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 Mar
§,
Anbanandam
Asokan
,
Bryan
Espiritu
,
Deok
Cheon
Yeh
,
Karine
Auclair¶, and
Paul R.
Ortiz de
Montellano¶
From the
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 |
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
-
-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
-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
-meso bridge, demonstrating that
the close placement of the distal helix over the heme exerts control of
stereospecificity by both blocking access to the
,
, and
-meso positions and tilting the axial ligand, a
proposed peroxide, toward the
-meso position.
 |
INTRODUCTION |
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,
-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
-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
-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
-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
-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-C
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 |
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
|
(Eq. 1)
|
using the heme for the
-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,
(
,
, and
), 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,
, is diagonal, i.e. (x, y, z) = (x', y', z')
(
,
,
), where
,
, and
are the three Euler angles
(26-29, 32, 35). Angle
dictates the tilt of the major magnetic
axis, z, from the heme normal z' (Fig. 1B), angle
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
~
+ b
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):
|
(Eq. 2)
|
where the calculated dipolar shift in the reference coordinate
system, x', y', z', or R,
'
', is given by:
|
(Eq. 3)
|
with 
ax and

rh as the axial and rhombic
anisotropies of the diagonal paramagnetic susceptibility tensor. The
observed dipolar shift,
dip(obs), is given by:
|
(Eq. 4)
|
where
DSS(obs) and
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
DSS(dia), it may be reliably estimated from the
available molecular structure:
|
(Eq. 5)
|
where
tetr,
sec, and
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 |
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
-
-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 m28
)
isomers are significantly broader than those of the major
(M3,M28
) 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.

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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
, -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 is
diagonal. The Euler angles in A (the angle between the
projection of the z axis on the x', y' plane and
the x' axis), in B (tilt of the major
magnetic axis from the heme normal), and in A (= + ) 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 in
A).
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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,
m28 , and h2 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), - (meso-H,
i.e. Ha = -meso-H), or the residue number and
proton (M28A = Ala-28
C 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-C
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
-helices (24, 25), we use the expected (40) characteristic
strong intra-residue Ni-
i, backbone
inter-residue strong Ni-Ni+1,
i-Ni+1, moderate
i-Ni+3,
i-
i+3,
and weak
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 H
s of the two
propionates (18). Here we can resolve the individual H
s
and confirm all previous heme assignments. The only sequence-specific
assignment offered previously was for the axial His-25
C
HC
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
-
-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 X
8-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
C
HC
H2C
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 C
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 C
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-H
(160 ms),
2H
c(~200 ms), 4H
(160 ms),
6H
(150 ms), 7H
(150 ms), and
-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 C
Hs (~80 ms),
Ser-142 C
1H (85 ms), Ser-142
C
2H (50 ms), Glu-29
C
1H (~80 ms), Gly-143 C
H
(~80 ms), and Ala-28 C
H3 (200 ms). The
paramagnetic contribution to the relaxation of heme substituents is
small to negligible for all pyrrole substituents. However, the
-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 2H
(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
C
HC
H2C
H2
fragment (Fig. 4B), which, in turn, exhibits NOESY cross-peaks to 3-CH3 (Fig.
4K) and 4H
(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
C
HC
H2 fragment (Fig. 3,
C and E), and the TOCSY-detected
C
HC
HC
H3
fragment exhibits the NEOSY cross-peak to Nj+2
(N26) characteristic of Thr-26 (Fig. 3E). The
C
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
C
H3s. A strong NOESY cross-peak from the
C
H of the low-field shifted
C
HC
H2C
H2
fragment (previously residue Q (18)) to the His-25 C
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)
i-Ni+3 and
i+
i+3 connection for i = 25 (data not shown) and 26 (Fig. 3B) and
22-
25 cross-peaks (Fig. 3E); the latter connections also provide stereospecific assignment of the
His-25 and Glu-29 C
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.

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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 i-Ni+1,
i-Ni+3, and
i- 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 N 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 - 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.
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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 N 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.
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Fig. 5.
Schematic summary of observed
Ni-Ni+1, i-Ni+1,
i-Ni+1, i-Ni+3,
and/or i- 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).
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The confirmed axial His-25 C
H and C
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 C
H (data not shown) and
C
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 C
Hs closer to the iron
in the crystal structure. The Thr-21
C
HC
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),

ax = 2.48 × 10
8 m3/mol and

rh =
0.58 × 10
8
m3/mol, as input, the orientation of the magnetic axes was
determined (26) by the three-parameter (
,
, and
) least square
search for the minimum in the error function in Equation 2. The results yield
= 234 ± 4,
= 20 ± 2, and
=
+
= 25° ± 13° when using the crystal molecule A
coordinates and
= 238 ± 11,
= 19 ± 3, and
=
+
= 11° ± 14° when using the crystal B
molecular coordinates. Both result in acceptably low residual error
functions and good correlation between
dip(obs) and
optimized
dip(calc) in Table I, as shown in Fig.
6, A and B. The
direction and magnitude of
that defines the rhombic axes are
qualitatively consistent with the expectation (43) based on the
orientation of the axial His-25 imidazole orientation angle
~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
dip are
expected only for the distal Thr-135, Leu-138, Gly-139, and Ser-142,
whereas significant (>1 ppm) low-field
dip are expected
only for distal Asp-140, Gly-143, Gly-144, and Leu-147. The detailed
predicted
dip values are listed in Table I. Differences
in
dip(calc) for the two molecules in the unit cell will
be considered when the relevant residue is assigned.

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Fig. 6.
Plot of
dip(obs) versus
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
dip(calc) versus 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.
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|
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
C
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
C
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
C
1H. This assignment is confirmed upon
assignment of Ser-142 below. The expected
138N139 cross-peak (Fig. 4D)
confirms the direction of the helical fragment. The side chain of
Asp-140 (whose C
H should exhibit a significant low-field
dip; see Table I) and Leu-141 (which does not exhibit
dip) were not located. The Leu-138 side chain is
expected to make extensive dipolar contact to 5-CH3
(8-CH3) and 6H
s (7H
s), and
such contacts are observed between the moderately relaxed C
Hs and 8-CH3 (Fig. 4I) and
7H
(Fig. 4H). The two C
Hs are
assigned stereospecifically based on the stronger relaxation of
C
H and the observed
'138-N139
NOESY cross-peak. Whereas both Leu-141 and Leu-147 are expected to
exhibit NOEs to 3-CH3 (2H
), 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
C
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
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 7H
and 7H
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 C
H, and to a labile proton peak e at 8 ppm
(Fig. 7A), its likely NH, and to the 6-propionate
H6
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
C
H at 2.5 ppm. The nearly identical T1s
(~50) for
-meso-H and Ser-142
C
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 C
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
C
Hs stereospecifically. Weak NOESY cross-peaks are
detected between the Ser-142 C
Hs and the proposed
C
H of Gly-139 (data not shown; see supplemental material
(Figs. 1S and 2S)), as expected (Fig. 5B).

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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).
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|
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 C
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
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), C
2H of Ser-143 (T1 = ~50 ms, RFe = ~4.6 Å), and
-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 C
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
dip for Distal Residues--
The
results of predicting
dip(calc) for the assigned distal
residues, using the magnetic axes according to either molecule A or B,
and the comparison with
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 C
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
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
Phe-167
Tyr-58
Tyr-137. The magnetic axes for
either molecule A or B predict moderate (~1-2 ppm) low-field
dip for the two Phe (and a large, upfield ring current
shift for one) and small to negligible
dip for the two Tyr, as observed (Table I). NMR had originally detected these four
aromatic rings (cluster 4A (18)) and their interactions, PheA
PheD
and AromC
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)
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 C
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
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 C
H of a complete AlaX (NH,
C
H, and C
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.

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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
C 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.
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 |
DISCUSSION |
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
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
dip. In this respect, the magnetic axes
and predicted
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
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
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
-
-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
-
-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
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
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
dip(calc) is due to
the position of Gly-143 NH near the magic angle of the magnetic axes
where
dip
0. The much larger
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
dip(obs). Such a small translation of the distal helix
would still result in a sterically hindered access to
-,
-, and
-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 (
= 230° ± 10°) of the
-meso position (
= 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
-meso position. The
strong tilt of the Fe-CN unit toward the
-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
,
, and
positions, as shown in the crystal structures (24, 25). On the
other hand, as documented herein, the bound ligand is sterically tilted
toward the
-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
-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), m28
(Ala-28
C
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 h2
s and H2
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
C
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 C
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
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.
 |
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