iron homeostasis and heme catabolism (5, 6), whereas the constitutive HO-2 has been proposed (7) to generate CO as a neural messenger. In higher plants, algae, and cyanobacteria, HO generates the open tetrapyrroles as light-harvesting pigments (8). HO has also been identified in several pathogenic bacteria, where its role appears to be the essential "mining" of iron from hemes in the host (9, 10). Plant and bacterial HOs are soluble and somewhat shorter (~200 residues) (9, 10) than mammalian HO (11). Among the characterized bacterial HOs, sequence homology to the more extensively studied mammalian HO varies from relative high (33% sequence identity/70% similarity) for HmuO from Corynebacterium diphtheriae (10) to low (<25%) for HemO from Neisseria meningitides (9).

The remarkable recent progress in understanding the functional properties of HO based on mutagenesis and spectroscopic studies (3, 4, 12-14), of a slightly truncated, soluble, and completely active recombinant mammalian HO, has been considerably enhanced by the successful x-ray crystallographic characterization of the substrate complexes of first human HO (hHO), followed by rat HO (15, 16). These structures shed light on a key determinant of the alpha -stereoselectivity, in that the distal helix covers the heme so as to sterically completely block access to the beta - and delta -meso positions and partially block access to the gamma -meso positions (15-17). Although no distal residue that would stabilize the hydroperoxy unit could be identified, the occurrence in the crystal of a localized water molecule H-bonded to the distal helix Asp140 carboxylate, together with the observation that mutating Asp140 to a non-anionic side chain abolishes HO activity (12, 14), has led to the proposal that the water molecule may be sufficiently stabilized in its crystallographically defined position to serve as the weak H-bond donor to stabilize the hydroperoxy unit.

Solution 1H NMR characterization of hHO and its substrate complex has contributed to the understanding of the structure/function relationship of HO (18-22). An annoying, but functionally irrelevant property of the mammalian HOs is that binding of the native substrate, protohemin (PH; R = vinyl in Fig. 1), leads to ~1:1 orientational isomerism about the alpha /gamma -axis (18-20), which leads to spectral congestion and limits both the range and reliability of structural characterization. Nevertheless, the pattern of dipolar shifts for the protons on the proximal helix allowed determination of the orientation of the major magnetic axis, which could be correlated with a ~20° tilt of the FeCN in the direction of the alpha -meso position (19, 20). The orientation of ligated azide in the rat HO·heme·N3 complex confirms such a steric influence (23).

Thus, both the position of the distal helix in blocking access to other meso positions (15, 16) and its influence on tilting (19-21, 24) the axial ligand toward the alpha -meso position contribute to the stereoselectivity of the reaction. Two-dimensional 1H NMR of a hHO complex with the 2-fold symmetric substrate 2,4-dimethyldeuterohemin (DMDH; R = CH3 in Fig. 1) (25) allowed sufficiently definitive and extensive assignments to identify (21) an unusual distal H-bond networks involving some extremely strong H-bonds (labile proton shifts between 17 and 10 ppm) whose acceptor could be identified in the hHO·PH·H2O crystal structure (15). Moreover, it was demonstrated that water molecules were in the immediate vicinity (~3 Å) of each of the strong H-bond donors (22). The strongest of these H-bonds is between a conserved Tyr58 serving as a donor to the catalytically critical Asp140. We proposed that this network has, as one of its primary roles, the stabilization of the Asp140 side chain and the H-bonded water molecules, one of which can interact with the heme ligand (4, 21).


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Fig. 1.   Schematic representation of hemin (where M = methyl, P = propionates, and R = vinyl (PH) or methyl (DMDH)). The orientation of the axial His20 ring plane is shown as a rectangle. The magnetic coordinate system, x,y,z, is related to the iron-centered reference coordinate system, x',y',z', by the Euler angles Gamma (alpha ,beta ,gamma ), where beta  is the tilt of the major magnetic axis (z) from the heme normal (z'), alpha  describes the direction of the tilt in the angle between the projection of z on the z',y' axes and the x' axis, and the rhombic axes (x,y) are related to the reference x',y' axis by the angle kappa  ~ alpha  + gamma .

We report herein on the extension of our 1H NMR investigation to HmuO, the 216-residue soluble bacterial HO from C. diphtheriae (10), using hHO as a homology model (21). Functional (26, 27) and spectroscopic (27, 28) studies, as well as mutagenesis (29, 30), have confirmed the same mechanism and stereospecificity as for mammalian HOs, although the turnover rate is slower (27); the enzyme has been crystallized (31), and the structure of the substrate hemin complex has been refined to 1.4-Å resolution.2 Our interests are to establish the degree to which the available extensive NMR data on hHO·DMDH·CN (19-22) and the crystal structure of hHO·PH·H2O (15) can be used to assign the resonances and to structurally interpret the NMR spectral parameters (32) of HmuO·PH·CN in terms of the orientation of the FeCN vector and the presence or absence of a distal H-bond network similar to that reported for hHO·DMDH·CN (21, 22). This 216-residue soluble HmuO enzyme has His20 as its axial ligand (30) and exhibits extensive sequence homology to the distal helix and the four fragments of HO shown (21) to participate in the H-bond network in hHO·DMDH·CN (Fig. 2). To provide a broader comparison with the NMR data on hHO complexes (19-22), we explore in parallel both the disordered HmuO·PH·CN complex and the homogeneous HmuO·DMDH·CN complex to show that this bacterial HO exhibits remarkable conservation of the distal steric effects on the axial ligand and distal H-bond network relative to hHO.


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Fig. 2.   Sequence portions of interest for HmuO (with the homologous residues in hHO in italics) and schematic representation of the observed sequential backbone NOESY contacts in hHO·PH·CN for portions of the proximal helix (I), the distal helix (II), two additional helical fragments (IV and V), and one non-helical fragment (VI), which participate in the distal H-bond network and aromatic cluster. Helix III, identified in hHO, was not located in HmuO·PH·CN.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein Sample-- HmuO was expressed and purified as reported previously (27). PH was purchased from Sigma. 2,4-Dimethyldeuteroporphyrin was purchased from Mid-Century Chemicals, and the iron was incorporated to yield DMDH by standard procedures (25). PH and DMDH were titrated into apo-hHO to a 1:1 stoichiometry in the presence of a 10-fold molar excess of KCN in a 90% H2O and 10% 2H2O solution buffered at pH 7.4 with 100 mM phosphate. The final concentrations of the HmuO·substrate·CN complexes were ~1.5 mM.

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 a temperature range of 10-40 °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 T1 values were determined in both 1H2O and 2H2O at 20, 25, 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 RHi = R*Fe(T1*/T1i)1/6, using the heme for the alpha -meso-H for H* (R*Fe = 4.6 Å and T1* = 50 ms) as reference (20, 21, 32). Steady-state NOEs from HmuO·DMDH·CN in 1H2O were recorded with and without saturation of the solvent resonance for 300 ns using 3:9:19 detection (33). NOESY spectra (mixing time of 40 ms, 10-40 °C) (34) and Clean-TOCSY spectra (25, 35 °C; spin lock of 15 and 30 ms) (35) using MLEV-17 (36) were recorded over a bandwidth of 14 kHz (or 28 kHz) (NOESY) and 14 kHz (TOCSY) with recycle times of 1 s (or 0.33 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 magnetic axes (Fig. 1) were determined by a least-squares search for the minimum in the error function (21, 32, 37) (Equation 1).


F/n=<LIM><OP>∑</OP><LL><UP>i=1</UP></LL><UL><UP>n</UP></UL></LIM><UP>‖&dgr;</UP><SUB><UP>dip</UP>(<UP>obs</UP>)</SUB><UP>−&dgr;</UP><SUB><UP>dip</UP>(<UP>calc</UP>)</SUB><UP>‖<SUP>2</SUP></UP> (Eq. 1)
The calculated dipolar shift in the reference coordinate system, x',y',z' (or R,theta ',Omega '), is given by Equation 2, with Delta chi ax = 2.48 × 10-8 m3/mol and Delta chi rh = -0.58 × 10-8 m3/mol as the axial and rhombic anisotropies of the diagonal paramagnetic susceptibility tensor taken from the isoelectronic met-cyano-myoglobin complex (37).
&dgr;<SUB><UP>dip</UP>(<UP>calc</UP>)</SUB><UP>=</UP>(<UP>24&pgr;N</UP>)<SUP><UP>−1</UP></SUP>(<UP>2&Dgr;&khgr;<SUB>ax</SUB></UP>(<UP>3cos<SUP>2</SUP>&thgr;′−1</UP>)R<SUP><UP>−3</UP></SUP> (Eq. 2)

<UP>+3&Dgr;&khgr;<SUB>rh</SUB></UP>(<UP>sin<SUP>2</SUP>&thgr;′cos2&OHgr;′</UP>)<UP>R<SUP>−3</SUP></UP>)<UP>&eegr;</UP>(<UP>&agr;,&bgr;,&ggr;</UP>)
alpha , beta , and gamma  are the Euler angles that rotate the reference system (x',y',z') into the magnetic coordinate system, x,y,z (R,theta ,Omega ), where beta  reflects the tilt of the major magnetic axis (z) from the heme normal; alpha  defines the projection of z onto the x',y' plane (tilt direction); and kappa  ~ alpha  + gamma  locates the rhombic axes, as shown in Fig. 1. The observed dipolar shift, delta dip(obs) is given by Equation 3,
<UP>&dgr;</UP><SUB><UP>dip</UP>(<UP>obs</UP>)</SUB><UP>=&dgr;</UP><SUB><UP>DSS</UP>(<UP>obs</UP>)</SUB><UP>−&dgr;</UP><SUB><UP>DSS</UP>(<UP>dia</UP>)</SUB> (Eq. 3)
where delta DSS(obs) is the chemical shifts (in ppm) referenced to DSS for the paramagnetic HmuO·PH·CN and hHO·DMDH·CN complexes. delta DSS(dia) may be estimated from the available molecular structure for the hHO·PH·H2O and HmuO·PH·H2O complexes (20, 21) via Equation 4.
<UP>&dgr;</UP><SUB><UP>DSS</UP>(<UP>dia</UP>)</SUB><UP>=&dgr;<SUB>tetr</SUB>+&dgr;<SUB>sec</SUB>+&dgr;<SUB>rc</SUB></UP> (Eq. 4)
The symbols delta tetr, delta sec, and delta rc are the chemical shifts of an unfolded tetrapeptide (38) relative to DSS and the effect of secondary structure (39) and ring currents (40) on the shift, respectively.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The initially assembled HmuO·PH·CN complex exhibits two sets of hyperfine shifted resonances (data not shown; see Supplemental Material), one set of which loses intensity over several days to yield an Mi:mi or Hj:hj isomer equilibrium ratio of ~3:1 (Mi and Hi represent a methyl and single proton of the major or only equilibrium species, respectively; and mi and hj reflect a methyl and hydrogen of the minor equilibrium species, respectively), as illustrated in Fig. 3B. The subscript i refers to heme pyrrole substituent positions 1-8, heme alpha /delta -meso positions, or the residue number and proton position. Hence, the substrate is initially bound disordered about the alpha /gamma -meso axis and, like mammalian HO complexes, equilibrates to a ~3:1 ratio with the more stable heme orientation depicted in Fig. 1 (18-20). This heterogeneity is absent in the complex with the symmetric DMDH substrate (Fig. 3C), as found previously for hHO·DMDH·CN (21). We will concern ourselves further only with the major isomer of HmuO·PH·CN and the single species HmuO·DMDH·CN.


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Fig. 3.   Resolved portions of the 600-MHz 1H NMR spectra in 1H2O at pH ~7.4. A, hHO·PH·CN; B, HmuO·PH·CN; C, HmuO·DMDH·CN; D, hHO·DMDH·CN. Mi and Hi indicate the major (or only) isomer for methyls and single protons, respectively, where i indicates heme pyrrole substituent positions 1-8, heme alpha /delta -meso positions, or the residue number and proton position. Peaks for the minor isomer of the PH complexes are mi and hi. The homologous assignments are connected by vertical dashed lines. Peaks labeled with dots are due to impurities.

The resolved portions of the 600-MHz 1H NMR spectra of equilibrated hHO·PH·CN and HmuO·PH·CN (19, 20) are compared in Fig. 3 (A and B, respectively). Similarly, the traces of HmuO·DMDH·CN and hHO·DMDH·CN (21) are compared in Fig. 3 (C and D, respectively). The very close similarity of the pattern of resolved resonances in the hHO and HmuO complexes is quite apparent. The homologous assignments are connected by dashed lines between the two hHO and the two HmuO complexes. The crowded region between 10 and 15 ppm for HmuO·DMDH·CN in 1H2O is expanded in Fig. 4A. The relevant homologous portions of the amino acid sequences for the two HOs are illustrated in Fig. 2. The nonselective T1 values for well resolved peaks of interest in hHO·DMDH·CN and HmuO·DMDH·CN (as well as in the PH complexes) are the same. In particular, the low-field labile proton peaks Gly139 NH and upfield Ser138 Cbeta 1H and Cbeta 2H exhibit T1 values indistinguishable from those of Gly143 NH and Ser142 Cbeta 1H and Cbeta 2H (~50, 85, and 50 ms, respectively) reported previously (20, 21).


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Fig. 4.   Expanded low-field portion of the 600-MHz 1H NMR 3:9:19 spectrum (carrier at 12.0 ppm) (33) illustrating the positions and intensities of labile protons of HmuO·DMDH·CN at 25 °C in 100 mM phosphate, pH 7.4, for 90% H2O and 10% 2H2O (A) and 20 min (B) and 4 h (C) after converting from 90% 1H2O and 10% 2H2O to ~5% 1H2O and 95% 2H2O. The trace in 1H2O upon saturating the 1H2O resonance (B') leads to intensity loss of numerous peaks relative to A, more apparent in the difference trace (C'). Assignments are given as described under `Results.` Peak a, a peptide NH, is not assigned. The peaks marked with asterisks are unassigned labile protons that exchange rapidly with bulk water even in 1H2O.

Comparison in Fig. 3 of the NMR spectra of the complexes of the two HOs shows that the patterns of shifts are so similar in the two proteins that it is highly advantageous to pursue assignments on the basis of the comprehensive and definitive assignments previously reported for hHO complexes (20-22). Hence, two-dimensional NMR data are presented only to define an important distal H-bond network/aromatic cluster as just recently characterized in hHO. We initially (and trivially) assign the heme, followed by locating hyperfine shifted protons that arise from TOCSY-detected side chains placed on sequentially assigned backbone via the standard Ni-Ni+1, alpha i-Ni+1, beta i-Ni+1, ai-Ni+3, and/or alpha i-beta i+3 NOESY connectivities characteristic of helices (41) as described for hHO (20, 21).

Heme Assignments-- Dipolar contacts were observed in a set of pyrrole substituents on both HmuO·PH·CN and HmuO·DMDH·CN (data not shown) that are identical to those reported in detail for the analogous hHO complexes (21). The remarkably similar hyperfine shifts for a given substituent in the HmuO and hHO complexes are in evidence in the data provided in Table I. The essentially identical shift pattern for the major isomer of the PH substrate is evidence for the same heme orientation in HmuO and hHO (see below).

                              
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Table I
Chemical shifts of heme protons in HmuO · PH · CN, HmuO · DMDH · CN, and the analogous hHO complexes
Shifts are in ppm from DSS in 1H2O and 100 mM phosphate, pH 7.3, at 25 °C.

Proximal and Distal Helices-- Standard backbone NOESY connectivities (data not shown; summarized in Fig. 2) among TOCSY-detected side chains locate two helical fragments (I and II) for which numerous side chains exhibit moderate-to-large hyperfine shifts. Fragment I is Alai-Zi+1-Alai+2-AMXi+3-Zi+4-Zi+5-Alai+6-Zi+7 (Z > four spins; AMX = three-spin system), which is unique for Ala17-Glu24 on the proximal helix. Consistent with the assignments are the large low-field contact shift for AMXi+3 of the axial His20 (His25 in hHO), the low-field dipolar shifts for Ala17 (Lys22 in hHO) and Ala23 (Ala28 in hHO), and the high-field dipolar shifts for Glu24 (Glu29 in hHO). The NOESY slices through the 3-CH3 (M3) of HmuO·PH·CN and hHO·PH·CN are compared in Fig. 5 (A and B), where it is clear that the contacts are remarkably conserved (21). Although only a part of Glu24 could be resolved, it exhibits similar shifts and NOESY cross-peaks to 3-CH3 in the two complexes. The conservation of the interaction of the proximal helix with the heme is apparent in the comparison of the NOESY slices through Ala23 Cbeta H3 (M23beta ; Ala28 in hHO) of the two PH complexes in Fig. 5 (C and D), which interacts in a similar fashion with the 2-vinyl/2-CH3 substituents; and the expected strong Ile211 contacts (21) in hHO (Fig. 5D) are replaced with a contact by the NH2 group (Fig. 5C) of the homologous His205.


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Fig. 5.   Dipolar connections involving the proximal helix and the heme. Shown is a comparison of slices of the identically recorded and processed 600-MHz 1H NMR NOESY spectra (mixing time of 40 ms) in 1H2O at 30 °C through the 3-CH3 (M3) peaks of HmuO·PH·CN (A) and hHO·PH·CN (B) and through the resolved methyl signals of Ala23 (M23beta ) of HmuO·PH·CN (C) and Ala28 (M23beta ) of hHO·PH·CN (D). The homologous residue peaks for hHO and HmuO are connected by vertical dashed lines. The intensities within each pair of slices for HmuO and hHO are normalized, allowing direct comparison of both relative and absolute intensities.

A moderately relaxed and weakly shifted aromatic ring in close contact with His20 and 8-CH3 (Phe207 in hHO) (20, 21) must arise from a similarly placed Phe201. The failure to detect a NOESY cross-peak between Phe201 and Ala23, as observed in hHO (20, 21), indicates that Phe201 is slightly shifted away from Ala23. Finally, note that 3-CH3 in HmuO·PH·CN exhibits weak NOESY cross-peaks to Phe208 (which also exhibits strong NOESY cross-peaks to the 2-vinyl group) (data not shown), whereas the small Phe214 cross-peak to 3-CH3 is not seen in hHO·PH·CN (20, 21) (but the strong 2-vinyl cross-peak to Phe214 is observed). This indicates that the conserved Phe208/Phe214 at the pyrrole A/B junction is slightly closer to position 2 in HmuO than in hHO. Moreover, NOESY cross-peaks of the labile protons for NH2 of Gln38 in hHO (Fig. 5B) (20, 21) are absent in the HmuO complex (Fig. 5A), but strong contacts with some aliphatic protons are present as expected because of the Gln38 right-arrow Leu33 replacement in HmuO. The chemical shifts of the two HmuO complexes, as well as of the two hHO complexes (20, 21), for these assigned residues are compared in Table II, where we also include the predicted dipolar shifts for the residues in the hHO complex. The observed inter-residue and heme-residue dipolar contacts are summarized schematically in Fig. 6.

                              
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Table II
Chemical shifts for assigned residues in HmuO · PH · CN, HmuO · DMDH · CN, and the analogous hHo complexes


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Fig. 6.   Schematic representation of the relative positions of the heme and assigned fragments I, II, and IV-VI in HmuO. Fragment III, which could not be assigned, but was concluded to occupy the same position, is shown by the dashed lines. Observed dipolar contacts (dashed lines) among these fragments and with the heme and the locations of strong H-bonds among these fragments (solid arrows from donor to acceptor) are shown. The arrangement is based on the similarly assigned fragments for hHO·DMDH·CN (20, 21) and comparison with the crystal structure of hHO·PH·H2O (15). The positions of two residue side chains, Phe42 and Gln46, which make key contacts with fragment IV, and two conserved aromatic rings, Phe201 and Phe208, which make contact with the heme, are also shown. The recent crystal structure of HmuO·PH·H2O (Footnote 2) confirms all observed contacts except that between the Tyr161 ring and Gly139 (labeled with an asterisk, where a distance >6 Å is predicted) and indicates that the likely acceptor for the strong H-bond by Tyr161 OH is Gln46.

The NOESY and TOCSY data (data not shown; summarized in Fig. 2) indicate that helical fragment II is represented by Vali-AMXi+2-Leui+3-Ni+4-AMXi+5-Nai+6-AMXi+7-Glyi+8 (Fig. 2), where AMXi+2 is in contact with a two-spin aromatic ring; Vali, Leui+3, and AMXi+7 exhibit moderate-to-large high-field dipolar shifts; and Glyi+8 exhibits strong low-field dipolar shifts. Both the sequence and the dipolar shift pattern identify (20, 21) this as a key portion of the distal helix Val131-Gly139 (analogous to Thr135-Gly143 in hHO). As shown in the slices through 8-CH3 in HmuO and hHO (Fig. 7, A and B), the strong contact with Calpha H of Val131 is conserved (relative to Calpha H of Thr135). However, the weak contacts between 8-CH3 and NH of the adjacent conserved Leu134 and Gly135 in HmuO (residues 138 and 139 in hHO) observed in the hHO complex (Fig. 7B) (20, 21) are not detectable in HmuO (Fig. 7A) and indicate a small movement of the distal helix near its kink away from 8-CH3 in HmuO relative to hHO. Finally, slices through the similarly relaxed (T1 ~ 85 ms) Ser138 Cbeta 1 in HmuO (Fig. 7C) and Ser142 in hHO (Fig. 7D) indicate that the Ser is slightly farther from heme 6-Halpha s in HmuO relative to the hHO complex. The chemical shifts for helix II residues, together with data from hHO complexes (20, 21), are listed in Table II. The observed inter-residue and heme-residue contacts are illustrated schematically in Fig. 6.


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Fig. 7.   Dipolar contacts involving the distal helix and the heme. Shown is a comparison of slices of identically recorded and processed 600-MHz 1H NMR NOESY spectra (mixing time of 40 ms) in 1H2O and 100 mM phosphate, pH ~7.3, at 30 °C through 8-CH3 (M8) of HmuO·PH· CN (A) and hHO·PH·CN (B) and through Ser138/Ser142 Cbeta 1H of HmuO·PH·CN (C) and hHO·PH·CN (D). The homologous peaks are connected by vertical dashed lines. The intensities in each pair of slices are normalized, allowing comparison of both relative and absolute intensities of cross-peaks.

H-bond Network/Aromatic Cluster-- The HmuO complexes, like the hHO complexes (21, 22), exhibit a set of strongly low-field shifted labile proton peaks (Figs. 3 and 4A), which (with the unique exception of HmuO Gly139 NH/hHO Gly143 NH) exhibit negligible paramagnetic relaxation, so their strong low-field bias must be attributed to strong hydrogen bonds (42). Three sequential fragments are easily recognized (summarized in Fig. 2) by their remarkably similar arrangements compared with the three characterized fragments labeled IV-VI in hHO·DMDH·CN (21). The analogous fragment III could not be recognized as easily in the HmuO complex (but see "Discussion"). The helical fragment IV, Zi-Glyi+1-AMXi+2-AMXi+3, exhibits strong low-field NH shifts for Glyi+1 and AMXi+2 (Fig. 8, B and D; summarized in Fig. 2), as found for Ala165-Phe166 in hHO (21). In agreement with the assignment of Leu159-Tyr161 to fragment IV, a three-spin TOCSY ring makes contact with AMXi+2 (Fig. 9D), and two spins of a three-spin aromatic ring make contact with AMXi+3 (Fig. 9C), which must arise from Tyr161. The three TOCSY/NOESY peaks of the AMXi+3 side chain exhibit the unusual pattern (Fig. 9, B-D) that one cross-peak becomes narrower, whereas the other broadens as the temperature is elevated. This behavior is consistent with a Tyr ring that reorients sufficiently slowly to resolve the individual Cdelta H or Cepsilon H, but leaves the two Cepsilon H shifts averaged. The NOESY cross-peak to its own backbone, as well as the cross-peak to a new low-field shifted labile proton with no TOCSY connectivity, identifies Tyr161 OH (Fig. 9D). Tyr161 Cepsilon H exhibits a weak-to-moderate intensity NOESY cross-peak to NH of Gly139 (data not shown; as also observed for the homologous Phe166 right-arrow Gly143 in hHO) (20, 21). A strong dipolar contact of Phe166 NH with a Met51 methyl in hHO (21) is lost in HmuO, but is replaced by a dipolar contact of Phe160 NH with an NH2 group (data not shown), which sequence comparison identifies as Gln46. A strong contact between the Tyr161 ring and a low-field, dipolar-shifted three-spin aromatic group (Fig. 9C) is analogous to the Phe167-Phe47 contact in hHO (21) and is readily rationalized by the homologous Tyr161-Phe42 contact observed here. This assignment is consistent with the observation (data not shown) of a Gln46 NH2 contact with Phe42. The dipolar contact for this fragment with the distal helix (II) and Phe42/Gln46 are summarized schematically in Fig. 6.


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Fig. 8.   Portions of the NOESY spectrum (mixing time of 40 ms) of HmuO·DMDH·CN illustrating the dipolar connections for fragments IV-VI, involved in the H-bond network.


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Fig. 9.   Portions of the 600-MHz 1H NMR NOESY spectra (mixing time of 90 ms) of HmuO·PH·CN in 1H2O and 100 mM phosphate at 30 °C illustrating contacts for the aromatic cluster (Phe160, Phe42, Tyr58, and Tyr161).

The extreme low-field labile proton exhibits no TOCSY peak, but displays strong NOESY cross-peaks to a two-spin aromatic ring (Fig. 9E) whose AMX backbone is readily identified. Ni-Ni+1 and beta -Ni+1 (Fig. 8A; summarized in Fig. 2) locate the adjacent residue as Thr, which identifies the Tyr53-Thr54 segment with shifts and dipolar contacts analogous to fragment V in hHO (21). The Tyr53 ring exhibits the NOESY cross-peak to the Phe160 ring (Fig. 9B) that was observed between the homologous Tyr58 or fragment V and Phe166 or fragment VI in hHO (21). The Tyr53 ring exhibits NOESY cross-peaks to Nepsilon H of Arg132 and OH of Tyr133 (data not shown) on the distal helix II in the same fashion as observed for the homologous residues in hHO (schematically shown in Fig. 6) (21). The dipolar contacts of fragment IV are summarized in Fig. 6.

Finally, the other two extreme low-field peptide NH groups are part of a 5-residue fragment, Zi-Alai+1-Zi+2-Vali+3-Zi+4 (Figs. 2 and 8, A, B, and D), which the sequence identifies as Arg79-Leu83, with the low-field NH groups occurring from the homologous residues i (Arg79) and i+1 (Ala80) in HmuO (as observed for Arg85 and Lys86 in hHO) (21). The contacts between fragment VI and the distal helix II and fragment V, viz. Asn78 and Arg79 to Tyr133 and Tyr53 (Fig. 6), expected on the basis of the contact in hHO, are clearly observed, as summarized in Fig. 6. Attempts to assign the fragment in HmuO analogous to fragment III on the basis of the NMR studies on hHO·DMDH·CN (21) failed, although the 1H NMR data available can be used to infer that the fragment is similarly highly conserved in HmuO relative to hHO (see below). The peak at 14.1 ppm exhibits equally intense cross-peaks to two non-labile protons in the aromatic window indicative of a His ring Nepsilon H, which, by analogy to hHO (19, 22), is His128; the expected strong NOESY cross-peak to Ala200 is observed (data not shown).

The low-field peak at 11.4 ppm exhibits properties consistent with its arising from Nepsilon H of Trp50 (which replaces Tyr55 in hHO) in NOESY cross-peaks to a TOCSY-detected (only two cross-peaks are resolved) aromatic ring and a weak NOESY cross-peak of the ring (Trp50 ring) to the rings of both Phe160 and Tyr161 (data not shown). Hence, we tentatively label it Nepsilon of Trp50. It should be noted that there remains one strongly low-field shifted NH (12.2 ppm; labeled a in Fig. 4A) that cannot be assigned at this time and that has no analog in hHO (21).

Acceptors for Strong H-bond Donors-- Having demonstrated for HmuO a remarkably conserved arrangement on the distal side of the heme for three of the four fragments involved in the H-bond network/aromatic cluster in hHO (21), one can speculate that the acceptors for these strong H-bond donors in hHO are also homologous in HmuO. Thus, sequence comparison indicates that the carboxylate of Glu57 (homologous to Glu62 in hHO) is the acceptor for NH groups of Arg79 and Ala80 and that the carboxylate of Asp136 (homologous to Asp140 in hHO) is acceptor for Tyr53 Oeta H. Similarly, His132 Nepsilon H as donor to Glu202 in hHO (22) indicates that Glu196 is the acceptor for His128 Nepsilon H in HmuO. Even though fragment III, identified in hHO (21), could not be assigned in HmuO, the sequence homology suggests that the Asp86 carboxylate (homologous to Asp92 in hHO) (Fig. 2) is the acceptor for the NH groups of Gly159 and Phe160, as depicted in Fig. 6. Finally, the acceptor for the completely new strong H-bond involving Tyr161 Oeta H (Phe167 in hHO) cannot be identified by comparison with hHO. The relative positions of the donors and acceptors in the strong H-bonds in HmuO and hHO (21) are compared in Table III.

                              
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Table III
Donor and acceptor residues in the distal H-bond network in the substrate DSS(obs)a complexes of HmuO and hHO

Labile Proton Exchange-- Comparison of the resolved low-field 1H NMR trace of HmuO·DMDH·CN in 1H2O in Fig. 4A with that of the complex 20 min (Fig. 4B) and 4 h (Fig. 4C) after transfer into 2H2O shows that, whereas Tyr53 OH has a half-life <5 min, other strong H-bonding protons exhibit long exchange half-lives not only for peptide NH groups (30 min for Arg79, ~30 days for Phe160, and 2 h for peak a), but also for side chain labile protons (~4 h for Nepsilon H of His128 and ~2 h for Nepsilon H of Trp50). Moreover, comparison of the 3:9:19 trace (33) without (Fig. 4A) and with (Fig. 4B') saturation of the bulk water resonance shows significant magnetization transfer to the low-field peaks (22, 43), as shown in the difference trace in Fig. 4C'. The magnetization transfer to the four labile protons shown to exchange slowly with water must arise from NOEs between these labile protons and "immobilized" water molecules (43), as we previously observed for hHO·DMDH·CN (22). The magnitude of the NOEs is ~10% for His128 Nepsilon H and ~25% for Phe160 NH, Trp50 Nepsilon H, and peak a. For the other peaks that exhibit magnetization transfer from water, it is not possible at this time to differentiate between chemical exchange and NOEs as the origin of the magnetization transfer (43).

Magnetic Axes and Cyanide Tilt-- The completely conserved pattern of large dipolar shifts for proximal helix residues (Table II) and conserved contacts with the heme (Fig. 6) in HmuO relative to the hHO complexes can arise only if HmuO·PH·CN and hHO·PH·CN possess very similar orientation for the major magnetic axis (32). A direct determination of the magnetic axes for HmuO·PH·CN using the present 1H NMR data and the recently available HmuO crystal coordinates2 leads to alpha  = 234 ± 16, beta  = 18 ± 2, and kappa  = 47 ± 12, which can be compared with reported values of alpha  = 234 ± 12, beta  = 20 ± 3, and kappa  = 25 ± 13 for hHO·DMDH·CN (21). The magnetic axes for both complexes yield similarly excellent correlation between the delta dip(obs) and delta dip(calc) for the input proximal side residues (data not shown; see Supplemental Material). In each case, z is tilted ~20° toward the gamma -meso position. The z direction is oriented toward the proximal side (20, 32); hence, the FeCN vector (-z direction) is tilted toward the alpha -meso position. Therefore, a direct contribution to stereoselectivity from the tilt due to direct distal steric interactions with the ligand in the direction of the alpha -meso position is operative in both hHO and HmuO.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PH Orientation-- The orientation of PH in both hHO (19, 20) and HmuO complexes is similarly rotationally disordered about the alpha /gamma -meso axis in the initially formed complex, with very similar ~3:1 ratios at equilibrium and with the same heme orientation dominating in each complex in solution. Notably, the heme orientation found in the HmuO·PH·H2O crystal structure2 is the same as the dominant isomer in solution, whereas that in the hHO·PH·H2O crystal structure (15) is the minor form in solution (19, 20). The resolution upon using DMDH rather than PH for HmuO is less dramatic than for hHO because, in contrast to hHO (20, 21), there are no detectable changes in the intrinsic line width of the signals in the DMDH relative to the PH complexes of HmuO, only the loss of the second set of minor compound signals. The narrower lines for the HmuO complex compared with the HO complex are attributed in part to a reduction in size (216 versus 265 residues) relative to hHO. An example of the narrower line widths in the HmuO complex compared with the hHO complex is the detection of the complete Ser138 TOCSY connections (data not shown; see Supplemental Material), including the NH-Calpha H correlation missing in hHO·DMDH·CN (21), despite unchanged paramagnetic relaxation.

Utility of hHO as a Homology Model-- The similarity in the 1H NMR spectra of hHO·PH·CN and HmuO·PH·CN in Fig. 3 is completely confirmed by the remarkable similarities in not only the positions of the various secondary structural elements represented by the homologous fragments I, II, and IV-VI, as reflected in dipolar contacts among each other and with the heme (Fig. 6), but also the pattern of paramagnetic relaxation and hyperfine shifts (Table II). The similar heme methyl contact shifts (Table I) reflect a similarly oriented axial His imidazole plane, and the conserved pattern of dipolar shifts for the proximal helix (Table II) confirms an ~20° steric tilt of the FeCN toward the alpha -meso position in both mammalian HO (20, 21) and this bacterial HO.

The close similarity of the environments of the individual heme methyls not only reflects the numerous completely conserved contacts, but allows the ready identification of the HmuO residues whose nature has been dramatically altered compared with hHO residues, i.e. hHO Ile211 right-arrow HmuO His205 in Fig. 5 (C and D) and hHO Gln38 right-arrow HmuO Leu33 in Fig. 5 (A and B). Fragment III (Fig. 2) could not be located in analogy with hHO because fragment III possesses three aromatic residues (Phe95, Trp96, and Tyr97) in hHO that could be easily identified (21) by their contacts with fragment IV, and these residues on fragment III are substituted by aliphatic residues (Lys89, Leu90, and Asn91) in HmuO. The cluster of aromatic side chains, i.e. those of HmuO/hHO Tyr53/Tyr58, Phe160/Phe166, Tyr161/Phe167, Tyr130/Tyr134, Tyr133/Tyr137, and Phe42/Phe47, are again largely conserved in the two HO proteins (Fig. 6). Finally, the acceptor for all but one (Tyr161 Oeta H) of the assigned strong H-bond donors could be identified in HmuO complexes solely on the basis of sequence homology. We therefore conclude that a structurally characterized HO complex will serve as a valuable homology model to facilitate the assignment of residues involved in many details of the active site structure in a related HO. The sequence homology between HmuO and hHO is relatively high (33% identity and 70% similarity if conservative substitutions are included) (10). Other bacterial HOs, such as HemO from N. meningitides (44), exhibit less sequence homology to mammalian HOs, but still exhibit a structure (45) that is related to that characterized in the two mammalian HOs (15, 16) and one bacterial HO (45) and exhibit different details of the active site. To date, 1H NMR data on HemO (44, 45) have not been reported to allow comparisons.

Comparison with the HmuO·PH·H2O Crystal Structure-- The 1H NMR data are consistent with the crystal structure2 for HmuO to the same degree previously found for the same hHO complexes (20, 21). The proximal helix is strongly conserved, but the distal helix exhibits dipolar shifts that deviate from those predicted by the relatively robust magnetic axes in the same fashion as found for hHO (see Supplemental Material) (20, 21). The loss in solution of dipolar contacts between the NH groups of Leu134 and Gly135 and 3-CH3 and the weakening of contacts between Ser138 Cbeta H and 6-Halpha (Fig. 7B) relative to predictions based on the crystal structure2 suggest possibly only a small (0.5-1.0 Å) movement of the distal helix near its kink. However, similar differences in the distal helix position have been observed in the two non-equivalent molecules in the hHO·PH·H2O crystal (15) and may simply represent the intrinsic mobility of the distal helix.

The distal H-bond network in the HmuO complex,2 as in the case of hHO·PH·H2O, is not readily discerned in the crystal structure (14, 15). However, once the donor NH and OH groups have been identified by 1H NMR, the crystal structure readily identifies the probable acceptors. The proposed acceptors for the strong H-bonds in HmuO are the Glu57, Asp86, Asp136, and Glu196 carboxylates, based solely on the homology to hHO·DMDH·CN NMR data (21) and the hHO·PH·H2O crystal structure (15) and completely confirmed in the HmuO crystal structure (Fig. 6).2 The crystallographic geometry (distance and angle)2 for these H-bonds in HmuO is summarized in Table III, where they can be compared with similar data on hHO·DMDH·CN and hHO·PH·H2O. Their dispositions are far from ideal (42) to allow the strong H-bond so obvious in the 1H NMR data, as shown in Table III. This non-ideal orientation of the donors and acceptors may be simply the result of the intrinsic uncertainties in the crystallographic positions of the two interacting units. The acceptor for the new strong H-bond from Tyr161 Oeta H is identified in the crystal2 as the side chain of Gln46 (Fig. 6). Similar strong H-bonds, as yet unassigned, appear in the 1H NMR spectra of apo-HO in both mammals and bacteria,3 indicating that the H-bond network plays a key role in the structures of both the apo-HO and substrate complexes.

A structural difference of possible significance between the HmuO cyanide-ligated complex and the crystal structure of the aquo-ligated complex2 is the relative position of fragment IV relative to the distal helix II. This same difference was previously observed in the hHO complex (20, 21). Thus, although moderate intensity NOEs are observed between the Tyr161 ring and Gly139 NH (i.e. rij ~ 4 Å), the crystal structure indicates rij > 6 Å. This same difference was previously observed for the analogous hHO complexes involving the homologous Phe167 ring and Gly143 NH (20, 21). Thus, the aromatic cluster appears to move ~2-3 Å closer to the distal helix in the cyanide complex in solution compared with the aquo complex in the crystal. This difference may be due to the different ligands used in the alternate studies in solution (CN-, a H-bond acceptor) and in the crystal (H2O, a H-bond donor).

Ordered Water Molecules-- NOEs indicative of nearby immobilized water molecules (Fig. 4, A, B', and C') (43) are observed for the NH groups of Arg79 and Phe160 and the Nepsilon H groups of His128 and Trp50. Water molecules are indeed observed close to the NH groups in the crystal structure.2 Similar water NOEs are observed for the NH groups of the homologous Arg85 and Phe166 in hHO and His132 Nepsilon H (the Tyr homolog of Trp50 was not assigned in hHO) (22). Hence, both HOs are characterized by ordered water molecules, particularly in the distal pocket. However, even these preliminary data indicate differences between the two HOs in the organization of these water molecules. Thus, the NOE for His Nepsilon H is weaker in HmuO (His128) than in hHO (His132) (21), but the NOEs for the NH groups of Arg85 and Phe160 are significantly larger (~25-30%) in the HmuO complex (Fig. 4C') than in the hHO complex (~10-15%) (22). These differences could be the result of differences in water-NH distances, the number of nearby water molecules, and/or the mobility of the ordered water molecules (43). The crystal structure2 of HmuO·PH·H2O reveals numerous water molecules in the distal side of the heme at positions similar to those detected by 1H NMR in the hHO complex (22). The different stages of refinement for the hHO and HmuO crystal structures and the availability of only preliminary NMR data in solution suggest that a discussion of differences in the occupation of water molecules be deferred until water NOEs can be more effectively studied in 15N-labeled HO.

Comparison of Dynamic Properties of HmuO and hHO-- The very close structural homology between HmuO and hHO apparent in both the solution 1H NMR data and crystal structures is, however, in contrast to the highly differential dynamic properties of the two enzymes. On the one hand, the rate of exchange with 2H2O of homologous labile protons involved in the strong H-bonds differs significantly for the two enzymes, with HmuO exhibiting significantly reduced rates. Comparison of the homologous NH groups shows that the half-lives for a residue are ~4 h for HmuO His128 Nepsilon H and 45 min for hHO His132 Nepsilon H (22), 30 min for HmuO Arg79 NH and 25 min for hHO Arg86 NH (22), and ~700 h for HmuO Phe160 NH and ~1 h for hHO Phe166 NH (22). Moreover, the Tyr53/Tyr58 OH groups, which exhibit saturation transfer due to exchange, exhibit a much smaller saturation factor (~10%) in HmuO than in hHO (~40%), dictating a much slower exchange rate in HmuO. The ~700 factor decrease in the Phe160/Phe166 NH exchange rate indicates that the dynamic stability near fragment IV is ~4 kcal greater in HmuO than in hHO (46). Although the extreme low-field shifts for the labile protons of the H-bond networks are very similar in the two HO complexes (Table III), indicating that the individual H-bonds are comparably strong, other factors that contribute to the stability of the folding in the environment of the network are clearly much weaker in hHO than in HmuO.

The 1H NMR data provide other indicators for a dynamically more stable (and hence, less flexible) HmuO than hHO. On the one hand, two new and strong H-bonds are observed, one of which could be uniquely attributed to Tyr161 Oeta H (which substitutes for Phe167 in hHO). The likely acceptor, Gln46, is suggested by the HmuO crystal structure,2 although the strong low-field bias due to H-bonding suggests a stronger or more than one acceptor. In fact, the crystal structure2 of HmuO·PH·H2O places a water molecule within 3 Å of this OH. The sequence origin of the other strong H-bond (peak a at 12.4 ppm in Fig. 4A) is not identified, but has no homolog in hHO. Nevertheless, these two strong H-bonds in HmuO are incremental over those conserved relative to hHO (21, 22). The second observation is that the Tyr161 ring, unlike the Phe167 ring, exhibits slow ring reorientation about the Cbeta -Cgamma bond, as evidenced by the resolution of two Cepsilon H peaks at low temperature, whereas an averaged Cdelta H peak is observed for Phe166 at all temperatures for hHO complexes (20, 21). The decreased mobility of Tyr161 in HmuO relative to the Phe167 ring in hHO (21) in 2-fold reorientation supports a tighter and more constrained distal environment in HmuO than in hHO.

Role of the H-bond Network-- The conservation of the H-bond network/aromatic cluster/ordered water molecules in HmuO relative to hHO argues for important functional roles. The existence of a network of water molecules that includes water molecules near the catalytically critical distal helix Asp136/Asp140 (12, 14) supports the notion that water provides the stabilizing H-bond to the novel hydroperoxy intermediate. Interaction of Asp140 with the distal ligand via two water molecules has been recently characterized in the crystal structure of the rat HO·PH·N3 complex (23). The presence of a water/H-bond network that extends from the distal pocket through the enzyme to its surface on the opposite side from the substrate-binding pocket (22)2 suggests that the channel may funnel the required nine protons to the active site in a controlled manner.

The greater dynamic stability of the pocket near the catalytically important Asp136/Asp140 is also apparent in two other observations. The greater dynamic stability of the distal pocket in HmuO relative to hHO, witnessed in both slower labile proton exchange and aromatic ring reorientation, may be responsible for the ~4 factor slower turnover rate in HmuO (30) than in mammalian HOs (2, 11). More extensive NMR studies of both the dynamic properties of the distal side and the distribution of oriented water molecules in HmuO and other HO complexes are in progress.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM62380 (to G. N. L.) and GM57272 (to M. I.-S.) and Grants-in-aid from the Ministry of Education, Culture and Sports, 12157201 and 14380300 (to M. I.-S.).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-4S.

§ Present address: Amgen, Inc., Thousand Oaks, CA 91320.

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

Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M211249200

2 S. Hirotsu, G. C. Chu, D.-S. Lee, M. Unno, T. Yoshida, S.-Y. Park, Y. Shiro, and M. Ikeda-Saito, manuscript in preparation (Protein Data Bank code 1WI0).

3 Y. Li, R. T. Syvitski, G. C. Chu, M. Ikeda-Saito, and G. N. La Mar, unpublished data.

    ABBREVIATIONS

The abbreviations used are: HO, heme oxygenase; hHO, human heme oxygenase; PH, protohemin; DMDH, 2,4-dimethyldeuterohemin; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate; NOE, nuclear Overhauser effect; NOESY, two-dimensional nuclear Overhauser effect spectroscopy; TOCSY, two-dimensional total correlation spectroscopy.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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