1
H NMR study of the molecular structure and magnetic properties
of the active site for the cyanomet complex of O
2
-avid hemoglobin
from the trematode
Paramphistomum epiclitum
Weihong Du
1
, Zhicheng Xia
1
, Sylvia Dewilde
2
, Luc Moens
2
and Gerd N. La Mar
1
1
Department of Chemistry, University of California, Davis, CA, USA;
2
Department of Biomedical Sciences,
University of Antwerp, Wilrijk, Belgium
The solution molecular and electronic structures of the active
site in the extremely O
2
-avid hemoglobin from the trematode
Paramphistomum epiclitum have been investigated by
1
H
NMR on the cyanomet form in order to elucidate the distal
hydrogen-bonding to a ligated H-bond acceptor ligand.

2
-binding proteins found widespread in
nature [1,2]. They exhibit an extraordinary range of ligation
rates and affinities, as well as autoxidation rates (conversion
to the nonfunctional ferric hemin) in spite of a highly
conserved folding topology (the Mb fold). The majority of
globins, which consists of % 150 residues, are arranged in a
compact globule consisting of eight (A–H) helices, with the
heme wedged between the E and F helices. A completely
conserved His F8 (eighth residue on helix F) provides the
only covalent bond to the protein, although a conserved
aromatic ring (CD1) (first residue and the loop between
helices C and D) provides considerable stabilization by
p-stacking on the heme [1–3]. Some recently discovered
ÔtruncatedÕ (% 100–120 residues) globins from bacteria
exhibit the general Mb fold but retain only four of the
helices, leaving a largely conserved active site with respect to
more conventional globins [4,5], and one has an unprece-
dented Tyr (CD1) [3]. The modulation of the extreme range
of ligation rates in monomeric globins appears to be
controlled primarily by limited sets of residues on the distal
(opposite side to the proximal His F8) side of the heme,
which determine the distal pocket polarity, provide stabi-
lizing H-bonds to ligands and/or sterically interfere with
ligand binding [6]. Among the extensively studied mono-
meric mammalian Mbs, the key residues have been identi-
fied at positions E11 and E7 (generally His, but occasionally
Gln [7]), where the latter provides the crucial H-bond to
stabilize O
2

Abbreviations: Hb, hemoglobin; Mb, myoglobin; WT, wild type; rWT,
recombinant WT; metHbCN, cyanide ligated ferric hemoglobin;
NOE, nuclear Overhauser effect; DSS, 2,2-dimethyl-2-silapentane-
5-sulfonate; WEFT, water-eliminated Fourier transform;
Pe, Paramphistomum epiclitum.
Note: a website is available at http://www.chem.ucdavis.edu/faculty/
(Received 20 December 2002, revised 24 March 2003,
accepted 28 April 2003)
Eur. J. Biochem. 270, 2707–2720 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03638.x
high O
2
affinity via an exceptionally slow off-rate. Their off-
rates are too slow to allow these globins to participate in
aerobic respiration, and their exact physiological role is
openly debated [14,15,20]. The nematode/trematode globins
share the property of a Tyr at position B10, which in
conjunction with or without the E7 H-bond donor, provides
an extremely strong H-bond to bound O
2
[11,12,16]. It must
be noted, however, that a Tyr B10 occurs in other globins
such as native Lucina pectinata (Lp) Hbs [21], with ÔordinaryÕ
O
2
affinity, where the B-helix is too far from the heme to
allow a strong H-bond. A Tyr B10 has been incorporated
into a mammalian Mb variant, where its relatively minor
influence on O
2
affinity was attributed to the B-helix being

have not been prepared to
date, the oxidized Pe wild type (WT) and recombinant
wild-type (rWT) metHb have crystallized in two different
forms [16,17], and the detailed structures provide import-
ant information on the novel Hb. The Tyr66(E7) ring is
oriented out of the heme pocket in both forms with the
Tyr32(B10) in one of the structures [16,17] serving as a
H-bond acceptor to a ligated water molecule. The two
structures of WT metHb and rWT metHbH
2
O, exhibit
significant differences in the interaction of the FG loop
with the heme and in the position of the B-helix, with
Tyr32(B10) closer to the iron by 1–2 A
˚
in WT than rWT
metHb, such that the ligated water is lost [16,17]. The
sizable structural accommodation to crystal forms for Pe
metHb is unprecedented and reflects a surprising Ôplasti-
cityÕ whose functional relevance is unknown. The differ-
ences in the two structures could be rationalized by
interactions between two molecules in the unit cell in one,
but not the other, crystal form [16,17].
Spectral congestion precludes more definitive
1
HNMR
structural studies of diamagnetic Pe WT HbO
2
at present
[23], and the molecule does not crystallize. Hence, we have

Protein samples
The monomeric wild-type (WT) hemoglobin, labeled WT
Hb, from the trematode Paramphistomum epiclitum (Pe)
and Isoparorchis hypselbagi (Ih) were isolated and purified
as described previously [14]. The cyanomet complexes were
prepared by adding approximately five molar equivalents of
KCN to the air-oxidized Hb. The final concentration of Pe
metHbCN complex was % 2m
M
and that of Ih metHbCN
was 0.2 m
M
.The
1
H
2
O solution was subsequently converted
to
2
H
2
O solution using an Amicon ultrafiltration cell.
Solution pH was adjusted with NaO
1
H(NaO
2
H) or
1
HCl
(

) from the iron, R
FeH
, was estimated from the relation:
R
FeÀH
¼ R
Ã
FeÀH
½T
Ã
1
=T
1i

1=6
ð1Þ
where R*
Fe-H
is the distance for a reference proton with T
1
*.
Using both the heme 18-CH
3
for H* (R*
Fe
¼ 5.88 A
˚
,
T
1

Fourier transformation.
2708 W. Du et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Magnetic axes determination
Experimental dipolar shifts for the structurally conserved
residues and backbone protons were used as input to search
for the Euler rotation angles a, b and c. These transform the
molecular pseudosymmetry coordinates x¢, y¢ and z¢ (Fig. 1)
obtained from crystal coordinates [16,17] into the magnetic
axes x, y and z, by minimizing the error function according
to the following equation [28–30,44]:
F=n ¼
X
½jd
dip
(obs) À d
dip
(calc)Fða; b; cÞj
2
ð2Þ
where
d
dip
ðcalcÞ¼ð12plN
o
Þ
À1
½2Dv
ax
ð3cos
2

(obs) are available from Pe
HbO
2
[23]; for other residues d
DSS
(dia) may be reliably
estimated from the available molecular structure [8,28,29]
as follows:
d
DSS
ðdiaÞ¼d
tetr
þ d
sec
þ d
rc
ð5Þ
where d
tetr
, d
sec
and d
rc
are the chemical shifts of an unfolded
tetrapeptide relative to DSS [45], the effect of secondary
Fig. 1. Schematic representation of the heme pocket of Pe Hb as found in the crystal structure of Pe metHbH
2
O [16] and as confirmed herein by
solution
1

H
2
O are illustrated in Fig. 2A,B. A WEFT spectrum [39]
designed to emphasize strongly relaxed signals is shown in
Fig. 2C. Comparison of the traces in Fig. 2A,B reveals
the presence of two strongly relaxed labile protons at
32.5 p.p.m. (T
1
% 10 ms) and 17.5 p.p.m. (T
1
% 35 ms),
as well as a weakly relaxed one at 11.4 p.p.m and several
inconsequentially relaxed peaks in the 11–9 p.p.m. win-
dow. The
2
H
2
O WEFT trace in Fig. 2C locates a broad
Fig. 2.
1
H NMR spectra (600 MHz) of Pe WT metHbCN at 30 °C, pH » 7.0. (A) Relaxed (repetition rate 1 s
)1
) reference trace in
1
H
2
O;
(B) relaxed, reference trace in
2
H

are listed in Table 2. T
1
values for predominantly
paramagnetically influenced protons are given in paren-
theses.
Heme assignments
TOCSY spectra (not shown) identify four (two three-spin
and two four-spin) hyperfine shifted and relaxed spin
systems with dipolar contacts (not shown) to four strongly
temperature-dependent methyl peaks [two resolved (Curie
behavior) and two nonresolved methyl peaks (antiCurie
behavior)], that uniquely identify the pyrrole substituents
[30]. Dipolar contacts to adjacent meso-Hs (5-H, 10-H,
15-H, 20-H), with their unique low-field intercept at
T
)1
¼ 0, locate the four meso-Hs (as listed in Table 1).
Sequence-specific assignments
The detection of the N
i
–N
i+1
, a
i
–Ni
+1
, b
i
–N
i+1

significant low-field dipolar shifts. The relaxed, low-field
labile proton at % 17 p.p.m. exhibits a NOE to the AMX
i+5
and its peptide NH that is unique for the proximal
His98(F8), and AMX
i+1
is in dipolar contact with a two-
spin aromatic ring, as expected for Tyr94(F4); this identifies
I as Gln93–His98 of the proximal F-helix(F3–F8). Further
backbone (nonhelical) dipolar connections (Fig. 3) allow
the adjacent assignments of Thr99–Val103, the residues that
constitute the FG corner (FG1–FG5), with expected dipolar
contacts to pyrroles B and C (Fig. 1) and residues in the
C and G helices (see below). Fragment II is represented
by AMX
i
-Ala
i+1
-Z
i+2
-Thr
i+3
-Leu
i+4
-Z
i+5
-Z
i+6
-Ala
i+7

AMX
i+4
-Thr
i+5
-Z
i+6
-AMX
i+7
-AMX
i+8
(Fig. 3) where
contacts of three-spin aromatic rings to AMX
i+4
and
AMX
i+8
and a two-spin aromatic ring to AMX
i+7
uniquely identify Gly111–Phe119 on the G-helix (G8–
G16). An additional AMX spin-system connected to a
hyperfine shifted aromatic ring identifies a Phe, and its
backbone exhibits the a
i-3
-N
i
cross peak to AMX
i
in III
(Gly111); this identifies it as Phe108(G5). The side chains
exhibit the expected NOESY cross peaks to the pyrrole A/B

-
Z
i+2
-Z
i+3
-AMX
i+4
(Fig. 3), where dipolar contacts of a
two-spin aromatic ring to AMX
i+1
(and to the 7-CH
3
and
8-vinyl; Fig. 1) identifies the Gln41–His45 fragment on the
C-helix (C3–C7), with the expected moderate dipolar shifts,
and which is in contact with the pyrrole B/C junction
(Fig. 1). Backbone NOESY connections allow the extension
of sequential assignment of fragment V to include AMX
i+5
-
Ser
i+6
that must arise from Phe46(CD1) and Ser47(CD2).
Table 1.
1
H NMR spectral parameters for the heme and His98(F8)
signals in Pe metHbCN. Chemical shifts in p.p.m. are referenced to
DSS in
1
H

8-H
a
8.83
8-H
b
s )1.22 [186], 0.29
13-H
a
14.32 [110], 5.44
13-H
b
s )3.34 [152], )2.63 [165]
17-H
a
s 13.29 [92], 4.86
17-H
b
s )3.80 [130], )169 [155]
Heme 5-H )1.02 [52]
10-H 8.41
15-H )0.09
20-H 6.23
His98(F8) NH 12.81 [129]
C
a
H 8.05
C
b
H 8.67
C

(dia)
Tyr32(B10) NH
8.50 7.41
C
a
H 5.04 3.27
C
b
H¢ 3.90 2.44
C
b
H 3.60 2.32
C
d
Hs 8.50 5.87
C
e
Hs 11.79 5.48
OH
32.51 8.12
Phe36(B14) NH
9.60 8.07
C
a
H 5.01 4.08
C
b
Hs
3.43, 3.32 3.12, 2.80
C

4.45
C
b
H 1.95 3.21, 2.98
C
d
H 5.89 7.50
Phe46(CD1)
NH
7.33 8.10
C
a
H 3.01 4.74
C
b
H 2.15 2.75, 2.67
C
d
Hs 6.15 7.09
C
e
Hs 8.88 6.04
C
f
H 11.42 6.44
Tyr66(E7)
NH
7.08 7.70
C
a

C
a
H 3.44 4.03
C
b
H 3.04 4.45
C
c
H
3
0.15 1.05
Leu70(E11)
NH
9.80 8.49
C
a
H
na
3.29
C
b
H 3.33 0.62
C
b
H¢ 6.08 0.91
C
c
H 2.8 0.56
C
d

DSS
(obs) d
DSS
(dia)
C
b
H¢ 3.25 2.76
C
d
Hs 7.40 7.71
C
e
Hs 6.57 7.16
Gly95(F5) NH 10.47 7.66
C
a
H 5.71 3.11
C
a
H¢ 6.80 2.26
Lys96(F6) NH 9.8 7.45
C
a
H 5.78 3.47
C
b
H 2.90, 2.79 1.62, 1.49
C
c
Hs 2.13 1.09

C
a
H 5.23 3.55
C
b
H 5.00 3.91
C
c
H
3
2.12 0.68
Val103(FG5) NH 6.81 8.06
C
a
H 3.50 4.08
C
b
H 0.79 2.67
C
c
H
3
0.54 )0.09
C
c
H
3
¢ )0.05 1.21
Phe108(G5) NH 8.08 8.14
C

Hs 6.52 7.10
C
e
Hs 6.82 6.96
C
f
H 8.96 5.82
Phe140(H16) NH 8.08 8.21
C
a
H 3.16 4.31
C
b
H 2.16 3.01, 3.31
C
d
Hs 6.19 7.39
C
e
Hs 4.95 7.46
C
f
H 4.38 7.90
Met143(H19) NH 7.49 7.89
C
a
H 4.49 3.64
C
b
Hs 2.28 0.98, 1.34

Fig. 1 and as found in the crystal structure. The C
f
Hof
Phe46(CD1) exhibits the strong relaxation (T
1
% 20 ms)
characteristic for this residue.
The remaining helical segment VI,Z
i
-Thr
i+1
-Gly
i+2
-
Z
i+3
-Gly
i+4
-Ala
i+5
-AMX
i+6
-AMX
i+7
-Ala
i+8
-Z
i+9
-
AMX

summarized in Fig. 1, unequivocally establish that the
Tyr66(E7) ring is oriented out of the heme pocket, exactly
as found in both Pe metHb crystal structures [16,17]. The
position is further supported by the calculated and
observed small d
dip
(and hence, negligible temperature-
dependence to its shifts) for the crystallographic orienta-
tion of the Tyr66(E7) ring (see below). While its hydroxyl
proton could not be located by its characteristic strong
NOE to the definitively assigned C
e
Hs, most likely due to
its lability, there is no orientation of the OH group that
can bring it close enough (>5 A
˚
)tointeractwiththe
bound cyanide.
The interresidue and residue-heme NOESY cross peak
pattern that led to the schematic representation of the Pe
metHbCN heme cavity structure in Fig. 1 is equally
consistent with qualitative expectations of either of the
two Pe metHb crystal structures [16,17]. It is only upon
quantitative consideration of cross peak intensities that
such detailed structural distinctions can be made. The
two X-ray structures, one of WT and the other of rWT
metHb, exhibit differences that include important por-
tions of the protein that we have characterized above.
Thus parts of the FG corner move away from the heme
and the B-helix moves closer to the heme in the WT

and E-helix backbone. Table 3 lists the alternate r
ij
for eight
sets of proton pairs in the two structures, as well as the
observed NOESY cross peak intensity (s, < 2.5 A
˚
;m,2.5–
4.6; w > 4.0 A
˚
). In each case, the distance in bold is the one
in better agreement with the experiment, and each of the
four distances dictate that the solution structure of WT
metHbCN is consistent only with the crystal structure of
rWT metHbH
2
O [16] (except for a labile proton on
Tyr32(B10), see below). The alternate structures predict
characteristic relaxation time differences for several proton
sets in the alternate crystal structure, i.e. Tyr32(B10),
Thr99(FG1), Val103(FG5), but in only one case is the key
resonance resolved so that its T
1
can be quantitated. Thus
the movement of the B-helix towards the heme in the WT
metHbrelativetothatintherWTmetHbH
2
Ocrystal
Fig. 3. Schematic representation of the sequential NOESY cross peak
pattern for the six characterized helical fragments I–VI that identify key
sections of the F, E, G, H, C and B helices, respectively.

2
O, but not
the WT metHb crystal structure.
Magnetic axes
The orientation of the magnetic axes was determined by
using the d
dip
(obs) via Eqns (4) and (5) for Pe metHbCN.
The anisotropies at 30 °C, which have been shown to be
highly conserved in a wide variety of cyanomet globins
[8,26,28–30], are Dv
ax
¼ 2:48 Â 10
À8
m
3
Áms
À1
and Dv
rh
¼
À0:58 Â 10
À8
m
3
Áms
À1
, as reported for sperm whale met-
MbCN. The coordinates that determine R, h¢ and W¢ in
Eqn (3) were taken alternatively from the Pe rWT

O [16] (case I) or the WT metHb [17] (case II)
crystal structures. In order to utilize the information in d
dip
for distinguishing between the two crystal structures, the
experimental shifts and crystal coordinates initially used to
determine the magnetic axes were only for those protons
where the residue exhibited the same position in the
alternate crystal structures. The results lead to equally well-
determined orientations of a ¼ 203 ± 10, b ¼ 9° ±1,
j ¼ 50 ± 10 and residual F/n ¼ 0.14 p.p.m.
2
for case I,
and a ¼ 206 ± 10, b ¼ 10 ± 1, j ¼ 40 ± 10° and resid-
ual F/n ¼ 0.20 p.p.m.
2
for case II. The plot of d
dip
(obs) vs.
the d
dip
(calc) (

,j) for each set of magnetic axes are given
in Fig. 5A (case I) and 5B (case II), and each represents a
good fit. The differences in b do not reflect differences in tilt
of the axis so much as a small difference in the reference
coordinate system x¢,y¢ and z¢ in the two structures (due to
different nonplanarity of the heme). The d
dip
(obs) and

tion, a ¼ 202 ± 10, b ¼ 9±1 and j ¼ 51 ± 10 for
the five-parameter search that yielded Dv
ax
¼ 2:36 Æ
0:04 Â 10
À8
m
3
Á mol
À1
and Dv
rh
¼À0:59 Æ 0:06  10
À8
m
3
Ámol
À1
which are within the uncertainties of the
respective determinations (not shown). The tilt of the
major magnetic axes is correlated with Fe-CN tilt [8,28,30]
(with the negative z axis), and indicates that the cyanide is
tilted % 10° in the direction of the 5-H position. The
rhombic axes are defined by j % 50° in Fig. 1. The
difference in the overall shift dispersion pattern of Pe
metHbCN relative to, for example, any of the mammalian
metMbCN where both the FG corner and PheCD1
residues exhibit large upfield and downfield shifts, respect-
ively, is due to the smaller tilt, b.
Fig. 5. Plot of the d

3
Æ mol
)1
as reported
for sperm whale metMbCN [29]. The solid
markers represent the input data for the
structurally conserved protons, while open
markers are for those protons whose positions
differ significantly in the two crystal structures.
Table 3. Comparison of predicted and observed NOESY cross peak
intensity for the two crystal structures of Pe metHb. Inter-proton
separation r
ij
(A
˚
). Pe rWT metMbH
2
O crystal structure [16], Pe WT
metHb crystal structure [17]. Observed NOESY cross peak intensities,
s (strong, r
ij
<2.5A
˚
), m (moderate, 2.5 < r
ij
<4.0A
˚
), weak (weak,
4.0 < r
ij

a
H(F6) 3.80 7.02 m
B-helix
C
a
H
2
(B10)-C
a
H(E8) 2.22 3.11 s
C
e
H2(B10)-C
b
H
3
(E8) 4.33/4.86 5.33 m
C
b
H
2
(B10)-C
b
H
1
(E7) 2.91 2.52 m
C
e
H
2

from
theironwithanangleof% 17° with the Tyr32(B10)
C
e
-C
f
-O-H plane; we define this angle w ¼ 0. In the
rWT metHbH
2
O crystal structure [16], the heme ligand
(water molecule) is an H-bond donor, while in metH-
bCN, it (cyanide) is an H-bond acceptor, so that a
significantly different OH orientation can be expected. A
plot of the effect of the angle, w, between the Tyr32(B10)
C
f
-OH and ring planes, on the three distinctive variables
that depend critically on the orientation of the OH group
is illustrated in Fig. 6. The shaded areas correspond to
the observed values of d
dip
(calc) (Fig. 6A), distance to the
iron, R
Fe
¼ 4.0–4.5 A
˚
(Fig. 6B), as indicated by
T
1
% 10 ms, and Tyr32(B10) OH to Tyr66(E7) C

interpretation that the structural differences between rWT
and WT Pe metHb in crystals result from the extensive
interaction between the two molecules in the unit cell for
WT metHb, rather than from significant structural
differences between isolated WT and rWT Pe Hb
molecules [17]. The distal Tyr66(E7) ring was found
oriented out of the heme pocket in both rWT metHbH
2
O
and WT metHb [16,17]. Our NMR data on Pe metHbCN
confirm that Tyr66(E7) is similarly oriented away from
the heme iron in a position essentially the same as in the
crystal structure with its O
g
H much too far removed
(>6 A
˚
) from the cyanide to provide a H-bond. The
failure to resolve the O
g
H signal for Tyr66(E7) can be
attributed to its expected rapid exchange with solvent.
While cyanide is a H-bond acceptor and a weak mimic of
O
2
, it does not induce a rearrangement of the Tyr66(E7)
ring into the heme pocket relative to the high-spin, metHb
complexes.
1
H NMR data on Pe WT HbO

Fe
, distance to Tyr66(E7)
C
d
H(via NOESY cross peak intensity) and /O-H-N angle as a function
of the C
f
O-H to aromatic plane dihedral angle, w, for the Tyr32(B10)
hydroxyl group, with the ring position as defined in the rWT metHbH
2
O
crystal structure [16] and confirmed for the WT metMbCN solution
structure described here. The shaded portions represent the observed
values (and their uncertainties) of the three variables. Note that a
simultaneous fit for all three variables occur only for
w % )140 ± 20°.
2716 W. Du et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Distal hydrogen bonding
It is noteworthy that for Pe WT metHbCN, only a single
labile proton (Tyr32(B10) (O
g
H) is found sufficiently near
the cyanide ligand to participate in H-bonding to the ligated
cyanide. The van der Waals surfaces for the Tyr32(B10) O
g
and the 10° tilted cyanide ligand are shown in Fig. 7 and
establish that the Tyr O
g
and cyanide N are in van der
Waals contact, with a (Tyr32(B10))O

H) composition [24,26,27,41,51] on the
heme electronic structure. Thus both the 18-CH
3
and 7-CH
3
exhibit two separate resonances in
1
H
2
O/
2
H
2
O mixtures,
whose relative intensities directly reflect the solvent isotope
composition. The splitting of the two methyls in 50%
1
H
2
O/
50%
2
H
2
O is illustrated in the insets to Fig. 2A, where the
larger heme methyl contact shifts result from a single
2
H
rather than the
1

3
Æmol
)1
,
Dv
rh
¼ )0.58 ± 0.04 · 10
)8
m
3
Æmol
)1
, and numerous of
its point mutants [26,30], and confirm the strong conserva-
tion of the magnetic anisotropies of low-spin hemin with
His/cyanide ligation. The 10° tilt of the major magnetic or
z-axis is shown to be consistent with the % 10° tilt of the
Fe-CN vector from the heme normal to avoid van der waals
overlap with the Tyr32(B10) O
g
. The location of the
rhombic axes, j % 50 ± 10°,isinagreementwiththe
expectations [30,35] of the counter-rotation principle which
dictates that the rhombic axes, j% 50°, rotate relative to an
N-Fe-N vector in opposite direction, but with the same
magnitude, as the axial His imidazole orientation relative to
the N-Fe-N vector, here given by / ¼ 52° (Fig. 1).
It has been proposed that the pattern of the meso-H
hyperfine shift is determined largely by dipolar shifts due
to the rhombic anisotropy [30,36,37]. The D(obs) ¼ 1/2[d

F8 imidazole is rotated % 45° clockwise relative to the
mammalian globin, and it is the His F8 rotation, not the
heme rotation, that leads to the low-field 7-CH
3
and 18-CH
3
signals. Hence it is clear that heme methyl assignments do
not yield information on heme orientation unless the axial
His orientation is known.
Dynamic properties
The remarkably slow autoxidation rate for Pe Hb has been
suggested [16] to result from a greater dynamic stability of
Pe Hb relative to other more autoxidizable globins. A
reliable indicator of the dynamic stability of distal heme
pockets in globins is the rate of reorientation of the aromatic
rings in the heme pocket [37,53]. The rate of reorientation
can be estimated by the excess line broadening at low-
temperature of the averaged C
e
H peaks if the chemical shift
differences are known [37,53]. Two such rings of interest are
Tyr32(B10) and Phe46(CD1). The TyrB10 ring of the
nematode Ascaris metHbCN with relatively ÔnormalÕ aut-
oxidation rate exhibits [37] % 450 Hz excess line broadening
at 15 °C that could be attributed to slow reorientation of the
ring. The difference in the TyrB10 C
e
H chemical shifts, as
Fig. 7. Model of the distal ligand environment as determined herein
which shows the van der Waals contact between the Tyr32(B10) side

the magnetic axes for sperm whale and Pe metHbCN,
indicating that the PheCD1 ring in Pe metHbCN reorients
>10 times faster than in the mammalian metMbCN
complexes [53,54]. Hence the heme pocket in Pe Hb does
not appear to be more dynamically stable than those of
other nematodes/trematodes or mammalian globins with
ÔordinaryÕ autoxidation rates. One possibility that cannot be
discounted is that the Pe Hb possesses a limited flexibility
that involves the position of the B-helix, as already
witnessed by the facility with which the position of the
B-helix and FG corner accommodates perturbations such
as interprotein contacts [17]. The limited ÔflexibilityÕ may be
required to allow the facile reorientation of the Tyr66(E7)
ring from ÔoutsideÕ the heme pocket in oxidized Pe globins to
ÔinsideÕ the pocket in Pe HbO
2
complexes, as observed [26] in
the
1
H NMR data of Pe HbO
2
. The static structure of
neither WT nor rWT metMb would allow the TyrE7 ring
reorientation without significant distortion of the heme
pocket.
Comparison to other nematode/trematode Hbs
The low-field portion of the
1
H NMR spectra of Pe
metHb-CN in Fig. 8A is compared with those for Ih

H NMR spectra. Dr S. Dewilde is a postdoctoral researcher of the
F.W.O. The results were supported by grants from the National
Institutes of Health, HL16087 (G.N.L.) and the Fund for Scientific
Research Flanders (F.W.O.), G0314.00 N (L.M.).
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)1
)in
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H
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O, 100 m
M
in phosphate, pH 6.8 at 35 °C, illustrating the E- and F-helix
N
i
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1
H NMR spectral parameters for active site res-
idues in Pe metHbCN.
2720 W. Du et al.(Eur. J. Biochem. 270) Ó FEBS 2003