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 inte
Trang 1microscopic thermodynamic properties of a multicentre redox protein
Carlos A Salgueiro1,2, Leonor Morgado1,2, Bruno Fonseca1,2, Pedro Lamosa1, Teresa Catarino1,2, David L Turner3and Ricardo O Louro1
1 Instituto de Tecnologia Quimica e Biolo´gica, Universidade Nova de Lisboa, Portugal
2 Departamento de Quimica da Faculdade de Cieˆncias 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 c3 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 c3 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 propprop-erties [17–19] These studies have shown that cytochromes c3 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 c3; 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 c3 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 c3, the small changes observed for the thermodynamic properties of cytochrome c3 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
DvHc3, Desulfovibrio vulgaris (Hildenborough) cytochrome c3; DvHc3:Pi, Desulfovibrio vulgaris cytochrome c3with phosphate; DvHc3:ZnRb, Desulfovibrio vulgaris cytochrome c 3 with zinc rubredoxin; EXSY, exchange spectroscopy.
Trang 2However, no experimental data exist on the effect of
binding small ligands or proteins on these properties
For cytochromes c3these effects have only been
inves-tigated in a theoretical study where cytochrome c3 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 c3 [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 c7 [24]
Also, the analysis of one-dimensional NMR
experi-ments showed that cytochrome c3and rubredoxin form
a complex with a binding constant > 104m)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 182 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 c3 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 c3and 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 c3 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 c3 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 c3 with phosphate (DvHc3:Pi) and Desulfovibrio vulgaris cyto-chrome c3 with zinc rubredoxin (DvHc3:ZnRb) It is
Fig 1 Two-dimensional EXSY NMR spectra
of DvHc3: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 DvHc3: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 CHII, 12 1 CH3IIIor 18 1 CH3IV) in different oxidation stages for DvHc 3 :Pi (solid lines) and DvHc3: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.
Trang 3apparent 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 DvHc3:Pi The fittings of both
NMR and visible spectroscopy data are reported in
Figs 2 and 3, respectively The thermodynamic
parame-ters obtained for DvHc3:Pi are listed in Table 1, together
with the macroscopic pKa values for the five stages of
oxidation
The pH dependence of the chemical shifts of the
haem methyl groups 21CH3I, 181CH3II, 121CH3III and
181CH3IV both Desulfovibrio vulgaris cytochrome c3
(DvHc3) and DvHc3: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 DvHc3: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 pKa values are within 0.2 units
Fig 2 The pH dependence of the chemical shift of haem methyl resonances 2 1 CH3I, 18 1 CHII, 12 1 CH3III and 18 1 CH3IV, of DvHc3: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 DvHc3: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 m M 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.
Trang 4of those measured for the isolated cytochrome As
pre-viously reported for cytochromes c3 from Desulfovibrio
gigas, Desulfomicrobium norvegicum and
Desulfomicro-bium baculatum[23], the presence of phosphate induced
a generalized narrowing of the line widths of the DvHc3
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 DvHc3:Pi to
be collected in a NMR spectrometer operating at
300 MHz, and establishing that the intermolecular
elec-tron exchange is < 340 s)1at 1 mm and 297.3 K
The paramagnetic chemical shifts of each haem
methyl group (21CHI
3, 181CHII
3 , 121CHIII
181CHIV
3 ), of DvHc3:ZnRb are plotted in Fig 4 and
the relative thermodynamic parameters together with
the macroscopic pKa 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 visposs-ible
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 DvHc3:ZnRb and DvHc3is 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 c3 [6] Table 2 shows that as for the case of phosphate binding, the association with
Table 1 Thermodynamic parameters determined for DvHc3[13] and DvHc3:Pi (Top) Diagonal terms (in bold) represent the oxidation ener-gies of the four haems and the enerener-gies 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.
DvHc3
DvHc3:Pi
Macroscopic pK a values Oxidation stage
Trang 5Table 2 Thermodynamic parameters determined for DvHc 3 [13] and DvHc 3 :ZnRb (Top) Relative thermodynamic parameters (Bottom) Mac-roscopic pKavalues 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.
DvHc3
DvHc3:ZnRb
Macroscopic pK a values Oxidation stage
Fig 4 The pH dependence of the chemical shift of haem methyl resonances 21CH3I, 181CHII, 121CH3IIIand 181CH3IV, of DvHc 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 DvHc3and the nearest label (1–4) indicates the oxidation stage represented by the line.
Trang 6Zn-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 pKavalues are absolute, and therefore
the macroscopic pKavalues 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 DvHc3 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 DvHc3 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 c3 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 c3 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 c3 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 c3 [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 DvHc3 is
> 104m)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 DvHc3: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 pKa values remain
Trang 7essentially 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
DvHc3, 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
DvHc3 Our results show that the equilibrium
thermo-dynamic data obtained for the isolated cytochrome c3
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 c3 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 ZnCl2(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 c3
Cells of D vulgaris (Hildenborough; Universidade Nova de Lisboa) were grown and the tetrahaem cytochrome c3 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, 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 saturacalibra-ted 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 c3 from
D vulgaris (Hildenborough) (DvHc3) 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 DvHc3in the presence of 100 mm phosphate
The protein was lyophilized twice with 2H2O (99.96% atom) and then dissolved in 500 lL2H2O (99.96% atom)
100 mm K3PO4.7H2O solution to a final concentration of
1 mm (this sample will be referred hereafter as DvHc3:Pi) Identical NMR spectra of the DvHc3(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 NaO2H or2HCl solutions In the reduced and intermediate
Trang 8stages 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
DvHc3with Zn-rubredoxin
The cytochrome and the Zn-rubredoxin were lyophilized
separately and a sample was prepared in 500 lL 2
H2O (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 DvHc3:ZnRb) The sample was
mani-pulated as described above for the sample containing
100 mm phosphate
NMR spectroscopy of partially oxidized samples
1H NMR spectra were obtained either in a Bruker
AMX300 for DvHc3:Pi or in a Bruker DRX500 Avance
spectrometer for DvHc3: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
(t2)· 1 k (t1) 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
DvHc3 [13] was applied to the data obtained for DvHc3:Pi
and DvHc3:ZnRb This model considers five interacting
cen-tres: four haems and one acid–base centre As shown in the
Results, DvHc3exhibits 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 contribuoxida-tions
As shown previously [26], the haem methyl groups 21CH3I,
181CHII
3 , 121CHIII
3 and 181CHIV
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 DvHc3:ZnRb), or to the NMR and UV-visible data sets simultaneously (for the case
of DvHc3: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)
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