The XANES (X‐ray absorption near edge structure) spectra of deoxy human adult haemoglobin (HbA) and myoglobin (Mb) have been measured at the wiggler beam line of the Frascati synchrotron radiation facility. The XANES are interpreted by the multiple scattering cluster theory. The variations in the XANES between HbA and Mb are assigned to changes in the Fe‐porphyrin geometry

A. Bianconi

A. Congiu-Castellano

A. Giovannelli

Bianconi

Collman

Cramer

Desbois

Durham

E. Burattini

Eisenberger

Fermi

Hopfield

Jameson

M. Barteri

M. Dell'Ariccia

Monod

P.J. Durham

Perutz

Takano

Traylor

English

Crossref

FEBS Letters

XANES study of iron displacement in the haem of myoglobin

AbstractThe XANES (X-ray absorption near edge structure) spectra of deoxy human adult haemoglobin (HbA) and myoglobin (Mb) have been measured at the wiggler beam line of the Frascati synchrotron radiation facility. The XANES are interpreted by the multiple scattering cluster theory. The variations in the XANES between HbA and Mb are assigned to changes in the Fe-porphyrin geometry

Bianconi, A.

Congiu-Castellano, A.

Dell'Ariccia, M.

Giovannelli, A.

Durham, P.J.

Burattini, E.

Barteri, M.

Elsevier - Publisher Connector 

XANES study of iron displacement in the haem of myoglobin 

Volume 178, number 1 FEBS 2039 December 1984 
XANES study of iron displacement in the haem of 
myoglobin 
A. Bianconi, A. Congiu-Castellano, M. Dell’Ariccia, A. Giovannelli, P.J. Durham”, E. Burattini+ 
and M. Barter? 
Dipartimento di Fisica, Universith di Roma ‘La Sapienza’, 00185 Roma, Italy, “SERC, Daresbury Laboratory, Daresbury, 
Warrington. WA4 4AD. England, +CNR-INFN, Laboratori Nazionali di Frascati, 00044 Frascati, and *Dipartimento di 
Chimica, Universita di Roma La Sapienza, 00185 Roma, Italy 
Received 10 October 1984 
The XANES (X-ray absorption near edge structure) spectra of deoxy human adult haemoglobin (HbA) 
and myoglobin (Mb) have been measured at the wiggler beam line of the Frascati synchrotron radiation 
facility. The XANES are interpreted by the multiple scattering cluster theory. The variations in the XANES 
between HbA and Mb are assigned to changes in the Fe-porphyrin geometry. 
Myoglobin Hemoglobin XANES X-ray absorption Synchrotron radiation Heme structure 
1. INTRODUCTION 
The displacement of the iron atom out of the 
plane of porphyrin-nitrogens (Fe-Ct) in the haem 
of haemoglobin has attracted large interest since 
the work in [l]; Fe, out of plane in deoxygenated 
haemoglobin (deoxy-Hb), moves into the plane 
upon oxygenation. This movement has been con- 
sidered to be the main pathway by which the infor- 
mation of 02 bonding to Fe is transmitted to the 
rest of the protein and it is associated with the tran- 
sition between the two states of haemoglobin (the 
low affinity, tense (T) state, and the high affinity, 
relaxed (R) state), first suggested by authors in [2] 
to provide a satisfactory description of the 
cooperative ligand binding in haemoglobin [3]. 
High energy electron storage rings provide in- 
tense synchrotron radiation in the X-ray range 
which makes possible measurements of X-ray ab- 
sorption spectra of proteins in solution. The ap- 
pealing aspect of X-ray absorption spectroscopy 
for molecular biology is that it can provide direct 
information on the local structure of proteins in 
solution and therefore makes feasible the studies 
of structural changes during the function of the 
proteins. 
The distance of Fe from the plane of porphyrin 
nitrogens (Fe-Ct) in deoxy-Hb has been related to 
the affinity for ligand binding of haemoglobin in 
the state T [4]. Although many recent papers have 
pointed out that the ligand affinity in haemoglobin 
[3] and in synthetic porphyrin models [5-71 is a 
complex phenomenon, involving also atoms of the 
globin and the rest of the protein, the iron 
displacement in the deoxy form is one of the key 
parameters in the control of ligand affinity. 
Above the continuum threshold for core (K 
level) excitation the absorption cross-section is 
modulated by diffraction from neighbour atoms of 
the photoelectron emitted in the continuum at the 
site of the absorbing atom. At the photoelectron 
kinetic energies (given by the photon energy less 
the energy of the absorption threshold) higher than 
50 eV (EXAFS regime) the photoelectron is scat- 
tered only once from each neighbour atom (single 
scattering) and the interference effects modulate 
the absorption cross-section giving the EXAFS 
oscillations which give the interatomic distances 
and coordination numbers. At lower energies 
(XANES regime) the photoelectron is generally in 
the multiple scattering regime and the X-ray ab- 
sorption near edge structure (XANES) can give 
Published by Elsevier Science Publishers B. V. 
00145793/84/$3.00 0 1984 Federation of European Biochemical Societies 165 
Volume 178, number 1 FEBS LETTERS December 1984 
more information on the local geometry: bonding 
angles and distortions of symmetry due to small 
atomic displacements [S-lo]. EXAFS has been 
successfully used to measure the mean distance of 
Fe from the porphyrin nitrogen (Fe-Np) of 
haemoglobin in solution with an accuracy of 
+ 0.01 A. Authors in [11,12] have found the same 
Fe-Np distance 2.055 A in the low affinity human 
deoxy-Hb A (T), in the high affinity deoxy-Hb 
kempsey and in the deoxy ‘picket-fence’ porphyrin 
Fe(TpivPP)(N-MeIm). They reach the conclusion 
that the difference between the high and low affini- 
ty forms of Hb is not associated with a Fe-Np bond 
strain. 
The iron displacement Fe-Ct cannot be 
measured directly from EXAFS because EXAFS 
data do not contain information on bonding 
angles, as pointed out in [13]. Therefore the con- 
clusion of authors in [12] that the iron displace- 
ment in deoxy HbA is the same as in 
Fe(TpivPP)(N-MeIm), Fe-Ct = 0.2% A cannot 
convincingly be inferred from their similar EXAFS 
spectra. 
Crystallographic data (see table 1) give different 
Fe-Ct displacements in deoxy-HbA, deoxy 
myoglobin [14] and in Fe(TpivPP)(N-MeIm) por- 
phyrin [12,15] (see table 1). We have performed 
this experiment o show that XANES, because of 
multiple scattering, contains information on the 
bonding angles and therefore on variations of the 
Fe displacement. 
2. EXPERIMENTAL 
The XANES measurements were performed at 
the Frascati ‘wiggler’ beam line using synchrotron 
radiation monochromatized with a Si(220) 
channel-cut crystal and 0.5-l mm exit slits were 
used. The absorption spectra were collected in 
transmission using solution samples of the proteins 
at concentrations ranging from 4 to 10 mM in 
haem. The zero of the energy scale was carefully 
fixed at the absorption threshold of the Fe metal 
K-edge defined as the first maximum of its 
derivative spectrum. This energy is very close to 
the absorption threshold of the deoxymyoglobin 
spectrum defined as the energy of the maximum of 
the first weak absorption peak. The pre-edge ab- 
sorption background was subtracted in all spectra. 
The absorption coefficient was normalized in all 
spectra to a~, the atomic absorption above the ab- 
sorption jump obtained by extrapolation toward 
lower energy of the linear fitting of EXAFS oscilla- 
tions in the range 50-150 eV above the absorption 
threshold. Because the Fe atomic absorption is the 
same for all spectra, this normalization procedure 
allows the quantitative comparison between dif- 
ferent spectra. The spectra have been obtained 
both by adding up to three scans and by single 
scans with integration time of 10 s/point. 
The experiment has been repeated in 3 different 
runs over a year using different samples. The 
energy resolution is about 1 eV and energy shifts 
Table 1 
Geometry of deoxy Fe-haem complexes from crystallographic dataa 
Fe-Ct (A) Fe-Np (A)b Fe-Cp (A) Fe-N, (A) 
Deoxy-HbA 0.434 2.06 (2.055)’ 
Deoxy-Mb (pH 5.75, 20°C) 0.42 2.03 
Deoxy-Mb (pH 8.41, - 12°C) 0.3 2.02 (5) 
Deoxy Fe(TpivPP) N-MeIm 0.25 2.055’ 
a Refined data communicated by G. Fermi and S.E. Phillips 
b Mean distance 
’ From EXAFS data 
0.54 2.12 (7)c 
0.47 2.22 
0.38 2.10 
Fe-Np, distance of Fe from the porphyrin nitrogen; Fe-N,, distance of Fe from the 
nitrogen of the proximal histidine F8 (His F8); Fe-Ct, distance of Fe from mean plane of 
porphyrin nitrogens, denotes displacement towards histidine F8 (His F8); Fe-Cp, distance 
of Fe from mean plane of porphyrin; deoxy, deoxygenated; HbA, human adult 
haemoglobin; Mb, myoglobin (sperm whale); TpivPP, meso-tetrakis ( LY,(Y,u,LY[(-O- 
pivaloy)amido]phenyl ) porphyrin; N-MeIm, N-methylimidazole 
166 
Volume 178, number 1 FEBS LETTERS December 1984 
larger than 0.2 eV can be measured. 
Deoxyhaemoglobin samples were prepared from 
human oxyhaemoglobin A flushing with nitrogen 
gas and adding dithionite before the beginning of 
the experimental run. Deoxymyoglobin (deoxy 
Mb) was prepared at pH 7.2 from sperm whale 
metmyoglobin from Sigma. Optical spectra of 
non-irradiated and irradiated samples were used to 
verify their quality. The samples, exposed to radia- 
tion in an inert He atmosphere, were kept at cons- 
tant temperature (1O’C). 
3. RESULTS AND DISCUSSION 
From figs 1 and 2 it appears that the features 
labeled A-E are present in all spectra and their 
relative intensity changes going from Mb to Hb 
while no significant changes are observed between 
Mb and the picket fence porphyrin. It is interesting 
to remark that while XANES spectra of Hb and of 
this porphyrin are different no differences were 
observed between their EXAFS spectra [12]. 
Fig.3 shows the results of theoretical calcula- 
tions of XANES of a porphyrin cluster by the 
multiple scattering theory [17] which has been suc- 
cessful in the determination of small geometrical 
distortions in ferro- and ferricyanide complexes. 
We have used the same scattering amplitude for 
1 -_-.I_IL_I 
a 10 20 30 40 50 
EHERO" ,*"I 
Fig. 1. Experimental Fe K-XANES of sperm whale 
deoxymyoglobin (Mb) (-) and of human adult 
deoxyhaemoglobin (HbA) (- - -). The spectra are 
normalized at the atomic absorption and the zero of the 
energy is fixed at the localized Fe 3d resonance at 
threshold. 
1.0. 
-Mb 
Fe lT,,,PPHN-Me Im) 
, __---L _._.-----L 
0 IO 20 30 40 50 
ENERGY(&) 
1 
Fig.2. Experimental Fe K-XANES of deoxymyoglobin 
(-) and of picket fence porphyrin Fe(TpivPP)(N- 
MeIm) from [16] (+ --). 
Fe, C and N atoms already tested in the analysis of 
XANES of K3Fe(CN)h [18]. The size of the cluster 
of atoms determining the XANES is limited by 
non-elastic photoelectron scattering with valence 
electrons and it has been empirically determined by 
successive XANES calculations including suc- 
cessive shells of neighbour atoms. A cluster 
formed by the 24 atoms of the porphyrin and the 
3 atoms of the proximal histidine F8 plus the cen- 
:ral Fe atom was necessary to reproduce all 
XANES features from A to E of the experimental 
spectra. At the same time, the non-elastic scatter- 
ing also gives rise to the large broadening of 
XANES, and it has been introduced into the 
calculations as an imaginary part in the energy. We 
found a good agreement [19] between the experi- 
mental spectrum of deoxy Hb and the averaged 
spectrum over the two polarizations of the photon 
beam (parallel to the normal to the porphyrin 
plane n’, and perpendicular to ii’, where the 
photoelectron is scattered in the porphyrin plane) 
using the atomic positions previously determined 
for Hb [12,13,20]. The structure used in the 
calculations was idealised in that the porphyrin 
ring was taken to be planar and 4-fold 
symmetrical. 
To relate the differences in the experimental 
XANES spectra to atomic displacements we have 
calculated the effect of Fe displacement out of the 
porphyrin plane by keeping the porphyrin planar. 
167 
number 1 
k 1 shell 
FEBS LETTERS 
Ii 
: 
: 
0 10 20 30 40 
ENERGY(eV) 
Fig.3. Theoretical Fe K-XANES of the haem cluster of 
deoxy haem-proteins. The unpolarized spectrum for a 
symmetrical idealized haem cluster including 27 
neighbour atoms is shown in the upper panel. The curves 
in the lower panel show the effect on calculated XANES, 
of a cluster including Fe and the atoms in the haem 
plane, of the size of the cluster. The spectra for small 
clusters including only the first shell (5 atoms), 1st and 
second shells (21 atoms) is shown. The zero of the energy 
scale for the photoelectron kinetic energy is fixed at the 
energy of the atomic Fe 3d resonance which corresponds 
in the experimental spectra to the defined absorption 
threshold. 
Fig.4 shows that as the Fe atom goes out of the 
porphyrin plane (Fe-Ct from 0 to 0.53 A = 1 a.u.) 
the intensity of peak C, due to multiple scattering 
of the photoelectron (E _L n) in the porphyrin 
plane, increases with the iron displacement (Fe- 
Ct). The agreement between the calculated 
XANES and the experimental spectra is very good 
for the energy positions of the spectral features. 
December 1 Y84 
L I I I I I 
0 10 20 30 40 50 
PHOTOELECTRON ENkRGY (eV) 
Fig.4. Theoretical XANES spectra for a cluster of 30 
atoms formed by a planar porphyrin where the Fe is 
moved out of plane: model (a): Fe-Ct = 1 a.u. (0.53 A), 
model (b): Fe-Ct = 0.5 a.u., model (c): Fe-Ct = 0 a.u., 
only the contribution of the scattering in the haem plane 
is shown. 
The lineshape of the features depends on the 
energy dependent non-elastic scattering which is 
approximated in the theory by a constant im- 
aginary part of the energy. Moreover, the por- 
phyrin plane is expected to be distorted in the pro- 
teins with some doming effect which is not taken 
into account here. Therefore, only a qualitative 
agreement for the lineshape between the 
theoretical and experimental XANES is expected. 
However, the XANES calculations indicate that 
the physical effect of the iron displacement (Fe-Ct) 
(keeping all atomic positions of the porphyrin 
fixed) on the XANES is an increase of peak C. The 
theory shows that the XANES spectra of haemo- 
proteins are determined by a large cluster of -30 
atoms but the XANES spectra features are only 5 
168 
Volume 178, number 1 FEBS LETTERS December 1984 
which can shift in energy, mainly following the 
variation of interatomic distance [8,9] and change 
their shape and relative intensities following the 
bonding angles. In deducing the variation of the 
structure from XANES in this class of proteins it 
is therefore necessary to keep in mind that several 
geometrical distortions such as movement of car- 
bon atoms in the periphery of the cluster as well as 
the tilt of pyrroles (the doming effect) and other 
distortions can affect the lineshape of XANES. 
The experimental XANES spectra of deoxy 
HbA and deoxy Mb in fig. 1 show variations in the 
energy region of peaks B and C. The XANES of 
deoxy human haemoglobin HbA exhibits a clear 
increase of the peak C in comparison with the 
XANES of myoglobin. 
The variation between the XANES of deoxy Mb 
and deoxy HbA are in agreement with the 
predicted spectral variation arising from the iron 
displacement oward the haem plane in Mb, by 
0.1 A, with respect to the placement in HbA 
(-0.4 A). Given the noise level of our data we 
estimate that we can appreciate the variation of 
0.05 A of the iron displacement. However, it is 
possible to use it only as a qualitative estimate of 
the error of the iron displacement in myoglobin 
(0.3 f 0.05 A) because other possible distortions 
of the haem can also affect the XANES in a similar 
way. 
The similarity observed in fig.2 between the 
XANES of deoxy Mb and of the picket fence por- 
phyrin where the Fe is -0.3 out of plane and where 
the EXAFS spectra of both systems [11,12] give 
the same Fe-Np distance, seems to confirm a 
similar iron displacement in the two systems. 
However, while the different atomic arrangements 
between Mb and HbA are different only because 
of atomic positions, in the picket fence porphyrin 
there are different side molecular groups on the 
periphery of porphyrin plane which can affect the 
XANES, and therefore the comparison between 
the two XANES spectra cannot be significative of 
variation or similarity in the local structure. 
4. CONCLUSION 
Our data indicate a smaller Fe displacement in 
myoglobin than in haemoglobin. This result is in 
agreement with Mossbauer data [21], from which 
a Fe displacement 0.1 A smaller in Mb than in 
HbA has been deduced, and with resonance 
Raman spectra [22]. Two diffraction studies of 
myoglobin at different temperatures and different 
pH have been carried out (see table 1) [14]. The 
diffraction results give two different Fe-Ct 
displacements 0.42 A at pH 5.7 and 0.3 A at pH 
8.5. The XANES of deoxymyoglobin taken in 
solution at 10°C and pH 7.2 seem to be in agree- 
ment with diffraction data of Mb taken at pH 8.4. 
It is interesting to note that the XANES spectra 
of deoxy a and ,0 chains of haemoglobin measured 
in [23] show a weak difference in the energy range 
of peaks A and B which, if it is confirmed by better 
signal-to-noise spectra, would indicate an iron 
displacement larger in the cy chain than in the fl 
chain. 
REFERENCES 
111 
PI 
I31 
I41 
I51 
161 
I71 
I81 
I91 
I101 
Ill1 
I121 
I131 
I141 
Perutz, M.F. (1970) Nature 228, 726-739. 
Monod, J., Wyman, J. and Changeux, J.P. (1965) 
J. Mol. Biol. 12, 88-118. 
Perutz, M.F. (1979) Annu. Rev. Biochem. 48, 
327-386. 
Hopfield, J.J. (1973) J. Mol. Biol. 77, 207-222. 
Traylor, T.G. (1981) Act. Chem. Res. 14, 102-109 
and references therein. 
Collman, J.P., Halbert, T.R. and Suslick, K.S. 
(1980) in: Metal Ion Activation of Dioxygen (Spiro, 
T.G. ed.) Wiley, New York, and references therein. 
Jameson, B.J. and Ibers, J.A. (1983) Comments 
Inorg. Chem. 2, 97-126. 
Bianconi, A., Alema, S., Castellani, L., Fasella, 
P., Giovannelli, A., Mobilio, S. and Oesh, B. 
(1983) J. Mol. Biol. 165, 125-138. 
Bianconi, A., Incoccia, L. and Stipcich, S. (1983) 
EXAFS and Near Edge Structure, Springer Series 
in Chemical Physics, ~01.27, Springer Verlag, 
Berlin. 
Bianconi, A. (1984) in: Principles, Techniques and 
Applications of EXAFS, SEXAFS and XANES 
(Prinz, R. and Koningsberger, D. eds) Wiley, New 
York. 
Eisenberger, P., Shulman, R.G., Brown, G.S. and 
Ogawa, S. (1976) Proc. Natl. Acad. Sci. USA 73, 
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Eisenberger, P., Shulman, R.G., Kincaid, B.M., 
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J.E. and Sessler, J.L. (1982) Nature 295, 535-538. 
Takano, T. (1977) J. Mol. Biol. 110, 537-568. 
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1151 Jameson, J.B., Molinaro, F.S., tbers, J.A., 1191 Durham, P.J., Bianconi, A., Congiu-Castellano, 
Collman, J.P., Brauman, J.I., Rose, E. and A., Giovannelli, A., Hasnain, S.S., Incoccia, L., 
Suslick, KS. (1980) J. Am. Chem. Sot. 102, Morante, S. and Pendry, J.B. (1983) EMBO J. 2, 
3224-3237. 1441-1444. 
116) Cramer, S.P. (1978) PhD Thesis, Stanford 
University, S.S.R.L. Report 78’07. 
[17] Durham, P.J., Pendry, J.B. and Hodges, C.R. 
(1982) Comput. Phys. Commun. 25, 193-205. 
[18] Bianconi, A., Dell’Ariccia, M., Durham, P.J. and 
Pendry, J.B. (1982) Phys. Rev. B26, 6502-6508. 
f20] Fermi, G. (1975) J .Mol. Biol. 97, 237-256. 
[22] Desbois, A., Lutz, M. and Banerjee, R. (1981) Bio- 
chim. Biophys. Acta 671, 177-183. 
[23] Pin, S., Alpert, B. and Michalowicz, A. (1982) 
FEBS Lett. 147, 106-110. 
170 


Antonio Bianconi

Mario Barteri

Open Access Repository

Volume 178, number 1 FEBS 2039 December 1984 XANES study of iron displacement in the haem of myoglobin A. Bianconi, A. Congiu-Castellano, M. Dell’Ariccia, A. Giovannelli, P.J. Durham”, E. Burattini+ and M. Barter? Dipartimento di Fisica, Universith di Roma ‘La Sapienza’, 00185 Roma, Italy, “SERC, Daresbury Laboratory, Daresbury, Warrington. WA4 4AD. England, +CNR-INFN, Laboratori Nazionali di Frascati, 00044 Frascati, and *Dipartimento di Chimica, Universita di Roma La Sapienza, 00185 Roma, Italy Received 10 October 1984 The XANES (X-ray absorption near edge structure) spectra of deoxy human adult haemoglobin (HbA) and myoglobin (Mb) have been measured at the wiggler beam line of the Frascati synchrotron radiation facility. The XANES are interpreted by the multiple scattering cluster theory. The variations in the XANES between HbA and Mb are assigned to changes in the Fe-porphyrin geometry. Myoglobin Hemoglobin XANES X-ray absorption Synchrotron radiation Heme structure 1. INTRODUCTION The displacement of the iron atom out of the plane of porphyrin-nitrogens (Fe-Ct) in the haem of haemoglobin has attracted large interest since the work in [l]; Fe, out of plane in deoxygenated haemoglobin (deoxy-Hb), moves into the plane upon oxygenation. This movement has been con- sidered to be the main pathway by which the infor- mation of 02 bonding to Fe is transmitted to the rest of the protein and it is associated with the tran- sition between the two states of haemoglobin (the low affinity, tense (T) state, and the high affinity, relaxed (R) state), first suggested by authors in [2] to provide a satisfactory description of the cooperative ligand binding in haemoglobin [3]. High energy electron storage rings provide in- tense synchrotron radiation in the X-ray range which makes possible measurements of X-ray ab- sorption spectra of proteins in solution. The ap- pealing aspect of X-ray absorption spectroscopy for molecular biology is that it can provide direct information on the local structure of proteins in solution and therefore makes feasible the studies of structural changes during the function of the proteins. The distance of Fe from the plane of porphyrin nitrogens (Fe-Ct) in deoxy-Hb has been related to the affinity for ligand binding of haemoglobin in the state T [4]. Although many recent papers have pointed out that the ligand affinity in haemoglobin [3] and in synthetic porphyrin models [5-71 is a complex phenomenon, involving also atoms of the globin and the rest of the protein, the iron displacement in the deoxy form is one of the key parameters in the control of ligand affinity. Above the continuum threshold for core (K level) excitation the absorption cross-section is modulated by diffraction from neighbour atoms of the photoelectron emitted in the continuum at the site of the absorbing atom. At the photoelectron kinetic energies (given by the photon energy less the energy of the absorption threshold) higher than 50 eV (EXAFS regime) the photoelectron is scat- tered only once from each neighbour atom (single scattering) and the interference effects modulate the absorption cross-section giving the EXAFS oscillations which give the interatomic distances and coordination numbers. At lower energies (XANES regime) the photoelectron is generally in the multiple scattering regime and the X-ray ab- sorption near edge structure (XANES) can give Published by Elsevier Science Publishers B. V. 00145793/84/$3.00 0 1984 Federation of European Biochemical Societies 165 Volume 178, number 1 FEBS LETTERS December 1984 more information on the local geometry: bonding angles and distortions of symmetry due to small atomic displacements [S-lo]. EXAFS has been successfully used to measure the mean distance of Fe from the porphyrin nitrogen (Fe-Np) of haemoglobin in solution with an accuracy of + 0.01 A. Authors in [11,12] have found the same Fe-Np distance 2.055 A in the low affinity human deoxy-Hb A (T), in the high affinity deoxy-Hb kempsey and in the deoxy ‘picket-fence’ porphyrin Fe(TpivPP)(N-MeIm). They reach the conclusion that the difference between the high and low affini- ty forms of Hb is not associated with a Fe-Np bond strain. The iron displacement Fe-Ct cannot be measured directly from EXAFS because EXAFS data do not contain information on bonding angles, as pointed out in [13]. Therefore the con- clusion of authors in [12] that the iron displace- ment in deoxy HbA is the same as in Fe(TpivPP)(N-MeIm), Fe-Ct = 0.2% A cannot convincingly be inferred from their similar EXAFS spectra. Crystallographic data (see table 1) give different Fe-Ct displacements in deoxy-HbA, deoxy myoglobin [14] and in Fe(TpivPP)(N-MeIm) por- phyrin [12,15] (see table 1). We have performed this experiment to show that XANES, because of multiple scattering, contains information on the bonding angles and therefore on variations of the Fe displacement. 2. EXPERIMENTAL The XANES measurements were performed at the Frascati ‘wiggler’ beam line using synchrotron radiation monochromatized with a Si(220) channel-cut crystal and 0.5-l mm exit slits were used. The absorption spectra were collected in transmission using solution samples of the proteins at concentrations ranging from 4 to 10 mM in haem. The zero of the energy scale was carefully fixed at the absorption threshold of the Fe metal K-edge defined as the first maximum of its derivative spectrum. This energy is very close to the absorption threshold of the deoxymyoglobin spectrum defined as the energy of the maximum of the first weak absorption peak. The pre-edge ab- sorption background was subtracted in all spectra. The absorption coefficient was normalized in all spectra to a~, the atomic absorption above the ab- sorption jump obtained by extrapolation toward lower energy of the linear fitting of EXAFS oscilla- tions in the range 50-150 eV above the absorption threshold. Because the Fe atomic absorption is the same for all spectra, this normalization procedure allows the quantitative comparison between dif- ferent spectra. The spectra have been obtained both by adding up to three scans and by single scans with integration time of 10 s/point. The experiment has been repeated in 3 different runs over a year using different samples. The energy resolution is about 1 eV and energy shifts Table 1 Geometry of deoxy Fe-haem complexes from crystallographic dataa Fe-Ct (A) Fe-Np (A)b Fe-Cp (A) Fe-N, (A) Deoxy-HbA 0.434 2.06 (2.055)’ Deoxy-Mb (pH 5.75, 20°C) 0.42 2.03 Deoxy-Mb (pH 8.41, - 12°C) 0.3 2.02 (5) Deoxy Fe(TpivPP) N-MeIm 0.25 2.055’ a Refined data communicated by G. Fermi and S.E. Phillips b Mean distance ’ From EXAFS data 0.54 2.12 (7)c 0.47 2.22 0.38 2.10 Fe-Np, distance of Fe from the porphyrin nitrogen; Fe-N,, distance of Fe from the nitrogen of the proximal histidine F8 (His F8); Fe-Ct, distance of Fe from mean plane of porphyrin nitrogens, denotes displacement towards histidine F8 (His F8); Fe-Cp, distance of Fe from mean plane of porphyrin; deoxy, deoxygenated; HbA, human adult haemoglobin; Mb, myoglobin (sperm whale); TpivPP, meso-tetrakis ( LY,(Y,u,LY[(-O- pivaloy)amido]phenyl ) porphyrin; N-MeIm, N-methylimidazole 166 Volume 178, number 1 FEBS LETTERS December 1984 larger than 0.2 eV can be measured. Deoxyhaemoglobin samples were prepared from human oxyhaemoglobin A flushing with nitrogen gas and adding dithionite before the beginning of the experimental run. Deoxymyoglobin (deoxy Mb) was prepared at pH 7.2 from sperm whale metmyoglobin from Sigma. Optical spectra of non-irradiated and irradiated samples were used to verify their quality. The samples, exposed to radia- tion in an inert He atmosphere, were kept at cons- tant temperature (1O’C). 3. RESULTS AND DISCUSSION From figs 1 and 2 it appears that the features labeled A-E are present in all spectra and their relative intensity changes going from Mb to Hb while no significant changes are observed between Mb and the picket fence porphyrin. It is interesting to remark that while XANES spectra of Hb and of this porphyrin are different no differences were observed between their EXAFS spectra [12]. Fig.3 shows the results of theoretical calcula- tions of XANES of a porphyrin cluster by the multiple scattering theory [17] which has been suc- cessful in the determination of small geometrical distortions in ferro- and ferricyanide complexes. We have used the same scattering amplitude for 1 -_-.I_IL_I a 10 20 30 40 50 EHERO" ,*"I Fig. 1. Experimental Fe K-XANES of sperm whale deoxymyoglobin (Mb) (-) and of human adult deoxyhaemoglobin (HbA) (- - -). The spectra are normalized at the atomic absorption and the zero of the energy is fixed at the localized Fe 3d resonance at threshold. 1.0. -Mb Fe lT,,,PPHN-Me Im) , __---L _._.-----L 0 IO 20 30 40 50 ENERGY(&) 1 Fig.2. Experimental Fe K-XANES of deoxymyoglobin (-) and of picket fence porphyrin Fe(TpivPP)(N- MeIm) from [16] (+ --). Fe, C and N atoms already tested in the analysis of XANES of K3Fe(CN)h [18]. The size of the cluster of atoms determining the XANES is limited by non-elastic photoelectron scattering with valence electrons and it has been empirically determined by successive XANES calculations including suc- cessive shells of neighbour atoms. A cluster formed by the 24 atoms of the porphyrin and the 3 atoms of the proximal histidine F8 plus the cen- :ral Fe atom was necessary to reproduce all XANES features from A to E of the experimental spectra. At the same time, the non-elastic scatter- ing also gives rise to the large broadening of XANES, and it has been introduced into the calculations as an imaginary part in the energy. We found a good agreement [19] between the experi- mental spectrum of deoxy Hb and the averaged spectrum over the two polarizations of the photon beam (parallel to the normal to the porphyrin plane n’, and perpendicular to ii’, where the photoelectron is scattered in the porphyrin plane) using the atomic positions previously determined for Hb [12,13,20]. The structure used in the calculations was idealised in that the porphyrin ring was taken to be planar and 4-fold symmetrical. To relate the differences in the experimental XANES spectra to atomic displacements we have calculated the effect of Fe displacement out of the porphyrin plane by keeping the porphyrin planar. 167 number 1 k 1 shell FEBS LETTERS Ii : : 0 10 20 30 40 ENERGY(eV) Fig.3. Theoretical Fe K-XANES of the haem cluster of deoxy haem-proteins. The unpolarized spectrum for a symmetrical idealized haem cluster including 27 neighbour atoms is shown in the upper panel. The curves in the lower panel show the effect on calculated XANES, of a cluster including Fe and the atoms in the haem plane, of the size of the cluster. The spectra for small clusters including only the first shell (5 atoms), 1st and second shells (21 atoms) is shown. The zero of the energy scale for the photoelectron kinetic energy is fixed at the energy of the atomic Fe 3d resonance which corresponds in the experimental spectra to the defined absorption threshold. Fig.4 shows that as the Fe atom goes out of the porphyrin plane (Fe-Ct from 0 to 0.53 A = 1 a.u.) the intensity of peak C, due to multiple scattering of the photoelectron (E _L n) in the porphyrin plane, increases with the iron displacement (Fe- Ct). The agreement between the calculated XANES and the experimental spectra is very good for the energy positions of the spectral features. December 1 Y84 L I I I I I 0 10 20 30 40 50 PHOTOELECTRON ENkRGY (eV) Fig.4. Theoretical XANES spectra for a cluster of 30 atoms formed by a planar porphyrin where the Fe is moved out of plane: model (a): Fe-Ct = 1 a.u. (0.53 A), model (b): Fe-Ct = 0.5 a.u., model (c): Fe-Ct = 0 a.u., only the contribution of the scattering in the haem plane is shown. The lineshape of the features depends on the energy dependent non-elastic scattering which is approximated in the theory by a constant im- aginary part of the energy. Moreover, the por- phyrin plane is expected to be distorted in the pro- teins with some doming effect which is not taken into account here. Therefore, only a qualitative agreement for the lineshape between the theoretical and experimental XANES is expected. However, the XANES calculations indicate that the physical effect of the iron displacement (Fe-Ct) (keeping all atomic positions of the porphyrin fixed) on the XANES is an increase of peak C. The theory shows that the XANES spectra of haemo- proteins are determined by a large cluster of -30 atoms but the XANES spectra features are only 5 168 Volume 178, number 1 FEBS LETTERS December 1984 which can shift in energy, mainly following the variation of interatomic distance [8,9] and change their shape and relative intensities following the bonding angles. In deducing the variation of the structure from XANES in this class of proteins it is therefore necessary to keep in mind that several geometrical distortions such as movement of car- bon atoms in the periphery of the cluster as well as the tilt of pyrroles (the doming effect) and other distortions can affect the lineshape of XANES. The experimental XANES spectra of deoxy HbA and deoxy Mb in fig. 1 show variations in the energy region of peaks B and C. The XANES of deoxy human haemoglobin HbA exhibits a clear increase of the peak C in comparison with the XANES of myoglobin. The variation between the XANES of deoxy Mb and deoxy HbA are in agreement with the predicted spectral variation arising from the iron displacement toward the haem plane in Mb, by 0.1 A, with respect to the placement in HbA (-0.4 A). Given the noise level of our data we estimate that we can appreciate the variation of 0.05 A of the iron displacement. However, it is possible to use it only as a qualitative estimate of the error of the iron displacement in myoglobin (0.3 f 0.05 A) because other possible distortions of the haem can also affect the XANES in a similar way. The similarity observed in fig.2 between the XANES of deoxy Mb and of the picket fence por- phyrin where the Fe is -0.3 out of plane and where the EXAFS spectra of both systems [11,12] give the same Fe-Np distance, seems to confirm a similar iron displacement in the two systems. However, while the different atomic arrangements between Mb and HbA are different only because of atomic positions, in the picket fence porphyrin there are different side molecular groups on the periphery of porphyrin plane which can affect the XANES, and therefore the comparison between the two XANES spectra cannot be significative of variation or similarity in the local structure. 4. CONCLUSION Our data indicate a smaller Fe displacement in myoglobin than in haemoglobin. This result is in agreement with Mossbauer data [21], from which a Fe displacement 0.1 A smaller in Mb than in HbA has been deduced, and with resonance Raman spectra [22]. Two diffraction studies of myoglobin at different temperatures and different pH have been carried out (see table 1) [14]. The diffraction results give two different Fe-Ct displacements 0.42 A at pH 5.7 and 0.3 A at pH 8.5. The XANES of deoxymyoglobin taken in solution at 10°C and pH 7.2 seem to be in agree- ment with diffraction data of Mb taken at pH 8.4. It is interesting to note that the XANES spectra of deoxy a and ,0 chains of haemoglobin measured in [23] show a weak difference in the energy range of peaks A and B which, if it is confirmed by better signal-to-noise spectra, would indicate an iron displacement larger in the cy chain than in the fl chain. REFERENCES 111 PI I31 I41 I51 161 I71 I81 I91 I101 Ill1 I121 I131 I141 Perutz, M.F. (1970) Nature 228, 726-739. Monod, J., Wyman, J. and Changeux, J.P. (1965) J. Mol. Biol. 12, 88-118. Perutz, M.F. (1979) Annu. Rev. Biochem. 48, 327-386. Hopfield, J.J. (1973) J. Mol. Biol. 77, 207-222. Traylor, T.G. (1981) Act. Chem. Res. 14, 102-109 and references therein. Collman, J.P., Halbert, T.R. and Suslick, K.S. (1980) in: Metal Ion Activation of Dioxygen (Spiro, T.G. ed.) Wiley, New York, and references therein. Jameson, B.J. and Ibers, J.A. (1983) Comments Inorg. Chem. 2, 97-126. Bianconi, A., Alema, S., Castellani, L., Fasella, P., Giovannelli, A., Mobilio, S. and Oesh, B. (1983) J. Mol. Biol. 165, 125-138. Bianconi, A., Incoccia, L. and Stipcich, S. (1983) EXAFS and Near Edge Structure, Springer Series in Chemical Physics, ~01.27, Springer Verlag, Berlin. Bianconi, A. (1984) in: Principles, Techniques and Applications of EXAFS, SEXAFS and XANES (Prinz, R. and Koningsberger, D. eds) Wiley, New York. Eisenberger, P., Shulman, R.G., Brown, G.S. and Ogawa, S. (1976) Proc. Natl. Acad. Sci. USA 73, 491-49s. Eisenberger, P., Shulman, R.G., Kincaid, B.M., Brown, G.S. and Ogawa, S. (1978) Nature 274, 30-34. Perutz, M.F., Hasnain, S.S., Duke, P.J., Hahn, J.E. and Sessler, J.L. (1982) Nature 295, 535-538. Takano, T. (1977) J. Mol. Biol. 110, 537-568. 169 Volume 178, number 1 FEBS LETTERS December 1984 1151 Jameson, J.B., Molinaro, F.S., tbers, J.A., 1191 Durham, P.J., Bianconi, A., Congiu-Castellano, Collman, J.P., Brauman, J.I., Rose, E. and A., Giovannelli, A., Hasnain, S.S., Incoccia, L., Suslick, KS. (1980) J. Am. Chem. Sot. 102, Morante, S. and Pendry, J.B. (1983) EMBO J. 2, 3224-3237. 1441-1444. 116) Cramer, S.P. (1978) PhD Thesis, Stanford University, S.S.R.L. Report 78’07. [17] Durham, P.J., Pendry, J.B. and Hodges, C.R. (1982) Comput. Phys. Commun. 25, 193-205. [18] Bianconi, A., Dell’Ariccia, M., Durham, P.J. and Pendry, J.B. (1982) Phys. Rev. B26, 6502-6508. f20] Fermi, G. (1975) J .Mol. Biol. 97, 237-256. [22] Desbois, A., Lutz, M. and Banerjee, R. (1981) Bio- chim. Biophys. Acta 671, 177-183. [23] Pin, S., Alpert, B. and Michalowicz, A. (1982) FEBS Lett. 147, 106-110. 170 

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XANES study of iron displacement in the haem of myoglobin

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