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

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.

Comparison of the strengths of dipolar interactions in
solution with the alternate crystal structures of methemo-
globin establish that the solution structure of wild-type Hb
more closely resembles the crystal structure of the recom-
binant wild-type than the true wild-type met-hemoglobin.
The distal Tyr66(E7) is found oriented out of the heme
pocket in solution as found in both crystal structures. Ana-
lysis of dipolar contacts, dipolar shift and paramagnetic
relaxation establishes that the Tyr32(B10) hydrogen proton
adopts an orientation that allows it to make a strong H-bond
to the bound cyanide. The observation of a significant iso-
tope effect on the heme methyl contact shifts confirms a
strong contact between the Tyr32(B10) OH and the ligated
cyanide. The quantitative determination of the orientation
and anisotropies of the paramagnetic susceptibility tensor
reveal that the cyanide is tilted %10° from the heme normal
so as to avoid van der Waals overlap with the Tyr32(B10)
Og. The pattern of heme contact shifts with large low-field
shifts for 7-CH
3
and 18-CH
3
is shown to arise not from the
180° rotation about the a-c-meso axis, but due to the %45°
rotation of the axial His imidazole ring, relative to that in
mammalian globins.
Keywords: hemoglobin; trematode; H-bonding; dipolar
shift; NMR.
Globins (hemoglobin, Hb, and myoglobin, Mb) are ferrous
heme-containing, O

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

. Val E11 makes van der Waals contact with the
ligand and, in certain cases, may sterically destabilize ligands
[6,7]. The B10 residue is generally a Leu, except in elephant
Mb [8,9], where a Phe at B10 (and a Gln at E7) results in an
Mb with a relatively reduced autoxidazibility but conserved
O
2
affinity relative to other mammalian Mbs.
The globins from invertebrates exhibit much more vari-
ability in both the nature of the distal residues that provide
the stabilizing H-bond to the O
2
and their positions in the
globin [10–17]. Thus the sea hare Aplysia limacina possesses
a Val E7, but H-bond stabilization of O
2
is provided by Arg
E10 [18]. The monomeric Hb from Glycera dibranchiata
possesses a Leu E7, and the absence of an alternate H-bond
donor leads to rapid O
2
off-rates [19]. Some of the most
unusual Hbs characterized to date are those from nematodes
and trematodes (mammalian parasites) such as the nematode
Ascaris suum (As) [11,12] and the trematode Paramphisto-
mum epiclitum (Pe) [14,15], which exhibit extraordinarily
Correspondence to G. N. La Mar, Department of Chemistry,
University of California, One Shields Avenue, Davis, CA 95616, USA.
Fax: + 1 530 752 8995, Tel.: + 1 530 752 0958,
E-mail:

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 />(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
too close to the heme to allow a robust H-bond to O

2
[22].
The extremely O
2
-avid monomeric Pe Hb is unique in that it
possesses Tyr at both the B10 and E7 positions [14]. Solution
1
HNMRonPe WT HbO
2
[23] had found two labile protons
in the vicinity of the bound O
2
, both arising from Tyr,
implying that both residues are oriented into the heme
pocket. However, mutagenesis on Pe Hb [16] has shown that
substituting Tyr E7 does not, while substituting Tyr B10
does, strongly reduce O
2
affinity via a faster O
2
off-rate.
These studies firmly establish that the O
2
avidity does not
require an H-bond by Tyr E7. However, these results do not
alone establish whether the Tyr E7 is oriented into or out of
the heme pocket.
While crystals of Pe HbO
2
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
embarked on a

1
H NMR study of the paramagnetic Pe WT
metHbCN form whose ligand, like O
2
,isan(albeitweaker)
H-bond acceptor [24–27], and whose tilting from the heme
normal reflects steric repulsive and/or H-bonding attractive
interactions in the distal pocket [24,28–30]. The large
hyperfine shifts, moreover, provide highly enhanced resolu-
tion to the active site residue protons that greatly facilitates
their assignment, and whose hyperfine shifts contain a
wealth of information on the detailed molecular structure
not readily obtained in an analogous diamagnetic complex
[30,31]. At the same time, the paramagnetic-induced relax-
ation is sufficiently weak so as not to interfere with effective
conventional 2D NMR experiments [32] that will confirm
whether the active site structure is closer to one or the other
crystal forms, or distinct from both. The hyperfine shifts of
the heme are sensitive to the presence of an H-bond to
ligated cyanide [24–26,33], and hence provide direct infor-
mation on distal H-bonding interactions. Lastly, a relatively
robust interpretive basis of the hyperfine shift of the heme
and active site residue for low-spin ferric globins in terms of
distal ligand tilt [28–30] and axial His orientation [26,34–38]
has been established on the basis of characterizing a variety
of hemoproteins with different properties. The Pe metHb
provides a novel His F8 [16] orientation that will allow
further testing of this procedure.
Experimental procedures
Protein samples

The monomeric wild-type (WT) hemoglobin, labeled WT
Hb, from the trematode Paramphistomum epiclitum (Pe)
and Isoparorchis hypselbagi (Ih) were isolated and purified
as described previously [14]. The cyanomet complexes were
prepared by adding approximately five molar equivalents of
KCN to the air-oxidized Hb. The final concentration of Pe
metHbCN complex was % 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
(
2

HCl) solution.
NMR spectroscopy
1
H NMR data were collected on a Brucker AVANCE 600
spectrometer, operating at 600 MHz for protein samples in
both
1
H
2
Oand
2
H
2
O over the temperature range 15–35 °C,
at a repetition rate of 1 s
)1
with presaturation of the solvent
signal. Water-eliminated Fourier transform (WEFT) [39]
spectra were recorded to detect broad, strongly relaxed proton
signals. Chemical shifts were referenced to 2,2-dimethyl-2-
silapentane-5-sulfonate (DSS) through the water resonance.
Non-selective T
1
s, with ± 15% uncertainty, were determined
for the resolved strongly relaxed protons at 25 °C from the
initial magnetization recovery of a standard inversion-
recovery pulse sequence. The distance of proton H (with
T
1
) 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
* ¼ 140 ms) and F8 N

d
H(R*
Fe
¼ 5.01 A
˚
,
T
1
* ¼ 37 ms) as reference. The 1 : 1 [40] and steady-state
NOE spectra were recorded at 30 °C as described previously
in detail [41]. NOESY [42] and TOCSY [43] spectra were
collected (512t
1
blocks, 2048t
2
points) at 25 °C,30°C and
35 °C in order to identify scalar and dipolar connectivities
among heme and amino acid residues. Spectral widths of
13 kHz and mixing times of 80 ms for NOESY and 50 ms
for TOCSY were used. Scans (192) were collected for each
block with a repetition time of 1.0 s
)1
. Two-dimensional
data sets were processed by
XWINNMR
software on a Silicon
Graphics Indigo workstation. Both NOESY and TOCSY
spectra were processed by a 30° shifted sine bell squared
apodization, and zero-filled to 2048 · 2048 points prior to
Fourier transformation.

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
h

0
À 1ÞR
À3
þ 3ðDv
rh
sin
2
h
0
cos 2X
0
ÞR
À3
ð3Þ
The observed dipolar shifts are given by:
d
dip
ðobsÞ¼d
DSS
ðobsÞÀd
DSS
ðdiaÞð4Þ
where ddip(obs) and d
DSS
(dia) are the chemical shifts (in
p.p.m.) referenced to DSS, for the paramagnetic Pe
metHbCN complex and an isostructural diamagnetic com-
plex, respectively. Limited d
DSS
(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
HNMRofPe metMbCN. Proximal and distal residues are shown as rectangles and circles, respectively, and arrows connecting heme

substituents and residues, and between residues, reflect NMR observed, and crystallographically expected, contacts. The reference, x¢,y¢,z¢,aswell
as the magnetic coordinate systems, x, y, z, are shown, where b represents the tilt of the major magnetic axis, z, away from the heme normal (z¢ axis),
a is the angle between the projection of the tilt of the z axis on the x¢,y¢ plane (defined direction of tilt of z and the x¢ axis), and j % a + c defines the
location of the rhombic (x, y) axes. The orientation of the axial His imidazole plane relative to the heme is given by /.
Ó FEBS 2003 Active site structure of trematode cyanomet Hb (Eur. J. Biochem. 270) 2709
structure [46] and the heme-induced ring current shift [47],
respectively.
Structural modeling
Protons were added to the crystal coordinates of recom-
binant WT Pe metHbH
2
O [16] and WT metHb [17] using
the program
INSIGHT-II
(Accelrys). This provided unique
coordinates for all protons of interest except the Tyr32(B10)
hydroxyl proton, as its position was determined from the
1
H
NMR spectral parameters.
Results
The
1
H NMR spectra of Pe WT metHbCN in
1
H
2
Oand
2
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
2

O; (C) WEFT spectrum (relaxation delay 30 ms, repetition rate 10 s
)1
)in
2
H
2
O which allows detection of strongly
relaxed, broad signs at 9 p.p.m.; and (D) steady-state NOE difference trace at 35 °C upon saturating the Tyr32(B10) OH signal (vertical arrow).
Heme resonances are labeled as shown in Fig. 1, and residues are labeled by position numbers and protons.
2710 W. Du et al.(Eur. J. Biochem. 270) Ó FEBS 2003
(% 300 Hz) and very strongly relaxed (T
1
<5ms) peak
on the low-field shoulder of the diamagnetic envelope.
Heme pocket residue assignments were pursued by
backbone connectivities as standard in a diamagnetic
protein [48], with the remainder of the target residues
assigned by detection of NOESY residue–heme and
interresidue cross-peaks solely by the standard Mb fold,
and the observation of relaxation effects and/or sufficient
TOCSY cross-peaks to identify the side chain uniquely.
The identification of hyperfine shifted and relaxed reso-
nances was greatly facilitated by variable temperature
studies to define unique scalar/dipolar connectivities as
described previously [49]. Assignments deduced herein are
given by the heme-labeling scheme shown in Fig. 1 and
the residue numbers and proton. The chemical shifts for
the heme and axial His98(F8) are listed in Table 1, and
those for assigned residues with significant dipolar shifts
are listed in Table 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
, a

i
–N
i+3
and/or a
i
–b
i+3
NOESY connection diagnostic of helical
fragments [48] with limited (but sufficient) TOCSY-identi-
fied side chains leads to the identification of the six segments
labeled I–VI (Fig. 3). Fragment I is represented by
Z
i
-AMXi
+1
-Gly
i+2
-Z
i+3
-AMX
i+4
-AMX
i+5
-, where Z is
>4 spin side chain, and AMX
i+1
and AMX
i+4
exhibit
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
,

which the sequence uniquely identifies as the expected
E-helix (E7–E16) segment Tyr66–Val75, with the ThrE10
and LeuE11 side chains exhibiting weak-to-moderate
upfield shifts. The detection of an inconsequentially shifted,
two-spin aromatic ring in contact with AMX
i
(Fig. 4E)
confirms Tyr66(E7). Key dipolar contacts that define the
orientation of the Tyr66(E7) ring include those to the
13-propionate H
b
s (Fig. 4B,C) and to the Phe46(CD1) ring,
and the NHs of Arg48(CD3) and Leu49(CD4) (Fig. 4G).
It was not possible to detect a labile proton in
dipolar contact with the Tyr66(E7) C
e
Hs signal that would
identify the side chain hydroxyl proton. The observed
heme-residue contacts are characteristic of the general Mb
fold (Fig. 1).
Fragment III is detected as Gly
i
-Z
i+1
-X
i+2
-Ile
i+3
-
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
junction and to the E-helix as depicted in Fig. 1. Frag-

ment IV,Val
i
-AMX
i+1
-X
i+2
-Z
i+3
-Z
i+4
-Ala
i+5
-Ala
i+6
-
Z
i+7
-Z
i+8
(Fig. 3), is unambiguously identified as Val139–
Ile147 on the H-helix (H15–H23). The dipolar contacts of
Phe140(H16) and Met143(H19) to pyrrole A, as well as
Ile147(H23) to the axial His98(F8), and the interresidue
contacts to the G-helix (Fig. 1) confirm their locations in
a standard H-helix.
The helical fragment V is represented by Z
i
-AMX
i+1
-

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
2

O100m
M
phosphate, pH 6.8, 30 °C. Non-selective T
1
,in
ms, in square brackets for resolved resonances.
Protons d
DSS
(obs) [T
1
]
Heme 2-CH
3
6.18
7-CH
3
23.64 [88]
12-CH
3
7.97
18-CH
3
14.10 [143]
3-H
a
12.40 [182]
3-H
b
s )4.88 [253], )4.23 [235]
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
b

H 10.59 [84]
N
d
H 17.51 [37]
Ó FEBS 2003 Active site structure of trematode cyanomet Hb (Eur. J. Biochem. 270) 2711
Table 2.
1
H NMR spectral parameters for strongly dipolar shifted
active site residues in Pe metHbCN. Observed chemical shifts,
d
DSS
(obs), in p.p.m., are referenced to DSS in
1
H
2
O, 100 m
M
phos-
phate, pH 6.8 at 30 °C. Diamagnetic chemical shifts, d
DSS
(dia), cal-
culated via Eqn (5) using the WT Pe metHb H
2
O crystal coordinates
[16]. na, Not assigned.
Residue Proton d
DSS
(obs) d
DSS
(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
d

Hs 7.61 6.99
C
e
Hs 7.78 6.08
C
f
H 7.34 5.81
Tyr42(C4)
NH
8.21 8.20
C
a
H 3.78 4.01
C
b
H 3.66 3.78, 3.36
C
d
Hs 6.73 7.04
C
e
Hs 6.35 6.85
His45(C7)
NH
7.62 8.25
C
a
H
4.14
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
H 4.85 3.51

C
b
H 6.27 1.86
C
b
H¢ 3.33 1.64
C
d
Hs 6.55 7.42
C
e
Hs 5.45 6.75
OH
na
8.50
Ala67(E8)
NH
9.32 7.78
C
a
H 5.19 3.87
C
b
H
3
2.37 1.06
Thr69(E10)
NH
8.17 7.79
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
H

3
2.63 )0.81
C
d
H
3
¢ 2.09 )1.10
Ala73(E14)
NH
7.10 7.81
C
a
H 3.84 4.60
C
b
H
3
)0.03 1.70
Tyr94(F4)
NH
8.62 7.93
C
a
H 6.44 3.94
C
b
H 3.87 3.03
Table 2. (Continued).
Residue Proton 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

d
Hs 2.30 1.47
C
e
Hs 2.47 2.73
Asp97(F7) NH 10.07 7.50
C
a
H 5.80 4.08
C
b
H 3.51 2.57
C
b
H¢ 6.54 1.50
His98(F8) NH 12.81 6.66
C
a
H 8.05 2.71
C
b
H 8.67 0.91
C
b
H¢ 10.59 0.67
N
d
H 17.51 6.42
Thr99(FG1) NH 9.35 7.45
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
a

H 1.34 4.57
C
b
H 2.43 2.87
C
b
H 1.75 2.30
C
d
Hs 4.22 5.63
C
f
H 3.76 6.09
Gly111(G8) NH 6.65 8.29
C
a
H 1.58 3.94
C
a
H¢ 2.92 3.50
Phe115(G12) NH 8.50 8.32
C
b
H 3.01, 2.82 3.69
C
b
H¢ 2.82 3.31
C
d
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
C

c
H 1.93 1.71
C
e
H
3
0.48 1.27
Ile147(H23) C
a
H 4.38 3.15
C
b
H 2.30 1.29
C
c
Hs 2.78 0.18
C
c
H¢ 1.22 )0.15
C
d
H
3
0.54 )2.44
2712 W. Du et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Dipolar contact to a strongly relaxed and moderately
dipolar shifted aromatic spin-system that is in contact with
pyrrole C (Fig. 4B,C) confirm both the assignment as
Phe(CD1) and the orientation of the heme, as depicted in
Fig. 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
i+10

-Thr
i+11
-Ala
i+12
(Fig. 3), is unique to residues
Glu26–Ala38 on the B-helix (B4–B16). The dipolar contact
of a weakly shifted, three-spin aromatic ring to AMX
i+10
,
and that of a strongly hyperfine shifted two-spin aromatic
ring to AMX
i+6
confirm the assignments of Phe36(B14)
and the key Tyr32(B10). These side chains do not exhibit
NOESY cross peaks to the heme (as expected), but exhibit
the expected contacts to helix E (Tyr32(B10) to Leu70(E11),
Fig. 4D; Ala67(E8), Fig. 4F; and Tyr66(E7), Fig. 4G, as
depicted in Fig. 1. A strong NOE to the assigned Tyr32(B10
C
e
Hs signal, upon saturating the extreme low-field, strongly
relaxed (T
1
% 10 ms) labile proton signal (Fig. 2D), locates
the residue side chain hydroxyl proton.
Comparison to the alternate crystal structures
The pattern of NOESY cross peaks of the Tyr66(E7) ring
to the heme (Fig. 4A–C), Tyr32(B10) (Fig. 4F), and in
particular, to the Phe46(CD1) backbone (Fig. 4E), as
summarized in Fig. 1, unequivocally establish that the

Tyr66(E7) ring is oriented out of the heme pocket, exactly
as found in both Pe metHb crystal structures [16,17]. The
position is further supported by the calculated and
observed small 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
metHbthanintherWTmetHbH

2
O crystal structure,
with the result that the ligated water is lost in the latter
complex [16,17].
Prior to determining the magnetic axes, which will allow
us to elaborate the tilt of the ligated cyanide and charac-
terize the H–bonding interaction of Tyr32(B10) with its
ligand, it is necessary to establish which crystal structure
better represents the solution structure. This distinction can
be made on the basis of three NMR observations, the
NOESY cross peak intensities between proton i and j
(/ r
i
j
)6
), the paramagnetism-induced relaxation,
T
À1
1i
/ R
À6
FeÀi;
, of proton i, and the magnetic axes themselves
(see next section).
Inspection of the two sets of crystal coordinates identifies
a series of proton pairs whose separations differ significantly
between the two structures [16,17] and which we have been
able to identify unambiguously. The contacts involve the
FG corner and the position of helix B relative to the heme
and E-helix backbone. Table 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.
Ó FEBS 2003 Active site structure of trematode cyanomet Hb (Eur. J. Biochem. 270) 2713

structure leads to the reduction of the R
Fe-i
for the two Tyr
B10 C
e
Hs of 5.8 and 7.9 A
˚
in rWT metHbH
2
O(with
expected T
1
% 20–30% shorter than that for a heme
methyl), to 4.5 and 5.8 A
˚
in WT metHb such that a T
1
is
expected closer to that of a meso-H (T
1
% 50 ms). The
observed T
1
(Tyr32(B10) (C
e
H) % 100 ms, is consistent with
the former, but not the latter distances, such that the
relaxation effects similarly confirm a WT metHbCN
solution structure similar to the rWT metHbH
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
Fig. 4. Portions of the 600 MHz

1
HNOESY
spectra (mixing time 80 ms) of Pe metHbCN in
1
H
2
O 100 m
M
in phosphate, pH 7.0 at 35 °C.
Dipolar contacts are illustrated, involving key
distal residues Phe46(CD1) and helical ring
cross peaks for Tyr32(B10) (F), Phe36(B14)
(G), Phe46(CD1) (E), Tyr66(E7) (G) and
Leu70(E11) (E), and to NHs of heme-residue
contacts Tyr66(E7) and Phe46(CD1) to have
propionates (A, B, C) and interresidue con-
tacts from Tyr66(E7) to Phe46(CD1) and NHs
of Arg48(CD3) Leu49(CD4) (G), and
Tyr32(B10) (D) contacts to Ala67(E8) (F).
The cross peak between Tyr32(B10) C
e
Hs and
Tyr66 (E7) C
d
Hs is observed only at a lower
contour level.
2714 W. Du et al.(Eur. J. Biochem. 270) Ó FEBS 2003
metHbH
2
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
d

dip
(calc) for those protons whose coordinates differed
significantly in the two structures are shown as s and h.
For the most part, in particular Thr99(FG1) and
Val103(FG5), residues with different geometries exhibit
reasonable fits for both cases in Fig. 5, in part because d
dip
is
small, but also because their position is not very sensitive to
dipolar shift. However, it is noted that the Tyr32(B10) C
e
Hs
exhibit a very reasonable fit for case I (Fig. 5A), but an
unacceptable fit for case II (Fig. 5B). Hence the magnetic
axes completely concur with the results of both NOESY
intensity analysis and paramagnetic relaxation effects in
finding the Pe metHbCN active site solution structure to
coincide with the crystal structure of rWT metHbH
2
O[16]
but not WT metHb [17].
Redetermination of the magnetic axes orientation (a, b,
c), from a large variety of available input data using only
the pertinent Pe rWT metH
2
O crystal structure [16] led to
a ¼ 202 ± 10°, b ¼ 9±1° and j ¼ 52 ± 10° for a
three-parameter search using the sperm whale metMbCN
anisotropies [29], and yielded minimally changed orienta-
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
dip

(obs) vs. d
dip
(calc) for the
magnetic axes of Pe WT metHbCN as based on
the crystal coordinates of rWT metHbH
2
O;
and WT metHb. (A) rWT metHbH
2
O; and (B)
WT metHb, using as input only the d
dip
(obs)
for protons whose positions are the same
in the two crystal structures [16,17], with
Dv
ax
¼ 2.48 · 10
)8
m
3
Æmol
)1
and
Dv
rh
¼ )0.58 · 10
)8
m
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
<5.0A

˚
). Distances in bold are in agreement with the NMR
observations.
r
ij
(A
˚
)
rWT metHbH
2
O WT metHb NOE
F-helix/FG-corner
NH(FG1)-C
a
H(F8) 3.49 2.45 m
C
a
H(FG1)-C
a
H(H23) 2.55 5.92 mÆs
)1
C
a
H(FG1)-C
c
H(H23) 3.47 6.22 m
C
b
H(FG1)-C
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
(B10)-C

b
H
2
(E11) 3.14 2.38 m
Ó FEBS 2003 Active site structure of trematode cyanomet Hb (Eur. J. Biochem. 270) 2715
Structural simulation of Tyr32(B10)
The good correlation between d
dip
(obs) and d
dip
(calc) for
the Tyr32(B10) ring in the magnetic axes based on the
rWT metHbH
2
O crystal structure [16] indicates that the
ring (and hence B-helix) occupies the same position as in
the crystal. This conclusion is supported by the relaxation
properties of the C
e
Hs signal (see above). The hydroxyl
proton position is not directly determined in the crystal
structure, but can be inferred by the position of other H-
bond acceptor/donors in the immediate vicinity. The
proposal that the Tyr32(B10) hydroxyl proton acted as a
donor to the carbonyl of Tyr66(E7) in the rWT
metHbH
2
O crystal structure [16] places it % 5.9 A
˚
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
d

H
distance, r
ij
of Tyr66(E7), Fig. 6C, as indicated by a
weak-to-moderate NOESY cross peak intensity. Inspec-
tion of Fig. 6 reveals a single orientation,
w ¼ )140 ± 20°, that essentially quantitatively and sim-
ultaneously accounts for the three observations. The
effect on the Tyr32(B10) O-H.N(cyanide) angle on Y is
illustrated in Fig. 6D. The position of the Tyr32(B10) O
g
relative to the cyanide ligand tilted by % 10° in the
direction of the 10-H position is illustrated in Fig. 7 and
reveals a van der Waals contact between Tyr32(B10) and
the cyanide.
Discussion
Active site structure
The combination of NOESY cross peak intensities,
paramagnetic relaxation and the magnetic axes data
provide compelling evidence that WT Pe metHbCN
much more closely resembles the structure found in the
crystal structure of rWT Pe metHbH
2
O [16] than of WT
Pe metHb [17]. Because the WT Pe metHbCN active site
structureisessentiallythesameasrWTmetHbH
2
O, and
different from WT metHb, the present data support the
interpretation that the structural differences between rWT

and WT Pe metHb in crystals result from the extensive
interaction between the two molecules in the unit cell for
WT metHb, rather than from significant structural
differences between isolated WT and rWT Pe Hb
molecules [17]. The distal Tyr66(E7) ring was found
oriented out of the heme pocket in both rWT 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
2

had shown that there are
two interacting labile protons from two Tyr in the distal
pocket capable of interacting with the bound O
2
[23].
Moreover, NOESY cross peaks between the Tyr66(E7) ring
and the terminus of Leu70(E11) indicated that the
Tyr66(E7) ring is oriented into the heme pocket. There
appears to be no obvious rationalization for these contra-
dictory results. It has been demonstrated that Tyr66(E7) can
be substituted without significantly affecting the extreme O
2
ligation dynamics/thermodynamics [16]. However, these
results do not alone determine that Tyr66(E7) is not
oriented into the heme pocket, they only demonstrate that
any interaction of the Tyr66(E7) ring with O
2
does not
incrementally increase the H-bond stabilization of bound
O
2
. A crystal structure or solution NMR structure of Pe
HbO
2
is clearly important.
Fig. 6. Plots of d
dip
(calc) derived from optimized magnetic axes, the
distance to iron via paramagnetic relaxation R
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
g

-N(cyanide) separ-
ation of 2.6 A
˚
. It appears, moreover, that the 10° tilt of the
cyanide results from a steric interaction between the
Tyr32(B10) O
g
and the nitrogen, because a zero tilt angle
wouldleadtoan% 0.2–0.3 A
˚
overlap of the two van der
Waals surfaces. A moderate-to-strong H-bond [50] can
occur when the donor/acceptor heteroatoms are separated
by % 2.5–3.0 A
˚
, and the O-H.N bond angle is close to 180°.
Figure 6D shows that the unique orientation of the
Tyr32(B10) O
g
H that optimally accounts for the NOESY,
relaxation and dipolar shift data, i.e. w ¼ )140° ±20,is
precisely what would lead to the strongest H-bond, with an
O-H.N angle of % 170°.
Experimental verification of a moderate-to-strong single
H-bond to bound cyanide is obtained from the effect of
solvent isotope (
1
H/
2
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
H in a single H-bond to a heme ligand. This

single H-bond is clearly that of the Tyr32(B10) OH
described above.
Magnetic axes
The magnetic axes could be determined to relatively high
accuracy on the basis of the rWT metHbH
2
Ocrystal
structure [16]. The optimized anisotropies of Dv
ax
¼
2.38 · 10
)8
m
3
Æmol
)1
and Dv
rh
¼ )0.56 · 10
)8
m
3
Æmol
)1
,
are within the uncertainties of those reported for sperm
whale metMbCN, [29] 2.48 ± 0.03 · 10
)8
m
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
hf

(5-H) ) d
hf
(10-H) + d
hf
(15-H) ) d
hf
(20-H)] ¼ 8.2 p.p.m.,
while D(calc) ¼ 1/2[ddip(5-H) ) ddip(10-H) + ddip(15-H)
) ddip(20-H)] ¼ 9 ± 2 p.p.m., which confirms the predic-
tion. Lastly, a pattern of heme hyperfine shift with the two
largest low-field shifts for 7-CH
3
and 18-CH
3
is generally
interpreted as indicating that the heme orientation is rotated
% 180° about the a-c-meso axis relative to mammalian
globins, as observed in Chironomus Hbs[26].Thisinter-
pretation has as its premise that the axial His orientation is
conserved relative to globins for which both
1
HNMRand
crystallographic characterization has been carried out. The
normal His F8 imidazole plane orientation for mammalian
globins is close to / % 0inFig.1[52].ForthepresentPe
Hb, the heme orientation is, in fact, identical to that in
mammalian globin (Phe CD1 near the 12-CH
3
) but its His
F8 imidazole is rotated % 45° clockwise relative to the

mammalian globin, and it is the His F8 rotation, not the
heme rotation, that leads to the low-field 7-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
chain and the » 10° tilt of the ligated cyanide. It is noted that the tilt of

the CN
–
is necessary to avoid steric interaction with the Tyr32(B10)
Og.
Ó FEBS 2003 Active site structure of trematode cyanomet Hb (Eur. J. Biochem. 270) 2717
determined from the magnetic axes [37], was found to be
6.0 p.p.m. at 600 MHz. For the present Pe metHbCN, the
Tyr32(B10) C
e
Hs signal exhibits <10 Hz excess linewidth
at 15 °C that could result from the role of ring reorientation.
The magnetic axes yield a chemical shift difference of
6.6 p.p.m., very similar to that in Ascaris metHbCN [37].
Hence the narrow Tyr32(B10) C
e
H signal dictates that the
rate of Tyr32(B10) ring reorientation is >10
2
times faster in
Pe metHbCN than As metHbCN.
The Phe46(CD1) averaged C
e
Hs peak in Pe metHbCN
is narrow, in contrast to some % 50 Hz excess linewidth due
to ring reoriented observed for Phe(CD1) in sperm whale
[53] and elephant [54] metMbCN. Again, very similar
chemical shift differences for the two C
e
Harepredictedby
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
metHbCN and Dicrocoelium dendriticum (Dd)metHbCN

[55]. In each case, the saturation of the strongly relaxed
low-field labile protons (not shown) results in NOEs to a
two-proton signal [55] (Fig. 2D; not shown for Ih
metHbCN) indicative of a hyperfine shifted Tyr. The
similar relaxation of the labile proton (T
1
% 10 ms) in all
three complexes [55] indicates that the Tyr is similarly
oriented with respect to the iron and positioned to
provide the H-bond to the bound cyanide, and by
implication, to the bound O
2
in the reduced form in each
of the three globins. The extensive sequence homology
for Pe and Ih Hbs [14] argues for very similar heme
pocket structures. For Dd Hb, for which a complete
sequence has never been reported [56], earlier NMR
studies [55] had proposed a distal Tyr at position E7 as
the source of the H-bond to ligand on the basis of a
partial sequence, which had indicated a Tyr on the distal
E-helix. The similarity in the
1
HNMRspectraofthe
three globin complexes in Fig. 8 dictates that the distal
Tyr in Dd metHbCN that results in the H-bond to the
ligand is Tyr(B10), rather than TyrE7.
Acknowledgements
The authors are indebted to Dr Yuyang Wu for obtaining preliminary
1
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.).
References
1. Antonini, E. & Brunori, M. (1971) Hemoglobin and Myoglobin and
Their Reactions with Ligands. Elsevier, North-Holland Publishing,
Amsterdam, the Netherlands.
2. Dickerson, R.E. & Geis, I. (1983) Hemoglobin: Structure, Func-
tion, Evolution and Pathology. Benjamin-Cummings, Menlo Park,
CA, USA.
3. Mukai, M., Savard, P Y., Ouellet, H., Guertin, M. & Yeh, S R.
(2002) Unique ligand–protein interactions in a new truncated
hemoglobin from Mycobacterium tuberculosis. Biochemistry 41,
3897–3905.
4. Pesce, A., Couture, M., Dewilde, S., Guertin, M., Yamauchi, K.,
Ascenzi, P., Moens, L. & Bolognesi, M. (2000) A novel two-over-
two a-helical sandwich fold is characteristic of the truncated
hemoglobin family. EMBO J. 19, 2424–2434.
5. Milani, M., Pesce, A., Ouellet, Y., Ascenzi, P., Guertin, M. &
Bolognesi, M. (2001) Mycobacterium tuberculosis hemoglobin n
displays a protein tunnel suited for O
2
diffusion to the heme.
EMBO J. 20, 3902–3909.
6. Springer, B.A., Sligar, S.G., Olson, J.S. & Phillips, G.N. (1994)
Mechanisms of ligand recognition in myoglobin. Chem. Rev. 94,
699–714.
7. Dene, H., Goodman, M. & Romero-Herrera, A.E. (1980) The
AminoAcidSequence(Elephas maximus) Myoglobin and
the Phylogen of Proboscidea. Proc. Royal Soc. London B207,

111–127.
8. Zhao, X., Vyas, K., Nguyen, B.D., Rajarathnam, K., La Mar.,
G.N., Li, T., Phillips, G.N.J., Eich, R.F., Olson, J.S., Ling, J. &
Bocian, D.F. (1995) A double mutant of sperm whale myoglobin
Fig. 8. Comparison of the low-field resolved portion of the
1
HNMR
spectra of the trematode/nematode globin complexes in
1
H
2
Oat30°.
(A) Pe metHbCN at pH 7.4; (B) Ih metHbCN at pH 7.5; (C) Dd
metHbCN [55] at pH 7.0. The similarly assigned (by helical position)
signals are connected by dashed lines.
2718 W. Du et al.(Eur. J. Biochem. 270) Ó FEBS 2003
mimics the structure and function of elephant myoglobin. J. Biol.
Chem. 270, 20763–20774.
9. Bisig, D.A., Di Iorio, E.E., Diederichs, K., Winterhalter, K.H. &
Piontek, K. (1995) Crystal structure of asian elephant (Elephas
maximus) cyano-metmyoglobin at 1.78-A
˚
resolution. J. Biol.
Chem. 270, 20754–20762.
10. Bolognesi, M., Coda, A., Frigero, F., Gatti, G., Ascenzi, P. &
Brunori, M. (1989) Aplysia limacina myoglobin – crystallographic
analysis at 1.6A
˚
resolution. J. Mol. Biol. 205, 529–544.
11. De Baere, I., Perutz, M.F., Kiger, L., Marden, M.C. & Poyart, C.

(1994) Formation of two hydrogen bonds from the globin to the
heme-linked oxygen molecule in Ascaris hemoglobin. Proc. Natl
Acad. Sci. USA 91, 1594–1597.
12. Yang, J., Kloek, A., Goldberg, D.E. & Mathews, F.S. (1995) The
structure of Ascaris hemoglobin domain I at 2.2 angstrom
resolution – molecular features of oxygen avidity. Proc. Natl Acad.
Sci. USA 92, 4224–4228.
13. Kapp,O.,Moens,L.,Vanfleteren,J.,Trotman,C.,Suzuki,T.&
Vinogradov, S. (1995) Alignment of 7–00 globin sequences –
extent of amino-acid substitution and its correlation with variation
in volume. Protein Sci. 4, 2179–2190.
14. Rashid, A.K., Van Hauwaert, M.L., Haque, M., Siddiqi, A.H.,
Lasters, I., De Mayer, M., Griffon, N., Marden, M.C., De Wilde,
S., Clauwaert, J., Vinogradov, S.N. & Moens, L. (1997) Trema-
tode myoglobins, functional molecules with a distal tyrosine.
J. Biol. Chem. 272, 2992–2999.
15. Rashid, A.K. & Weber, R.E. (1999) Functional differentiation in
trematode hemoglobin isoforms. Eur. J. Biochem. 260, 717–725.
16. Pesce, A., Dewilde, S., Kiger, L., Milani, M., Ascenzi, P.,
Marden,M.C.,Hauwaert,M L.V.,Vanfleteren,J.,Moens,L.
& Bolognesi, M. (2001) Very High Resolution Structure of a
Trematode Hemoglobin Displaying a TyrB10-TyrE7 Heme
Distal Residue Pair and High Oxygen Affinity. J. Mol. Biol. 309,
1153–1164.
17. Milani, M., Pesce, A., Dewilde, S., Ascenzi, P., Moens, L. &
Bolognesi, M.(2002)Structural plasticity in the eight-helix fold of a
trematode haemoglobin. Acta Crystallogr. D-Biol Cryst. D58,1–4.
18. Cutruzzola, F., Allocatelli, C.T., Brancaccio, A. & Brunori, M.
(1996) Aplysia limacina myoglobin cDNA cloning – an alternative
mechanism of oxygen stabilization as studied by active-site

mutagenesis. Biochem. J. 314, 83–90.
19. Parkhurst, L.J., Sima, P. & Goss, D.J. (1980) Kinetics of oxygen
and carbon monoxide binding to the hemoglobins of Glycera
dibranchiata. Biochemistry 19, 2688–2692.
20. Goldberg, D.E. (1995) The enigmatic oxygen-avid hemoglobin of
Ascaris. Bioessays. 17, 177–182.
21. Rizzi,M.,Wittenberg,J.G.,Coda,A.,Fasano,M.,Ascenzi,P.&
Bolognesi, M. (1994) Structure of the sulfide-reactive hemoglobin
from the clam Lucina pectinata. Crystallographic analysis at 1.5 A
˚
resolution. J. Mol. Biol. 244, 86–89.
22. Zhang, W., Cutruzzola
´
, F., Travaglini Allocatelli, C., Brunori, M.
& La Mar., G.N. (1997) A myoglobin mutant designed to mimic
the oxygen-avid Ascaris suum hemoglobin: elucidation of the distal
hydrogen bonding network by solution NMR. Biophys. J. 73,
1019–1030.
23. Zhang, W., Rashid, K.A., Haque, M., Siddiqi, A.H., Vinogradov,
S.N., Moens, L. & La Mar, G.N. (1997) Solution of
1
HNMR
structure of the heme cavity in the oxygen-avid myoglobin from
the trematode Paramphistonum epiclitum. J. Biol. Chem. 275,
3000–3006.
24. Lecomte, J.T.J. & La Mar., G.N. (1987)
1
H NMR probe for
hydrogen bonding of distal residues to bound ligands in heme
proteins: isotope effect on heme electronic structure of myoglobin.

J. Am. Chem. Soc. 109, 7219–7220.
25. Thanabal, V., de Ropp, J.S. & La Mar., G.N. (1988) Proton
NMR characterization of the catalytically relevant proximal and
distal hydrogen-bonding networks in ligated resting state horse-
radish peroxidase. J. Am. Chem. Soc. 110, 3027–3035.
26. Zhang, W., La Mar., G.N. & Gersonde, K. (1996) Solution
1
H
NMR structure of the heme cavity in the low-affinity state for the
allosteric monomeric cyano-met hemoglobins from Chironomus
thummi thummi. Comparison to the crystal structure. Eur. J.
Biochem. 237, 841–853.
27. Yamamoto, Y. (1998) H-1-NMR investigation of the influence of
the heme orientation on functional properties of myoglobin.
Biochim. Biophys. Acta. 1388, 349–362.
28. Emerson, S.D. & La Mar., G.N. (1990) NMR determination of
the orientation of the magnetic susceptibility tensor in cyano met-
myoglobin: a new probe of steric tilt of bound ligand. Biochemistry
29, 1556–1566.
29. Nguyen, B.D., Xia, Z., Yeh, D.C., Vyas, K., Deaguero, H. & La
Mar., G. (1999) Solution NMR determination of the anisotropy
and orientation of the paramagnetic susceptibility tensor as a
function of temperature for metmyoglobin cyanide; implications
for the population of excited electronic states. J. Am. Chem. Soc.
121, 208–217.
30. La Mar., G.N., Satterlee, J.D. & de Ropp, J.S. (1999) NMR of
hemoproteins. The Porphyrins Handbook (Kadish, K.M., Guilard,
R. & Smith, K.M., eds), pp. 185–298. Academic Press, San Diego,
USA.
31. Bertini, I. & Luchinat, C. (1996) NMR of paramagnetic sub-

stances. Coord. Chem. Rev. 150, 1–296.
32. La Mar., G.N. & de Ropp, J.S. (1993) NMR methodology for
paramagnetic proteins. Biol. Magn. Reson. 18, 1–79.
33. Lecomte, J.T.J., La Mar., G.N., Smit, J.D.G., Winterhalter, K.H.,
Smith, K.M., Langry, K.C. & Leung, H K. (1987) Structural and
electronic properties of the liver fluke hemoglobin heme cavity by
nuclear magnetic resonance: isotope labeling. J. Mol. Biol. 197,
101–110.
34. Peyton, D.H., La Mar., G.N., Pande, U., Ascoli, F., Smith, K.M.,
Pandey, R.K., Parish, D.W., Bolognesi, M. & Brunori, M. (1989)
Proton nuclear magnetic resonance study of the molecular and
electronic structure of the heme cavity in Aplysia cyano-
metmyoglobin. Biochemistry 28, 4880–4887.
35. Shokhirev, N.V. & Walker, F.A. (1998) The effect of axial ligand
plane orientation on the contact and pseudocontact shifts of
low-spin ferriheme proteins. J. Biol. Inorg. Chem. 3, 581–594.
36. Wu, Y., Chien, E.Y.T., Sligar, S.G. & La Mar., G.N. (1998)
Influence of proximal side mutations on the molecular and
electronic structure of cyanomet myoglobin: a
1
H NMR study.
Biochemistry 37, 6979–6990.
37. Xia, Z., Zhang, W., Nguyen, B.D., Kloek, A.P., Goldberg, D.E. &
La Mar, G.N. (1999)
1
H NMR Investigation of the distal
hydrogen bonding network and ligand tilt in the cyanomet
complex of oxygen-avid Ascaris hemoglobin. J. Biol. Chem. 274,
31819–31826.
38. Nguyen, B.D., Xia, Z., Cutruzzola

´
, F., Travaglini Allocatelli,
C., Brancaccio, A., Brunori, M. & La Mar, G.N. (2000)
Solution
1
H NMR study of the active site of Aplysia limacina
cyanomet myoglobin mutant Val (E7) His/Thr (E10) Arg
designed to mimic the sperm whale myoglobin pocket. influence
of distal hydrogen-bonding and N-terminus acetylation on the
heme electronic and molecular structure. J. Biol. Chem. 275,
742–751.
39. Gupta, R.K. (1976) Dynamic range problem in fourier trans-
form nmr. modified weft pulse sequence. J. Magn. Reson. 24,
461–465.
40. Plateau, P. & Gueron, M. (1982) Exchangeable proton NMR
without base-line distortions using new strong-pulse sequences.
J. Am. Chem. Soc. 104, 7310–7311.
41. Thanabal, V., de Ropp, J.S. & La Mar, G.N. (1987) Identification
of the catalytically important amino acid residues resonances in
Ó FEBS 2003 Active site structure of trematode cyanomet Hb (Eur. J. Biochem. 270) 2719
ferric low-spin horseradish peroxidase using nuclear overhauser
effect measurements. J. Am. Chem. Soc. 109, 7516–7525.
42.Jeener,J.,Meier,B.H.,Bachmann,P.&Ernst,R.R.(1979)
Investigation of exchange processes by two dimensional NMR
spectroscopy. J. Chem. Phys. 71, 4546–4553.
43. Griesinger, C., Otting, G., Wu
¨
thrich, K. & Ernst, R.R. (1988)
Clean TOCSY for
1

H spin system identification in macro-
molecules. J. Am. Chem. Soc. 110, 7870–7872.
44. Williams, G., Clayden, N.J., Moore, G.R. & Williams, R.J.P.
(1985) Comparison of the solution and crystal structures of
mitochondrial cytochrome c. Analysis of paramagnetic shifts in
the nuclear magnetic resonance spectrum of ferricytochrome c.
J. Mol. Biol. 183, 447–460.
45. Bundi, A. & Wu
¨
thrich, K. (1979)
1
H NMR parameters of the
common amino acid residues measured in aqueous solutions of
the linear tetrapeptides H-Gly-Gly-X-L-Ala-OH. Biopolymers 18,
285–297.
46. Wishart, D.S., Sykes, B.D. & Richards, F.M. (1991) Relationship
between nuclear magnetic resonance chemical shift and protein
secondary structure. J. Mol. Biol. 222, 311–333.
47. Cross, K.J. & Wright, P.E. (1985) Calibration of ring-current
models for the heme ring. J. Magn. Reson. 64, 220–231.
48. Wu
¨
thrich, K. (1986) NMR of Proteins and Nucleic Acids.Wiley&
Sons, New York, USA.
49. Qin, J. & La Mar, G.N. (1992) Complete sequence-specific
1
hnmr
resonance assignment of hyperfine-shifted residues in the active
site of a paramagnetic protein: application to Aplysia cyano-
metmyoglobin. J. Biomol. NMR 2, 597–618.

50. Jeffrey, G.A. (1997) An Introduction to Hydrogen Bonding.Oxford
University Press, New York, USA.
51. Thanabal, V., de Ropp, J.S. & La Mar, G.N. (1987)
1
HNMR
study of the electronic and molecular structure of the heme cavity
in horseradish peroxidase. Complete heme resonance assignments
based on saturation transfer and nuclear Overhauser effects.
J. Am. Chem. Soc. 109, 265–272.
52. Kuriyan, J., Wilz, S., Karplus, M. & Petsko, G.A. (1986) X-ray
structure and refinement of carbon-monoxy (Fe-II)-myoglobin at
1.5 A
˚
resolution. J. Mol. Biol. 192, 133–154.
53. Emerson, S.D., Lecomte, J.T.J. & La Mar, G.N. (1988)
1
HNMR
resonance assignment and dynamic analysis of phenylalanine cd1
in a low-spin ferric complex of sperm whale myoglobin. J. Am.
Chem. Soc. 110, 4176–4182.
54. Vyas,K.&Rajarathnam,K.,Yu,L.P.,Emerson,S.D.&LaMar,
G.N. (1993) 1H NMR investigation of the heme cavity of elephant
(E7 GLN) metMbCN: evidence for a B–helix phenylalanine
interaction with bound ligand. J. Biol. Chem. 268, 14826–14835.
55. Lecomte, J.T.J., Smit, J.D.G., Winterhalter, K.H. & La Mar,
G.N. (1989) Structural and electronic properties of the liver fluke
heme cavity by nuclear magnetic resonance and optical spectro-
scopy. J. Mol. Biol. 209, 235–247.
56. Tuchschmid, P.E., Kuntz, P.A. & Wilson, K.J. (1978) Isolation
and characterization of hemoglobin from Lanceolate fluke

Dicrocoelium dendriticum. Eur. J. Biochem. 88, 387–394.
Supplementary material
The following material is available from http://www.
blackwellpublishing.com/products/journals/suppmat/EJB/
EJB3638/EJB3638sm.htm
Figure S1. Portions of the 500 MHz TOCSY spectrum
(mixing time 30 ms, repetition rate 2 s
)1
)in
1
H
2
O, 100 m
M
in phosphate, pH 7.2 at 25 °C, showing the heme proton spin
systems.
Figure S2. Portions of the 500 MHz NOESY spectrum
(mixing time 60 ms, repetition rate 2 s
)1
)in
2
H
2
O, 100 m
M
in phosphate, pH 7.1 at 30 °C, illustrating heme proton
dipolar contacts.
Figure S3. Portion of the 600 MHz NOESY spectrum
(mixing time 80 ms, repetition rate 1 s
)1

)in
1
H
2
O, 100 m
M
in phosphate, pH 6.8 at 35 °C, illustrating the E- and F-helix
N
i
–N
i+1
connections (solid lines and arrows), the F-helix a
i
–
N
i+1
connections (dashed lines), and the dipolar contacts
between Tyr32(B10), Tyr66(E7) and Phe46(CD1).
Table S1.
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