Tài liệu Báo cáo khoa học: Binding of ligands originates small perturbations on the microscopic thermodynamic properties of a multicentre redox protein pptx

Binding of ligands originates small perturbations on the
microscopic thermodynamic properties of a multicentre
redox protein
Carlos A. Salgueiro
1,2
, Leonor Morgado
1,2
, Bruno Fonseca
1,2
, Pedro Lamosa
1
, Teresa Catarino
1,2
,
David L. Turner
3
and Ricardo O. Louro
1
1 Instituto de Tecnologia Quimica e Biolo
´
gica, Universidade Nova de Lisboa, Portugal
2 Departamento de Quimica da Faculdade de Cie
ˆ
ncias e Tecnologia da Universidade Nova de Lisboa, Portugal
3 School of Chemistry, University of Southampton, UK
The structural aspects of protein complexes have
received considerable attention and several experimen-
tal and computational methods for the structural
determination of complexes exist [1]. Redox proteins
usually form transient complexes that can be studied
using NMR methods, which, in addition to the struc-

tural characterization, also provide information on the
lifetime and dynamics of the bound forms [2,3]. Trans-
fer of electrons between redox proteins at rates com-
patible with metabolic processes requires the proper
orientation of the partners for close approximation of
the redox centres of the donor and acceptor, and that
the reduction potentials ensure a favourable driving
force, which is one of the main determinants of the
rate of electron transfer [4]. Experimental measure-
ments of the reduction potentials of proteins involved
in complexes have been reported [5–7], but the effect
of partner binding on the microscopic properties of the
redox centres in proteins with multiple centres has not
been addressed in detail yet.
Cytochromes c
3
from sulfate-reducing bacteria are
small soluble proteins containing four haems, and have
been assigned a fundamental role in the bioenergetic
metabolism of these organisms, mediating the flow of
electrons from periplasmic hydrogenases to respiratory
transmembrane electron transfer complexes coupled to
the transfer of protons [8–11]. Several cytochromes c
3
have been isolated and characterized in great detail
with respect to structure (for a recent revision of struc-
tural work see [12]), equilibrium thermodynamic prop-
erties [9,13–17] and transient kinetic properties [17–19].
These studies have shown that cytochromes c
3

have
the required thermodynamic properties to perform a
coordinated transfer of two electrons coupled to the
transfer of protons in agreement with their proposed
physiological role as partners of hydrogenase [8,20,21].
Keywords
cytochrome c
3
; electron transfer; NMR;
protein docking; thermodynamic properties
Correspondence
R. O. Louro, Instituto de Tecnologia
Quimica e Biolo
´
gica, Universidade Nova de
Lisboa, Rua da Quinta Grande 6,
2780-156 Oeiras, Portugal
Fax: 351-21-4428766
Tel: 351-21-4469848
E-mail:
(Received 15 December 2004, revised 15
February 2005, accepted 7 March 2005)
doi:10.1111/j.1742-4658.2005.04649.x
NMR and visible spectroscopy coupled to redox measurements were used
to determine the equilibrium thermodynamic properties of the four haems
in cytochrome c
3
under conditions in which the protein was bound to lig-
ands, the small anion phosphate and the protein rubredoxin with the iron
in the active site replaced by zinc. Comparison of these results with data

for the isolated cytochrome shows that binding of ligands causes only small
changes in the reduction potentials of the haems and their pairwise inter-
actions, and also that the redox-sensitive acid–base centre responsible for
the redox–Bohr effect is essentially unaffected. Although neither of the lig-
ands tested is a physiological partner of cytochrome c
3
, the small changes
observed for the thermodynamic properties of cytochrome c
3
bound to
these ligands vs. the unbound state, indicate that the thermodynamic prop-
erties measured for the isolated protein are relevant for a physiological
interpretation of the role of this cytochrome in the bioenergetic metabolism
of Desulfovibrio.
Abbreviations
DvHc
3
, Desulfovibrio vulgaris (Hildenborough) cytochrome c
3
; DvHc
3
:Pi, Desulfovibrio vulgaris cytochrome c
3
with phosphate; DvHc
3
:ZnRb,
Desulfovibrio vulgaris cytochrome c
3
with zinc rubredoxin; EXSY, exchange spectroscopy.
FEBS Journal 272 (2005) 2251–2260 ª 2005 FEBS 2251

However, no experimental data exist on the effect of
binding small ligands or proteins on these properties.
For cytochromes c
3
these effects have only been inves-
tigated in a theoretical study where cytochrome c
3
and
a redox partner were docked in silico [22].
Experimental data reported in the literature argue in
favour of a specific binding of phosphate to cyto-
chrome c
3
[23] instead of a simple electrostatic effect
of increased ionic strength at least up to 0.2 m concen-
tration. The region of positively charged amino acid
residues at the surface of the cytochrome surrounding
haem IV, provides ample opportunity for binding a
small anion such as phosphate, as was found for chro-
mate for the homologous trihaem cytochrome c
7
[24].
Also, the analysis of one-dimensional NMR experi-
ments showed that cytochrome c
3
and rubredoxin form
a complex with a binding constant > 10
4
m
)1

, and that
the most downfield shifted signal in the NMR spec-
trum of the ferricytochrome displays the most obvious
modification upon binding [25]. This signal was
assigned to the methyl 18
2
of haem IV [26] (methyl
nomenclature according to IUPAC-IUB recommenda-
tions [27] and Roman numerals designate the order of
attachment of the haem to the polypeptide chain),
which confirms the extensive work of molecular dock-
ing models for cytochrome c
3
with physiological and
nonphysiological protein partners [6,22,28–30] showing
always the positively charged region around haem IV
as the most favoured docking site.
The complex between cytochrome c
3
and rubredoxin
is not physiological because the two proteins are
located in different cellular compartments, but it pro-
vides a convenient model for studying the effect of
partner binding on the thermodynamic properties of
the haems of cytochrome c
3
. Because the rubredoxin is
a very acidic protein and binds to the cytochrome
close to haem IV, it has the electrostatic characteristics
that mimic the physiological partners such as the

Fe-hydrogenase and the membrane associated multi-
haem cytochromes [6,22,31,32].
This work reports the first determination of the equi-
librium thermodynamic properties of a cytochrome c
3
when bound to phosphate and to an engineered form
of rubredoxin where the iron was replaced by zinc.
Results
Figure 1 shows the comparison of representative two-
dimensional exchange spectroscopy (EXSY) NMR
spectra of Desulfovibrio vulgaris cytochrome c
3
with
phosphate (DvHc
3
:Pi) and Desulfovibrio vulgaris cyto-
chrome c
3
with zinc rubredoxin (DvHc
3
:ZnRb). It is
Fig. 1. Two-dimensional EXSY NMR spectra
of DvHc
3
:Pi (above the diagonal) and
DvHc
3
:ZnRb (below the diagonal) at pH 7.6
showing the pattern of reoxidation in both
cases. The spectrum for DvHc

3
:Pi is slightly
more oxidized and therefore does not have
signals for stage 1. The lines connect sig-
nals of one particular methyl group (2
1
CH
I
3
,
18
1
CH
II
3
,12
1
CH
III
3
or 18
1
CH
IV
3
) in different
oxidation stages for DvHc
3
:Pi (solid lines)
and DvHc

3
:ZnRb (dashed lines). Some
signals are not easily visible at the level of
cut-off used to prepare the figure and were
boxed for clarity. Roman and Arabic num-
bers indicate the haem groups and the
oxidation stages, respectively.
Thermodynamic parameters in ligated proteins C. A. Salgueiro et al.
2252 FEBS Journal 272 (2005) 2251–2260 ª 2005 FEBS
apparent that the spectra are very similar with respect to
chemical shifts of the signals in intermediate stages of
oxidation, and that formation of the complex does not
lead to a marked decrease of the spectral quality in the
experimental conditions used, where most of the cyto-
chrome is bound to the Zn-rubredoxin (Discussion).
The pH dependence of the paramagnetic chemical
shifts of each haem methyl group and the data obtained
for redox titrations followed by visible spectroscopy
at pH 7.0 and 8.1, were used to monitor the thermo-
dynamic properties of DvHc
3
:Pi. The fittings of both
NMR and visible spectroscopy data are reported in
Figs 2 and 3, respectively. The thermodynamic parame-
ters obtained for DvHc
3
:Pi are listed in Table 1, together
with the macroscopic pK
a
values for the five stages of

oxidation.
The pH dependence of the chemical shifts of the
haem methyl groups 2
1
CH
I
3
,18
1
CH
II
3
,12
1
CH
III
3
and
18
1
CH
IV
3
both Desulfovibrio vulgaris cytochrome c
3
(DvHc
3
) and DvHc
3
:Pi are reported in Fig. 2 by

dashed and solid lines, respectively. Figure 2 shows
that the major differences in chemical shifts of the sig-
nals relative to the data obtained in the absence of
phosphate occur for the intermediate oxidation stages
of haems III and IV. However, these differences are
small and give rise to only a small modification on the
calculated thermodynamic properties of DvHc
3
:Pi as
indicated in Table 1 with all differences < 12 meV.
Also, Fig. 2 and Table 1 both show that the acid–base
centre and the redox–Bohr interactions are almost
undisturbed by the presence of phosphate and the
resulting macroscopic pK
a
values are within 0.2 units
Fig. 2. The pH dependence of the chemical shift of haem methyl resonances 2
1
CH
I
3
,18
1
CH
II
3
,12
1
CH
III

3
and 18
1
CH
IV
3
,ofDvHc
3
:Pi at
297.3 K. Squares correspond to stage 1 of oxidation, circles to stage 2, downward pointing triangles to stage 3, and upward pointing trian-
gles to stage 4. The chemical shifts of the haem methyl groups in the fully reduced stage 0 are not plotted because they are unaffected by
the pH. The solid lines represent the best fit of the shifts for DvHc
3
:Pi to the model of five interacting centres using the parameters listed in
Table 1. Dashed lines represent the best fit for the DvHc
3
and the nearest label (1–4) indicates the oxidation stage represented by the line.
Fig. 3. Reduced fraction of DvHc
3
in the presence of 100 mM phos-
phate determined from redox titrations followed by visible spectros-
copy performed at pH 7.0 and 8.1. Continuous lines are the fit of
the model to the data.
C. A. Salgueiro et al. Thermodynamic parameters in ligated proteins
FEBS Journal 272 (2005) 2251–2260 ª 2005 FEBS 2253
of those measured for the isolated cytochrome. As pre-
viously reported for cytochromes c
3
from Desulfovibrio
gigas, Desulfomicrobium norvegicum and Desulfomicro-

bium baculatum [23], the presence of phosphate induced
a generalized narrowing of the line widths of the DvHc
3
haem methyl signals at intermediate redox stages
when compared with the experiments performed in the
absence of phosphate (data not shown). These obser-
vations show that the intermolecular electron exchange
is slower, which allowed the data from DvHc
3
:Pi to
be collected in a NMR spectrometer operating at
300 MHz, and establishing that the intermolecular elec-
tron exchange is < 340 s
)1
at 1 mm and 297.3 K.
The paramagnetic chemical shifts of each haem
methyl group (2
1
CH
I
3
,18
1
CH
II
3
,12
1
CH
III

3
and
18
1
CH
IV
3
), of DvHc
3
:ZnRb are plotted in Fig. 4 and
the relative thermodynamic parameters together with
the macroscopic pK
a
values for the five stages of oxi-
dation determined from the fitting are listed in
Table 2. Absolute potentials and interactions are not
reported for these experiments because it is not poss-
ible to perform redox titrations followed by visible
spectroscopy under conditions that ensure a similar
proportion of bound vs. unbound state of the cyto-
chrome to those obtained in the NMR tube. This is a
consequence of the very strong absorption bands of
the cytochrome requiring very dilute solutions to per-
form visible absorption measurements, and the possi-
bility of interference from the redox mediators on
complex formation. Therefore, haem I and the inter-
action between haems I and IV were chosen as refer-
ences because these are the most distant pair of haems
in the structure and are therefore expected to have the
weakest interaction [33].

The pH dependence of the chemical shifts of the
NMR signals of the haem methyls obtained for
DvHc
3
:ZnRb and DvHc
3
is also reported in Fig. 4 and
indicated by continuous and dashed lines, respectively.
The figure shows that the effect of binding Zn-rubre-
doxin on the NMR signals of the haem methyls vs. the
results obtained for the isolated cytochrome is very
small. As observed for the case of phosphate binding,
the signals for intermediate redox stages 2 and 3 of
haems III and IV are the more affected. This suggests
that the binding of phosphate and Zn-rubredoxin occur
in a similar location on the surface of the cytochrome,
which is in agreement with the fact that both are neg-
atively charged molecules despite the dramatic differ-
ence in size, and in agreement with previous
comparative work of binding inorganic and protein
partners to cytochromes c
3
[6]. Table 2 shows that as
for the case of phosphate binding, the association with
Table 1. Thermodynamic parameters determined for DvHc
3
[13] and DvHc
3
:Pi. (Top) Diagonal terms (in bold) represent the oxidation ener-
gies of the four haems and the energies for deprotonating the acid–base centre for the fully reduced and protonated state of the protein and

have standard errors < 5 meV. The off-diagonal elements represent the redox- and redox–Bohr interactions between the centres. All para-
meters are reported in units of meV, making them numerically equal to the values of redox potentials and interactions reported in units of
mV [DG (meV) ¼ nE (mV)]. (Bottom) Macroscopic pK
a
values for the five stages of oxidation, from the fully reduced protein (stage 0) to the
fully oxidized (stage 4) measured from the data.
Haem I Haem II Haem III Haem IV Ionizable centre
DvHc
3
Haem I ) 245 ) 43 20 ) 4 ) 70
Haem II – 267 ) 88) 30
Haem III ) 334 32 ) 18
Haem IV – 284 –6
Ionizable centre 439
DvHc
3
:Pi
Haem I – 247 ) 38 26 6 ) 67
Haem II – 275 316) 25
Haem III – 335 35 ) 15
Haem IV – 293 –6
Ionizable centre 428
Macroscopic pK
a
values
Oxidation stage
01234
DvHc
3
7.4 7.1 6.4 5.6 5.3

DvHc
3
:Pi 7.2 6.9 6.3 5.6 5.3
Thermodynamic parameters in ligated proteins C. A. Salgueiro et al.
2254 FEBS Journal 272 (2005) 2251–2260 ª 2005 FEBS
Table 2. Thermodynamic parameters determined for DvHc
3
[13] and DvHc
3
:ZnRb. (Top) Relative thermodynamic parameters. (Bottom) Mac-
roscopic pK
a
values for the five stages of oxidation, from the fully reduced protein (stage 0) to the fully oxidized (stage 4). The table was pre-
pared as Table 1 using the energy of oxidation of haem I and the interaction between haems I and IV as reference values.
Haem I Haem II Haem III Haem IV Ionizable centre
DvHc
3
Haem I 0 ) 47 24 0 ) 70
Haem II ) 22 ) 412) 30
Haem III ) 89 36 ) 18
Haem IV ) 39 ) 6
Ionizable centre 439
DvHc
3
:ZnRb
Haem I 0 ) 34 18 0 ) 69
Haem II ) 30 27) 29
Haem III ) 93 37 ) 13
Haem IV ) 48 2
Ionizable centre 422

Macroscopic pK
a
values
Oxidation stage
01234
DvHc
3
7.4 7.1 6.4 5.6 5.3
DvHc
3
:ZnRb 7.2 6.9 6.2 5.5 5.3
Fig. 4. The pH dependence of the chemical shift of haem methyl resonances 2
1
CH
I
3
,18
1
CH
II
3
,12
1
CH
III
3
and 18
1
CH
IV

3
,ofDvHc
3
:ZnRb at
297.3 K. Squares correspond to stage 1 of oxidation, circles to stage 2, downward pointing triangles to stage 3, and upward pointing trian-
gles to stage 4. The chemical shifts of the haem methyl groups in the fully reduced stage 0 are not plotted since they are unaffected by the
pH. The solid lines represent the best fit of the shifts for DvHc
3
:ZnRb to the model of five interacting centres using the parameters listed in
Table 2. Dashed lines represent the best fit for the DvHc
3
and the nearest label (1–4) indicates the oxidation stage represented by the line.
C. A. Salgueiro et al. Thermodynamic parameters in ligated proteins
FEBS Journal 272 (2005) 2251–2260 ª 2005 FEBS 2255
Zn-rubredoxin gives rise to a small perturbation of the
relative reduction potentials and redox interactions
among the various centres. Furthermore, because the
pH of the samples could be measured inside the NMR
tube the values for the redox–Bohr interactions and
the macroscopic pK
a
values are absolute, and therefore
the macroscopic pK
a
values of the various redox stages
show only very small modifications relative to the data
obtained for the isolated cytochrome [13]. This obser-
vation is in agreement with the experimentally
observed binding of rubredoxin close to haem IV
because the acid–base centre has been assigned to

propionate D of haem I [26] which is on the opposite
pole of the cytochrome and therefore should be only
weakly affected by the docking.
Discussion
Our results demonstrate that at 100 mm phosphate
binds to DvHc
3
causing narrower NMR signals and
perturbing the chemical shifts of the haem methyl sig-
nals in intermediate redox stages. The contraction of
the line widths of the NMR signals in intermediate
redox stages shows that the intermolecular electron
exchange is slower than in the absence of phosphate.
Given that DvHc
3
is a very basic protein, with its iso-
electric point above 10, this result is contrary to the
expected increase in intermolecular electron exchange
rate for proteins of equal charge as the ionic strength
is increased, and indicates a specific binding of phos-
phate to the cytochrome [34,35]. Moreover, an increase
in reduction potentials of the centres with ionic
strength would be expected on electrostatic grounds
for a negatively charged protein [36]. This is not
observed in the current case and was also not observed
for some haems in the highly homologous cyto-
chrome c
3
from D. vulgaris (Miyazaki) in the presence
of increased phosphate concentration [37]. The results

presented in Table 1 show that the interactions among
the centres in the protein are subject to small modifica-
tions in the presence of phosphate, in agreement with
arguments in the literature that for nonsurface resi-
dues, such as the haems, the presence of counter ions
should have a small effect on pair-wise charge–charge
interactions between redox centres in a protein [33].
To measure the detailed equilibrium thermodynamic
parameters of the redox and redox–Bohr interactions
of the haems of cytochrome c
3
when forming a com-
plex with a partner protein, several experimental
requirements had to be met in addition to maintaining
slow intermolecular- and fast intramolecular-electron
transfer among the cytochrome c
3
molecules: (a) The
complex had to be sufficiently small so that line broad-
ening from a slower tumbling complex would not pre-
vent observation of the signals at the various redox
stages; (b) Bound and unbound states had to be in fast
exchange in the NMR time scale so that a single signal
is observed at a position that is weighted by the relat-
ive proportions of these states; (c) The partner should
not contain a paramagnetic centre to avoid excessive
broadening of the lines, as observed for the complex
between the native iron rubredoxin and cytochrome c
3
[25]; (d) The redox state of the partner should not

change under the various experimental conditions
probed to avoid distorting the results of the param-
eters measured for the cytochrome with varying elec-
trostatic interactions caused by diferent redox states of
the partner.
The use of Zn-rubredoxin as docking partner ful-
filled all these criteria: (i) the complex has a combined
mass of  20 kDa; (ii) the number of signals observed
in the intermediate stages of oxidation shows that the
exchange between the bound and unbound form is fast;
(iii) Zn(II) is diamagnetic; and (iv) Zn(II) has a d10
electronic configuration and therefore does not present
redox chemistry in the range explored in this work.
The binding constant of rubredoxin to DvHc
3
is
>10
4
m
)1
[25], which is assumed to be essentially
undisturbed by the replacement of Fe by Zn in the
rubredoxin given the identical structures of the two
protein forms [38,39]. Therefore, at the concentrations
used in the NMR experiments over 90% of the
cytochrome is bound to the rubredoxin and the effect
observed in the signals of the haem methyls of
DvHc
3
:ZnRb complex vs. the results in the isolated

cytochrome is close to the full effect of complex forma-
tion. Because the thermodynamic parameters for the
haems calculated from the NMR data are relative, it
could be argued that all haem potentials had been
modified to a similar degree but to an unknown extent
making their absolute values completely different from
those measured for the isolated cytochrome. However,
this scenario is unlikely because the rubredoxin is a
smaller protein than the cytochrome, it binds to a spe-
cific location close to haem IV, and the phenomena
giving rise to such a widespread modification of the
redox properties should also affect the acid–base centre
for which absolute thermodynamic parameters were
measured and that shows very small modifications
caused by the binding of Zn-rubredoxin.
Overall, the two sets of results reported show that
phosphate binding and docking with the Zn-rubredox-
in has a very limited effect on the redox properties
of the haem groups in the cytochrome. In fact, the
haem reduction potentials, redox interactions, redox–
Bohr interactions and macroscopic pK
a
values remain
Thermodynamic parameters in ligated proteins C. A. Salgueiro et al.
2256 FEBS Journal 272 (2005) 2251–2260 ª 2005 FEBS
essentially unaffected (Tables 1 and 2). These results
are in agreement with theoretical expectations that sur-
face charges interact with redox centres with a very high
effective dielectric constant and therefore their contri-
bution to the reduction potential of the centres is small

[40,41], and also with the experimental observation for
monohaem cytochromes that the effect of anion binding
or complex formation on the reduction potential is
small [7,42]. However, this is the first time that the net-
work of pairwise interactions in a multicentre protein
has been explored when forming a complex, and these
interactions also show small modifications relative to
the isolated protein, indicating that the intramolecular
dielectric environment is essentially undisturbed by lig-
and binding or complex formation.
Conclusions
The presence of small negatively charged ligands such
as phosphate, causes little perturbation on the equilib-
rium thermodynamic properties of the haems in
DvHc
3
, despite the evidence that electrostatic forces
are the main drive for complex formation, and the
haems are very exposed to the solvent. Also, mimics of
the physiological partners such as the Zn-rubredoxin
cause only small modifications in the relative reduction
potentials and redox interactions among the haems in
DvHc
3
. Our results show that the equilibrium thermo-
dynamic data obtained for the isolated cytochrome c
3
are similar to those measured when the protein is
bound to a small anion or to a mimic of physiological
partners and may be discussed within the framework

of their functional relevance for the role of cyto-
chrome c
3
in maintaining efficiency in the bioenergetic
metabolism of Desulfovibrio bacteria.
Experimental procedures
Bacterial growth and protein purification
Zn-rubredoxin
Cells of Escherichia coli BL21(DE; Universidade Nova de
Lisboa, Portugal) were transformed with the plasmid
pMSPL1 [43] to produce the rubredoxin from Desulfovibrio
gigas. Twenty millilitres of an overnight culture were ino-
culated in 1 L of the medium described in the literature [43]
that was allowed to grow up to an absorbance of 0.5. The
cells were induced with isopropyl thio-b-d-galactoside
(25 mgÆL
)1
) and supplemented with 4 mLÆL
)1
of glycerol
87% and ZnCl
2
(5 mgÆL
)1
; final concentration) to increase
the amount of the zinc form of rubredoxin present in the cell
cultures. After 6–10 h the cells were centrifuged and collected
in 50 mm Tris with 1 mm phenylmethanesulfonyl fluoride
and the Zn-rubredoxin purified as described in [44]. Purity
was checked by SDS ⁄ PAGE and UV-visible spectroscopy.

Cytochrome c
3
Cells of D. vulgaris (Hildenborough; Universidade Nova de
Lisboa) were grown and the tetrahaem cytochrome c
3
was
purified as previously described [13].
Redox titrations followed by visible spectroscopy
Anaerobic redox titrations followed by visible spectroscopy
were performed as described previously [45] with  2 lm pro-
tein solutions in 100 mm phosphate buffer at pH 7.0 and 8.1.
For each pH value the redox titrations were repeated at least
twice, both in the oxidative and reductive directions to check
for hysteresis. Reproducibility between the runs was typically
better than 5 mV. To ensure a good equilibrium between the
redox centres and the working electrode [46], a mixture of the
following redox mediators was added to the protein solution
at pH 7.0: indigo tetrasulfonate, indigo trisulfonate, indigo
disulfonate, anthraquinone-2-7-disulfonate, anthraquinone-
2-sulfonate, safranine O, diquat, neutral red, phenosafranine,
and methylviologen, all at a ratio of 100 : 8 of cytochrome
vs. mediator to avoid interference caused by specific binding
of mediators to the protein. For the redox titrations per-
formed at pH 8.1 the mediators gallocyanine and methylene
blue were added to the previous mixture.
The solution potential was measured using a combined
Pt|Ag ⁄ AgCl electrode (Crison, Barcelona, Spain), calibra-
ted against saturated quinhydrone ⁄ hydroquinone solutions
at pH 4 and 7, and the visible spectra were recorded at
297 ± 1 K in a Shimadzu UV-1203 spectrophotometer,

placed inside an anaerobic glove box (Mbraun MB 150 I).
The reduced fraction of the cytochrome c
3
from
D. vulgaris (Hildenborough) (DvHc
3
) was determined using
the a band peak at 552 nm. The optical contribution of the
mediators was subtracted by measuring the height of the
peak at 552 nm relative to the straight line connecting the two
isosbestic points (542 and 560 nm) flanking the a band
according to the method described in the literature [45].
NMR sample preparation
DvHc
3
in the presence of 100 mm phosphate
The protein was lyophilized twice with
2
H
2
O (99.96%
atom) and then dissolved in  500 lL
2
H
2
O (99.96% atom)
100 mm K
3
PO
4

.7H
2
O solution to a final concentration of
1mm (this sample will be referred hereafter as DvHc
3
:Pi).
Identical NMR spectra of the DvHc
3
(data not shown) were
obtained before and after the lyophilization, showing that
the protein structure was not affected.
The pH of the samples was adjusted using small volumes of
NaO
2
Hor
2
HCl solutions. In the reduced and intermediate
C. A. Salgueiro et al. Thermodynamic parameters in ligated proteins
FEBS Journal 272 (2005) 2251–2260 ª 2005 FEBS 2257
stages of oxidation the pH was adjusted inside the an-
aerobic glove box with argon circulation to avoid the
reoxidation of the sample. The pH values reported are
direct meter readings without correction for the isotope
effect [47,48]. Complete reduction of the sample was
achieved by the reaction with gaseous hydrogen in the
presence of catalytic amounts of the enzyme Fe-hydroge-
nase isolated from D. vulgaris (Hildenborough). Partially
oxidized samples were obtained by first flushing out the
hydrogen from the reduced sample with argon and then
adding controlled amounts of air in the microlitre range

into the NMR tube with a syringe through serum caps.
DvHc
3
with Zn-rubredoxin
The cytochrome and the Zn-rubredoxin were lyophilized
separately and a sample was prepared in  500 lL
2
H
2
O
(99.96% atom) at a final concentration of 0.5 mm cyto-
chrome and 0.8 mm Zn-rubredoxin (this sample will be
referred hereafter as DvHc
3
:ZnRb). The sample was mani-
pulated as described above for the sample containing
100 mm phosphate.
NMR spectroscopy of partially oxidized samples
1
H NMR spectra were obtained either in a Bruker
AMX300 for DvHc
3
:Pi or in a Bruker DRX500 Avance
spectrometer for DvHc
3
:ZnRb equipped with 5 mm inverse
detection probe heads.
To establish the complete pattern of oxidation for each
haem methyl group at each pH, several two-dimensional
EXSY NMR experiments, with 25 ms mixing time, were

collected at various degrees of oxidation. The spectra were
recorded at 297.3 K in the pH range 5.0–8.2, measuring 4 k
(t
2
) · 1k (t
1
) data points, and water presaturation was
achieved by selective, low-power pulses of 500–800 ms.
Chemical shifts values are reported in p.p.m. relative to
trimethylsilyl and the spectra were calibrated using the
residual water signal as internal reference [49].
Modelling the thermodynamic parameters
The model used for the thermodynamic characterization of
DvHc
3
[13] was applied to the data obtained for DvHc
3
:Pi
and Dv Hc
3
:ZnRb. This model considers five interacting cen-
tres: four haems and one acid–base centre. As shown in the
Results, DvHc
3
exhibits fast intramolecular and slow inter-
molecular electron exchange on the NMR time scale, in the
presence of both Zn-rubredoxin and 100 mm phosphate.
Therefore, each haem substituent displays five discrete NMR
signals corresponding to each of the five possible macro-
scopic oxidation stages of the cytochrome, connected by four

steps of one-electron uptake or release. The unpaired electron
in the oxidized haem causes a paramagnetic shift on its
signals that is directly proportional to the fractional oxida-
tion in the absence of extrinsic paramagnetic contributions.
As shown previously [26], the haem methyl groups 2
1
CH
I
3
,
18
1
CH
II
3
,12
1
CH
III
3
and 18
1
CH
IV
3
have large paramagnetic
shifts and negligible extrinsic dipolar contributions, hence
they are suitable for monitoring the thermodynamic proper-
ties of the cytochrome. The paramagnetic chemical shift of
the haem methyl resonances is a very sensitive probe of the

haem environment. The fact that the shifts in the fully oxi-
dized state of the protein are essentially identical to those
measured for the isolated protein indicates that the binding
of phosphate or the Zn-rubredoxin does not disturb the
structure of the haem core. Therefore, the same methyl
groups were followed here.
The NMR data provide only relative values for the
reduction potentials and interactions [13,50,51] and deter-
mination of absolute values require the use of data from
redox titrations followed by visible spectroscopy. A compu-
ter program was written to fit the thermodynamic model to
the NMR data (for the case of DvHc
3
:ZnRb), or to the
NMR and UV-visible data sets simultaneously (for the case
of DvHc
3
:Pi) using the Marquardt method for parameter
optimization. The half-height widths of the NMR signals
were used as a measure of the uncertainty of each NMR
data point and an experimental uncertainty of 2% was
assumed for the experimental points of the redox titrations.
Acknowledgements
The authors are grateful to Professor A.V. Xavier, for
many fruitful suggestions and discussions, and to
Professor Helena Santos for kindly providing the
Zn-rubredoxin. The assistance of Isabel Pacheco dur-
ing protein purification is gratefully acknowledged.
Financial support was provided by FCT-POCTI, Co-
financed by FEDER (POCTI ⁄ 42902 ⁄ QUI ⁄ 2001 to

CAS, POCTI ⁄ 43435 ⁄ QUI ⁄ 2001 to TC, and BPD ⁄
11511 ⁄ 2002 to PL).
References
1 Russell RB, Alber F, Aloy P, Davies FP, Korkin D,
Pichaud M, Topf M & Sali A (2004) A structural per-
spective on protein–protein interactions. Curr Opin
Struct Biol 14, 313–324.
2 Prudeˆ ncio M & Ubbink M (2004) Transient complexes
of redox proteins: structural and dynamic details from
NMR studies. J Mol Recogn 17, 524–539.
3 Hansen DF, Hass MAS, Christensen HM, Ulstrup J &
Led JJ (2003) Detection of short-lived transient protein–
protein interactions by intermolecular nuclear paramag-
netic relaxation: plastocyanin from Anabaena variabilis.
J Am Chem Soc 125, 6858–6859.
Thermodynamic parameters in ligated proteins C. A. Salgueiro et al.
2258 FEBS Journal 272 (2005) 2251–2260 ª 2005 FEBS
4 Marcus RA & Sutin N (1985) Electron transfers in
chemistry and biology. Biochim Biophys Acta 811, 265–
322.
5 Drepper F, Hippler M, Nitschke W & Haehnel W
(1996) Binding dynamics and electron transfer between
plastocyanin and photosystem I. Biochemistry 35, 1282–
1295.
6 Mus-Veteau I, Chottard G, Lexa D, Guerlesquin F &
Bruschi M (1992) Cytochrome c
3
–heteropolytungstate
complex: a model for the interaction of the tetraheme
cytochrome with its redox partners, ferredoxin and

rubredoxin. Biochim Biophys Acta 1102, 353–359.
7 Burrows AL, Guo LH, Hill AO, McLendon G & Sher-
man F (1991) Direct electrochemistry of proteins: inves-
tigations of yeast cytochrome c mutants and their
complex with cytochrome b
5
. Eur J Biochem 202, 543–
549.
8 Louro RO, Catarino T, LeGall J & Xavier AV (1997)
Redox–Bohr effect in electron ⁄ proton energy transduc-
tion: cytochrome c
3
coupled to hydrogenase works as a
‘proton thruster’ in Desulfovibrio vulgaris. J Biol Inorg
Chem 2, 488–491.
9 Louro RO (1998) Proton-thrusters: Energy transduction
performed by tetrahaem cytochromes c
3
and its physiolo-
gical relevance. PhD Thesis, Universidade Nova de
Lisboa, Portugal.
10 Pereira IAC, Roma
˜
o CV, Xavier AV, LeGall J & Teix-
eira M (1998) Electron transfer between hydrogenases
and mono- and multiheme cytochromes in Desulfovibrio
sp. J Biol Inorg Chem 3, 494–498.
11 Valente FM, Saraiva LM, LeGall J, Xavier AV, Teixe-
ira M & Pereira IA (2001) A membrane-bound
cytochrome c

3
: a type II cytochrome c
3
from Desulfovi-
brio vulgaris Hildenborough. Chembiochem 2, 895–905.
12 Araga
˜
o D, Fraza
˜
o C, Sieker L, Sheldrick GM, LeGall J
& Carrondo MA (2003) Structure of dimeric cyto-
chrome c
3
from Desulfovibrio gigas at 1.2 A
˚
resolution.
Acta Crystallogr D Biol Crystallogr 59, 644–653.
13 Turner DL, Salgueiro CA, Catarino T, LeGall J &
Xavier AV (1996) NMR studies of cooperativity in the
tetrahaem cytochrome c
3
from Desulfovibrio vulgaris .
Eur J Biochem 241, 723–731.
14 Park JS, Ohmura T, Kano K, Sagara T, Niki K,
Kyogoku Y & Akutsu H (1996) Regulation of the redox
order of four hemes by pH in cytochrome c
3
from D.
vulgaris Miyazaki F. Biochim Biophys Acta 1293, 45–54.
15 Salgueiro CA, Turner DL, LeGall J & Xavier AV

(1997) Reevaluation of the redox and redox–Bohr coop-
erativity in tetrahaem Desulfovibrio vulgaris (Miyazaki
F) cytochrome c
3
. J Biol Inorg Chem 2, 343–349.
16 Louro RO, Bento I, Matias PM, Catarino T, Baptista
AM, Soares CM, Carrondo MA, Turner DL & Xavier
AV (2001) Conformational component in the coupled
transfer of multiple electrons and protons in a monomeric
tetraheme cytochrome. J Biol Chem 276, 44044–44051.
17 Correia IJ, Paquete CM, Coelho A, Almeida CC,
Catarino T, Louro RO, Fraza
˜
o C, Saraiva LM,
Carrondo MA, Turner DL et al. (2004) Proton-assisted
two-electron transfer in natural variants of tetraheme
cytochromes from Desulfomicrobium sp. J Biol Chem
279, 52227–52237.
18 Catarino T & Turner DL (2001) Thermodynamic con-
trol of electron transfer rates in multicenter redox pro-
teins. Chembiochem 2, 416–424.
19 Correia IJ, Paquete CM, Louro RO, Catarino T,
Turner DL & Xavier AV (2002) Thermodynamic and
kinetic characterization of trihaem cytochrome c
3
from
Desulfuromonas acetoxidans. Eur J Biochem 269, 5722–
5730.
20 Yagi T, Honya M & Tamiya N (1968) Purification and
properties of hydrogenases of different origins. Biochim

Biophys Acta 153, 699–705.
21 Aubert C, Brugna M, Dolla A, Bruschi M & Giudici-
Orticoni MT (2000) A sequential electron transfer from
hydrogenases to cytochromes in sulfate-reducing bac-
teria. Biochim Biophys Acta 1476, 85–92.
22 Teixeira VH, Baptista AM & Soares CM (2004) Model-
ing electron transfer thermodynamics in protein com-
plexes: interaction between two cytochromes c
3
. Biophys
J 86, 2773–2785.
23 Moura JJG, Xavier AV, Cookson DJ, Moore GR &
Williams RJ (1977) Redox states of cytochrome c
3
in
the absence and presence of ferredoxin. FEBS Lett 81,
275–280.
24 Assfalg M, Bertini I, Bruschi M, Michel C & Turano P
(2002) The metal reductase activity of some multiheme
cytochromes c: NMR structural characterization of the
reduction of chromium (VI) to chromium (III) by
cytochrome c (7). Proc Natl Acad Sci 99, 9750–9754.
25 Stewart DE, LeGall J, Moura I, Moura JJG, Peck HD
Jr, Xavier AV, Weiner PK & Wampler JE (1989)
Electron transport in sulfate-reducing bacteria. Mole-
cular modeling and NMR studies of the rubredoxin–
tetraheme–cytochrome c
3
complex. Eur J Biochem 185,
695–700.

26 Salgueiro CA, Turner DL & Xavier AV (1997) Use of
paramagnetic NMR probes for structural analysis in
cytochrome c
3
from Desulfovibrio vulgaris . Eur J
Biochem 244, 721–734.
27 Moss GP (1988) IUPAC–IUB joint commission on
biochemical nomenclature. Nomenclature of tetrapyr-
roles. Tentative rules (1969). Eur J Biochem 178, 277–328.
28 Stewart DE, LeGall J, Moura I, Moura JJG, Peck HD,
Xavier AV, Weiner PK & Wampler JE (1988) A
hypothetical model of the flavodoxin–tetraheme cyto-
chrome c
3
complex of sulfate-reducing bacteria.
Biochemistry 27, 2444–2450.
29 Morais J, Palma PN, Fraza
˜
o C, Caldeira J, LeGall J,
Moura I, Moura JJ & Carrondo M-A (1995) Structure
of the tetraheme cytochrome from Desulfovibrio desul-
C. A. Salgueiro et al. Thermodynamic parameters in ligated proteins
FEBS Journal 272 (2005) 2251–2260 ª 2005 FEBS 2259
furicans ATCC 27774: X-ray diffraction and electron
paramagnetic resonance studies. Biochemistry 34,
12830–12841.
30 El Antak L, Morelli X, Bornet O, Hatchikian C, Czjzek
M, Dolla A & Guerlesquin F (2003) The cytochrome
c
3

– [Fe]-hydrogenase electron-transfer complex: struc-
tural model by NMR restrained docking. FEBS Lett
548, 1–4.
31 Matias PM, Saraiva LM, Soares CM, Coelho AV,
LeGall J & Carrondo MA (1999) Nine-haem cyto-
chrome c from Desulfovibrio desulfuricans ATCC 27774:
primary sequence determination, crystallographic
refinement at 1.8 and modelling studies of its interaction
with the tetrahaem cytochrome c
3
. J Biol Inorg Chem 4,
478–494.
32 Matias PM, Coelho AV, Valente FM, Placido D, Le-
Gall J, Xavier AV, Pereira IA & Carrondo MA (2002)
Sulfate respiration in Desulfovibrio vulgaris Hildenbor-
ough. structure of the 16-heme cytochrome c HmcA AT
2.5-A resolution and a view of its role in transmem-
brane electron transfer. J Biol Chem 277, 47907–47916.
33 Louro RO, Catarino T, Paquete CM & Turner DL
(2004) Distance dependence of interactions between
charged centres in proteins with common structural fea-
tures. FEBS Lett 576, 77–80.
34 Santos H (1984) Relac¸a
˜
o estrutura-func¸a
˜
o em citocromos
multihe
´
micos: caracterizac¸a

˜
o por RMN dos mecanismos
de transfere
ˆncia
electro
´
nica em citocromos c
3
de bacte
´
rias
reductoras de sulfato. PhD Thesis, Universidade Nova
Lisboa, Portugal.
35 Cudd A & Fridovich I (1982) Electrostatic interactions
in the reaction mechanism of bovine erythrocyte super-
oxide dismutase. J Biol Chem 257, 11443–11447.
36 Reid LS, Mauk MR & Mauk AG (1984) Role of heme
propionate groups in cytochrome b
5
electron transfer.
J Am Chem Soc 106, 2182–2185.
37 Ohmura T, Nakamura H, Niki K, Cusanovich MA &
Akutsu H (1998) Ionic strength-dependent physicochem-
ical factors in cytochrome c
3
regulating the electron
transfer rate. Biophys J 5, 1483–1490.
38 Dauter Z, Wilson KS, Sieker LC, Moulis JM & Meyer
J (1996) Zinc- and iron-rubredoxins from Clostridium
pasteurianum at atomic resolution: a high-precision

model of a ZnS
4
coordination unit in a protein. Proc
Natl Acad Sci 93, 8836–8840.
39 Lamosa P, Brennan L, Vis H, Turner D & Santos H
(2001) NMR Structure of Desulfovibrio gigas rubredoxin
– A model for studying protein stabilisation by compat-
ible solutes. Extremophiles 5, 301–311.
40 Cutler RL, Davies AM, Creighton S, Warshel A, Moore
GR, Smith M & Mauk AG (1989) Role of arginine-38
in regulation of the cytochrome c oxidation–reduction
equilibrium. Biochemistry 28, 3188–3196.
41 Zhou H-X (1994) Effects of mutations and complex for-
mation on the reduction potentials of cytochrome c and
cytochrome c peroxidase. J Am Chem Soc 116, 10362–
10375.
42 Battistuzzi G, Borsari M, Dallari D, Lancellotti I &
Sola M (1996) Anion binding to mitochondrial
cytochromes c studied through electrochemistry.
Effects of the neutralization of surface charges on the
redox potential. Eur J Biochem 241, 208–214.
43 Lamosa P, Turner D & Santos H (2003) Protein
stabilization by compatible solutes: effect of diglycerol
phosphate in the backbone dynamics of
Desulfovibrio gigas rubredoxin. Eur J Biochem 270,
4606–4614.
44 Lamosa P, Burke A, Peist R, Huber R, Liu M-Y, Silva
G, Rodrigues-Pousada C, LeGall J, Maycock C & San-
tos H (2000) Thermostabilisation of proteins by digly-
cerol phosphate. A new compatible solute from the

hyperthermophile Archaeoglobus fulgidus. Appl Environ
Microbiol 66, 1974–1979.
45 Louro RO, Catarino T, LeGall J & Xavier AV (2001)
Cooperativity between electrons and protons in a mono-
meric cytochrome c
3
: the importance of mechano-chemi-
cal coupling for energy transduction. Chembiochem 2,
831–837.
46 Dutton PL (1978) Redox potentiometry: determination
of midpoint potentials of oxidation–reduction compo-
nents of biological electron-transfer systems. Methods
Emzymol 54, 411–435.
47 Glasoe PK & Long FA (1960) Use of glass electrodes
to measure acidities in deuterium oxide. J Phys Chem
64, 188–191.
48 Delgado R, Frausto da Silva JJR, Amorim MTS,
Cabral MF, Chaves S & Costa J (1991) Dissociation
constants of Bronsted acids in D
2
O and H
2
O: studies
on polyaza and polyoxa–polyaza macrocycles and a
general correlation. Anal Chim Acta 245, 271–282.
49 Pierattelli R, Banci L & Turner DL (1996) Indirect
determination of magnetic susceptibility tensors in per-
oxidases: a novel approach to structure elucidation by
NMR. J Biol Inorg Chem 1, 320–329.
50 Coletta M, Catarino T, LeGall J & Xavier AV (1991) A

thermodynamic model for the cooperative functional
properties of the tetraheme cytochrome c
3
from Desulfo-
vibrio gigas. Eur J Biochem 202, 1101–1106.
51 Santos H, Moura JJ, Moura I, LeGall J & Xavier AV
(1984) NMR studies of electron transfer mechanisms in
a protein with interacting redox centres: Desulfovibrio
gigas cytochrome c
3
. Eur J Biochem 141, 283–296.
Thermodynamic parameters in ligated proteins C. A. Salgueiro et al.
2260 FEBS Journal 272 (2005) 2251–2260 ª 2005 FEBS