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
ˆncias 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: louro@itqb.unl.pt
(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 ª2005 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 mconcen-
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
3or 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 ª2005 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
3and
18
1
CH IV
3both 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
3and 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 mMphos-
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 ª2005 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 mmand 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
3and
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) ¼nE (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 ª2005 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
3and 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 ª2005 FEBS 2255
