A series of mutant forms was generated to span the possible combinations of charge neutralization by mutation to alanine and charge inversion by mutation to lysine and aspartate, respect
Trang 1and structural stability of the cytochrome c6 from Nostoc
sp PCC 7119
Christian Lange1, Irene Luque2, Manuel Herva´s1, Javier Ruiz-Sanz2, Pedro L Mateo2and
Miguel A De la Rosa1
1 Instituto de Bioquı´mica Vegetal y Fotosı´ntesis, Centro de Investigaciones Cientı´ficas Isla de la Cartuja, Seville, Spain
2 Departamento de Quı´mica Fı´sica e Instituto de Biotecnologı´a, Universidad de Granada, Spain
In the oxygenic photosynthesis of cyanobacteria and
many unicellular algae, cytochrome c6 acts as a soluble
electron carrier between the cytochrome bf complex and
photosystem I (PS I) [1,2] In recent years, considerable
efforts have been undertaken to gain insight into the
structure–function relationships of this small heme
protein [3–7] The structures of the cytochromes c6from
various organisms have been determined [8–14] Study
of the mutant series has contributed to our
understand-ing of the functional roles of important individual
amino acid residues within the protein [15–17] NMR studies with mutant bacterial c-type cytochromes [18– 20] have provided important insights into the role of heme–protein interactions for the structural stability of this class of proteins
The mutant D72K of cytochrome c6 from Nostoc (formerly Anabaena) sp PCC 7119 (cyt c6) [21], was found to be a more reactive electron donor towards
PS I than the wild-type [16] However, D72K was shown to have considerably reduced stability against
Keywords
cytochrome c 6 ; electron transfer;
electrostatic interactions; protein folding;
protein stability
Correspondence
Christian Lange, Martin-Luther-Universita¨t
Halle ⁄ Wittenberg, Institut fu¨r
Biotechnologie, Kurt-Mothes-Str 3,
06120 Halle (Saale), Germany
Fax: +49 345 552 7013
Tel: +49 345 552 4948
E-mail: christian.lange@biochemtech.
uni-halle.de
(Received 10 February 2005, revised 21
April 2005, accepted 3 May 2005)
doi:10.1111/j.1742-4658.2005.04747.x
We investigated the role of electrostatic charges at positions D72 and K8
in the function and structural stability of cytochrome c6 from Nostoc sp PCC 7119 (cyt c6) A series of mutant forms was generated to span the possible combinations of charge neutralization (by mutation to alanine) and charge inversion (by mutation to lysine and aspartate, respectively) in these positions All forms of cyt c6 were functionally characterized by laser flash absorption spectroscopy, and their stability was probed by urea-induced folding equilibrium relaxation experiments and differential scan-ning calorimetry Neutralization or inversion of the positive charge at position K8 reduced the efficiency of electron transfer to photosystem I This effect could not be reversed by compensating for the change in global charge that had been introduced by the mutation, indicating a specific role for K8 in the formation of the electron transfer complex between cyt c6 and photosystem I Replacement of D72 by asparagine or lysine increased the efficiency of electron transfer to photosystem I, but destabilized the protein D72 apparently participates in electrostatic interactions that stabil-ize the structure of cyt c6 The destabilizing effect was reduced when aspar-tate was replaced by the small amino acid alanine Complementing the mutation D72A with a charge neutralization or inversion at position K8 led to mutant forms of cyt c6 that were more stable than the wild-type under all tested conditions
Abbreviations
cyt c 6 , cytochrome c 6 from Nostoc sp PCC 7119; DSC, differential scanning microcalorimetry; E mpH 7, midpoint redox potentials at pH 7 and
at 25 C; DG int , interaction Gibbs energies; kbim, bimolecular reaction rate constant; N, native folded state; PS I, photosystem I; U, ensemble
of unfolded states; [urea] 50 , transition midpoint for urea-induced unfolding.
Trang 2unfolding by urea [22], and preliminary experiments
indicated that its thermal stability was affected in a
similar way Electrostatic interactions play a critical
role in guiding and stabilizing functional
protein–pro-tein interactions [6], as well as in the structural stability
of proteins [23–25]
Inspection of a preliminary structural model of
cyt c6, based on the known structure of cytochrome c6
from Synechococcus elongatus [11] and NMR data,
revealed a spatial proximity between the side chain of
D72, which is located in the C-terminal a helix, and a
positively charged residue in the protein’s N-terminal
helix, K8 In S elongatus cytochrome c6, the conserved
residues K8 and D72 are located near the crossing
region of the helices, and the Nx atom of K8 is found
at a distance of 6.2 A˚ from the nearest side-chain
oxy-gen of D72
Taking the functionally and structurally interesting
mutant D72K as a material starting point, we aimed
to study the contribution of the electrostatic charges at
positions D72 and K8 to the interaction of cyt c6 with
PS I, as well as to its structural stability For this
pur-pose, a series of mutant forms of cyt c6 was generated
that spans the possible combinations of charge
neutral-ization (by mutation to alanine) and charge inversion
(by mutation to lysine and aspartate, respectively) at
positions D72 and K8 After functional
characteriza-tion by laser-flash absorpcharacteriza-tion spectroscopy,
urea-induced folding equilibrium relaxation experiments
and differential scanning microcalorimetry (DSC) were
used to assess the changes in the stability against
denaturant-induced folding⁄ unfolding as well as against
thermal unfolding
Results and Discussion
Functional characterization of wild-type cyt c6
and its mutants
Wild-type cyt c6 and its mutant forms were
success-fully expressed in Escherichia coli strain GM119 and
purified to homogeneity The UV⁄ Vis spectra of
wild-type and mutants were virtually identical (not shown)
Their midpoint redox potentials at pH 7 (EmpH 7) were
very similar throughout (Table 1), as expected for the
mutation of surface residues located far from the heme
moiety Thus, the thermodynamic driving force for the
reaction of cyt c6 with PS I was essentially unaltered
by the introduction of the mutations Eventual
differ-ences in reactivity, therefore, have to be ascribed to
structural factors that influence the formation and
productivity of the encounter complex Indeed, such
differences were observed All forms of cyt c6 were
highly active electron donors to PS I (Fig 1) The determined values for the bimolecular rate constant (kbim) for wild-type cyt c6 and the mutant D72K were
in excellent agreement with the values that had been reported previously [16] Overall, a good correlation (R¼ 0.83) between the proteins’ net charge and their reactivity towards PS I was observed (Fig 1) This confirms the importance of long-range charge interac-tions for the electron carrier function of cyt c6 How-ever, closer inspection of the data revealed interesting deviations from the general trend
A reversal of the charge effect of the mutation K8D
in the mutant D72K⁄ K8D did not lead to recovery of wild-type reactivity The same holds for the mutant pair K8A and D72A⁄ K8A All three variants carrying the mutation K8D showed very similar kbim values, and the same was observed for all variants carrying
Table 1 Redox potentials of wild-type cyt c6and its mutant forms.
EmpH 7 (mV)
0 5 10 15 20 25 30
WT
K8D
D72K
D72N D72A KA KD
AA AD
k bim
7 M
-1 s
-1 )
relative nominal charge
K8A
Fig 1 Bimolecular rate constants, k bim , for the reduction of PS I The kbimvalues were determined as described in the text Data points are marked with the name of the corresponding mutants Error bars represent errors from the fit to determine k bim The solid line represents a linear fit to the data points, dashed lines mark the 95% confidence intervals Double mutants are abbreviated with the first letter representing the residue at position 72 and the second letter the one at position 8.
Trang 3the mutation K8A This clearly indicates a specific
functional significance of the charge in amino acid
position 8 for the formation of a productive encounter
complex during the electron transfer from cyt c6 to
PS I K8 is located at the edge of the contact surface
of cyt c6 that directly interacts with PS I [26], and a
positive charge in this position might be expected to
play an important role by establishing specific
electro-static interactions during complex formation with PS I
The variant carrying the single mutation D72N
showed an increased kbim value compared with cyt c6
wild-type, whereas for the mutant D72A, which has
the same net charge, no change in kbim was observed
This result implies that changes in amino acid position
72 have additional specific effects on the interaction of
cyt c6with PS I, independent of the influence of global
net charge
Urea-induced folding⁄ unfolding of cyt c6:
equilibrium stability
Preliminary experiments had shown that cyt c6 has
maximum stability against unfolding by heat and
denaturants around pH 5.5, in its physiological
work-ing range [27] Oxidized cyt c6 could not be fully
unfol-ded by urea at pH 5 and 30C Therefore, we chose
to observe the urea-induced folding⁄ unfolding of
oxi-dized cyt c6 and its mutant forms at pH 7 The study
was carried out by performing folding equilibrium
relaxation experiments In Fig 2, the results of typical
experiments with wild-type cyt c6 and the mutant
forms D72K and D72A⁄ K8D are shown as examples,
along with the derived equilibrium unfolding curves
(Fig 2D) Analysis of all performed experiments
yielded a set of parameters for comparison of the
sta-bilities of wild-type and mutants (Table 2)
As previously reported [22], the transition midpoint
for urea-induced unfolding ([urea]50) of the mutant
D72K was shifted to significantly lower urea
concen-trations ([urea]), and its Gibbs energy of unfolding at
0 [urea] (DGU0) was significantly reduced (D[urea]50¼
)2.4 ± 0.2 m, DDGU0¼)2.7 ± 1.6 kJÆmol)1) The mutant protein D72N was similarly affected (D[urea]50¼ )1.9 ± 0.1 m, DDGU0¼)3.1 ± 2.9 kJÆmol)1) All other single mutations, including D72A, had a comparably minor effect Charge neutralization at position K8 has no significant effect on the structural
0 1 2 3 4
wild type
A
B
C
D
0 1 2 3
AD
D72K
0.0 0.3 0.6 0.9 1.2 1.5 1
2 3 4
time (s)
0 2 4 6 8 10 0
20 40 60 80 100
[urea] (M)
Fig 2 Folding equilibrium relaxation experiments The represented
experiments were performed with wild-type cyt c 6 (A), D72K (B)
and D72A ⁄ K8D (AD) (C) Arrows on the right indicate increasing
[urea] final (0–9.8 M ) Percentages of unfolded protein at equilibrium
as determined from the experiments shown in (A) to (C) for
wild-type cyt c6(squares), D72K (circles) and AD (triangles) are plotted
as a function of [urea] in (D) The lines represent fits of two state
transitions to the data for wild-type cyt c 6 (solid), D72K (dashed)
and AD (dotted) Double mutants are abbreviated with the first
let-ter representing the residue at position 72 and the second letlet-ter
the one at position 8.
Trang 4stability of the protein, whereas inversion of the charge
at position 72 considerably reduces the stability against
urea-induced unfolding, i.e electrostatic interactions of
the negative charge at position D72 seem to play an
important role for the stability of cyt c6 It is interesting
to note that charge neutralization with a
hydrogen-bond-forming asparagine residue leads to
destabiliza-tion of the protein, whereas neutralizadestabiliza-tion with a small
hydrophobic alanine residue does not
Partial or total neutralization of the effect of the
mutation D72K on the protein’s global charge was not
sufficient to fully reverse the negative effect on the
stability of cyt c6 The apparent stability of the
mu-tants D72K⁄ K8A (D[urea]50¼)1.7 ± 0.1 m, DDGU0¼
)1.4 ± 2.1 kJÆmol)1) and D72K⁄ K8D (KD) (D[urea]50¼
)1.4 ± 0.1 m, DDGU0¼ +0.2 ± 2.8 kJÆmol)1) against
urea-induced unfolding was still reduced when
com-pared with wild-type cyt c6 This indicates that the
elec-trostatic interactions of the side-chain of D72 that
contribute to the stability of cyt c6 are at least partially
local, and that the adverse effects of the mutations
D72K and D72N cannot be entirely ascribed to global
electrostatic repulsion within the positively charged
protein
However, although mutation D72A did not lead to
a significant change in stability, the additional
elimin-ation of the positive charge K8 in the double mutants
D72A⁄ K8D and D72A ⁄ K8A resulted in an increased
apparent stability against urea-induced unfolding, as
well as an increased Gibbs energy of unfolding of
these mutants (D[urea]50¼ +0.2 ± 0.1 m, DDGU0¼
+3.5 ± 2.4 kJÆmol)1 and D[urea]50¼ +0.2 ± 0.1 m,
DDGU0¼ +0.5 ± 1.7 kJÆmol)1, respectively)
The equilibrium urea interaction parameter (meq),
which represents the steepness of the unfolding
transition with respect to [urea] and may be interpreted
as a measure of the change in solvent-accessible area
upon unfolding [28], was affected by changes at position D72, but not at position K8 It was significantly increased for all mutants in which position 72 had been modified, with the exception of D72A⁄ K8A This find-ing might be partially explained by a prevalence of more extended conformations in the unfolded ensemble of states due to local electrostatic repulsion in the vicinity
of the mutated position 72
Urea-induced folding⁄ unfolding of cyt c6: kinetic parameters
The performed folding relaxation experiments also allowed for the determination of kinetic parameters (Table 2) The most thermodynamically unstable mutants, D72N and D72K, were found to have the highest unfolding rate constant in absence of denatu-rant (kU0¼ 0.075 and 0.080 s)1, respectively), while the mutant with the highest DGU0 value, D72A⁄ K8D, was found to show the lowest unfolding rate (kU0¼ 0.023 s)1) The value for wild-type cyt c6 lay between these extremes (kU0¼ 0.035 s)1) (Table 2) In general,
a good correlation (R¼ –0.73) was found between log
kU0 and the Gibbs energy of unfolding at 0 [urea] (DGU0) In the mutant series, the overall height of the energy barrier for the rate-determining step of the fold-ing⁄ unfolding transition is mainly determined by the overall difference in Gibbs energy between the native state (N) and the ensemble of unfolded states (U), in agreement with a single transition state with a disor-dered (unfolded-like) structure [28] This indicates that the folding⁄ unfolding transition proceeds along a sim-ilar pathway for wild-type cyt c6and for all mutants When the folding⁄ unfolding traces were analysed individually, and the apparent rate constants (k) for the individual traces were plotted against [urea] in Chevron plots, pronounced deviations from linearity
Table 2 Evaluation of urea-induced folding equilibrium relaxation experiments Data represent means ± SD from 2–4 experiments for each protein.
[Urea]50 (M)
DG U0 (kJÆmol)1)
meq (kJÆmol)1Æ M )1)
log (k U0 Æs)1)
mk1 (kJÆmol)1Æ M )1)
mk2 (kJÆmol)1Æ M )2)
Trang 5(‘roll over’ and ‘roll down’) were observed (not
shown) These deviations were interpreted as indicative
of a Hammond shift [29] in the structure of the
trans-ition state with increasing denaturant concentration
For the global analysis, this was taken into account by
introducing the parameter mk2 From the fit
parame-ters, a values were calculated (as a function of urea)
according to Eqn (3) (see Experimental procedures)
These values may be interpreted as a measure of
native-like structure in the transition state [22,28]
Whereas a¼ 1 would indicate an all native-like
trans-ition state, a¼ 0 would indicate an all unfolded-like
one Wild-type cyt c6 and all mutant forms showed a
transition state shift to higher a-values with increasing
[urea] (not shown) The single mutants D72K and
D72N, as well as the double mutants D72K⁄ K8D and
D72K⁄ K8A, showed the steepest increase in a with
[urea] (i.e a higher mk2 value), and therefore
signifi-cantly higher a-values at high denaturant
concentra-tions than wild-type cyt c6 and the other mutant
forms Their kinetic properties were clearly more
sus-ceptible to the effect of urea and the structure of their
transition state shifted more strongly towards the
native conformation under the influence of
destabiliza-tion of the latter
Microcalorimetry studies
In order to obtain the thermodynamic parameters of
the thermal unfolding of cyt c6 and its mutants, DSC
was performed
At pH 7, oxidized cyt c6 was found to be
redox-unstable in the absence of oxidizing agents, whose
presence would affect the calorimetric trace of the
DSC experiment It was also observed that the heating
of reduced protein at pH 7 gave rise to complex
calori-metric traces, probably due to an oxidative process of
the protein upon thermal unfolding It was found that
at lower pH values ranging from 3.0 to 5.0, close to
the physiological working range of cyt c6, the oxidized
form was redox-stable in absence of oxidizing agents
during DSC experimental time Consequently, DSC
experiments were carried out at pH 3.0, 4.0 and 5.0
for all cyt c6variants In all cases, heat-induced
unfold-ing was highly reversible and independent of both
the scanning rate and the protein concentration All
calorimetric traces could be fitted very well to a
two-state equilibrium model (NfiU)
The experimental DSC curves, together with their
best fits, for the thermal unfolding of wild-type cyt c6,
and for the mutants D72K and D72A⁄ K8D at pH 5.0
are shown in Fig 3A as typical examples The effect
of pH on thermal unfolding of wild-type cyt c6 is
shown in Fig 3B The Gibbs energy of unfolding as a function of temperature, DGU(T), could be obtained using the thermodynamic parameter values from the multiple fits of the DSC traces at the studied pH con-ditions The values of DGU(T) at 30C (DG30 C
U ) for wild-type were 54.8 kJÆmol)1 at pH 5.0, 49.3 kJÆmol)1
at pH 4.0 and 34.5 kJÆmol)1at pH 3.0 It is clear from these results that there is a pronounced pH effect on the stability of cyt c6 Taking into account the value of 21.4 kJÆmol)1 obtained at pH 7.0 by the urea-induced folding⁄ unfolding experiments (Table 2), it could be confirmed that the maximum stability of wild-type cyt c6 is around pH 5.0 As it is shown in Table 3, this behaviour was common to all studied mutants
A similar dependence on the pH was observed for the Tm values obtained for all proteins (Table 3) By contrast, the effect of pH on the unfolding enthalpies
30 50 70 90 110 10
20 30 40 50 60
temperature (ºC)
temperature (ºC)
30 50 70 90 110 10
20 30 40 50 60
A
B
C p
-1 ·mol
-1 )
C p
-1 ·mol
-1 )
Fig 3 Temperature dependence of the partial molar heat capacity
of cyt c6 (A) DSC traces at pH 5.0 for wild-type (h), D72K (s) and D72A ⁄ K8D (n) (B) DSC traces for wild-type at pH 5.0 (h), pH 4.0 (s) and pH 3.0 (n) Symbols correspond to experimental data, for clarity every third data point is plotted Solid lines correspond to best fits to the two-state equilibrium model.
Trang 6seemed to be more complex (Fig 4) For many
pro-teins the dependence of DHU,m on Tmis linear,
indica-ting an entropic origin of the differences in stability
[30–32] The slope of such a representation
corres-ponds to DCp,U In our case, as shown in Fig 4, this
dependence was not linear for many of the studied
forms of cyt c6 Moreover, in the mutants for which
a linear dependence was observed, e.g K8D, the
obtained slope values were much greater than the
DCp,U values calculated from the global analysis of
the calorimetric traces As may be observed in Fig 4,
the enthalpic variation with pH is most pronounced
between pH 3 and 4 for the wild-type and most
mutants, with the exception of D72A, for which the
biggest pH effect was observed between pH 4 and 5
These results indicate that the stability differences
found are not only of entropic nature but that there is
a considerable enthalpic contribution Because the
ionic strength was fixed in all experiments, the pH
effects are exclusively due to changes in the
electro-static interactions The enthalpic contributions of
pro-tonation⁄ deprotonation events are small compared
with the changes in enthalpy observed in these mutants
(up to 60 kJÆmol)1) So, it is likely that these ionization
events are responsible for some kind of structural
rear-rangement that is associated with enthalpic changes
The changes in the thermodynamic parameters of
unfolding of each mutant with respect to the wild-type
protein are summarized in Fig 5 To avoid
extrapola-tion over a large temperature range, the values of
DDGU and DDHU are reported at 75C, which is at
approximately the median unfolding temperature At
all studied pH conditions, wild-type cyt c6had a
signi-ficantly higher Gibbs energy of unfolding than the
mutants, with the exception of D72A at pH 4 and 3,
and of the double mutants D72A⁄ K8D and
Table 3 Thermal unfolding transition of wild-type cyt c6and its mutant forms For each protein, thermodynamic parameters were deter-mined by global fits of the two-state equilibrium model to the DSC traces recorded at all three pH conditions Tmvalues refer to the trans-ition midpoint and DG 30 C
U values represent Gibbs energies of unfolding extrapolated to 30 C The reported values are estimated to be accurate within ±0.4 C for T m and ±10% for DG 30 C
U The DG U0 values for pH 7.0 from Table 2 are included for comparison.
pH 7.0 DG U0
(kJÆmol)1) Tm(C)
pH 5.0 DG 30 C
U (kJÆmol)1) Tm(C)
pH 4.0 DG 30 C
U (kJÆmol)1) Tm(C)
pH 3.0 DG 30 C
U (kJÆmol)1)
65 70 75 80 85 300
330 360 390 420
H U,m
-1 )
H U,m
-1 )
65 70 75 80 85 300
330 360 390 420
A
B
Fig 4 Temperature dependence of DH U,m Temperature depend-ence of the unfolding heat effect for wild-type cyt c 6 and its mutant forms Symbols correspond to the DH U,m and Tm values obtained
by multiple fits of the DSC traces at the three pH conditions for (A) wild-type ( ), K8A (s), D72A ⁄ K8D (n), D72K ⁄ K8D (,) and D72A ⁄ K8A (e), and for (B) K8D ( ), D72K (s), D72N (n), D72A (,) and D72K ⁄ K8A (e) The lines correspond to the DH U (T) functions obtained by multiple fits for wild-type (A) and for K8D (B) at pH 5.0 (solid line), pH 4.0 (dashed line) and pH 3.0 (dotted line).
Trang 7D72A⁄ K8A at all three pH values (Fig 5A) This
ten-dency was also observed when comparing the Tm
val-ues (Fig 5B) These results are in good agreement
with the apparent order of stabilities obtained from
the urea-induced unfolding experiments (Table 3) It is
interesting to note that the stabilizing effect of
substi-tuting D72 to alanine (in single and double mutants)
was neither observed when the position 72 was
mutated to a charged lysine residue, nor when it was
changed to the neutral and hydrogen bond forming
asparagine
With respect to DDHU75C values (Fig 5C) two
groups of mutants could be distinguished The point
mutations at position 8, for which no significant
enthalpic difference was observed, and the rest (where
position 72 is mutated), which presented lower
enthal-pies of unfolding than the wild-type protein
Inspection of the values obtained for the Gibbs
ener-gies of unfolding of the mutant forms of cyt c6
revealed significant nonadditive effects in the stabilities
of the double mutants when compared with the
stabili-ties of the respective single mutants The interaction
Gibbs energies (DGint) at 30C for the different double
mutants were estimated by double-mutant cycle
analy-sis [33,34] and are summarized in Table 4 At pH 5.0,
theDGint values obtained when K8 was substituted by
aspartate were 5 kJÆmol)1higher than when K8 was
substituted by alanine, independent of the nature of
the substitution at amino acid position 72 This
addi-tional interaction energy was reduced to 2 kJÆmol)1
(for the double mutants of D72K⁄ K8D and
D72K⁄ K8A) or even abolished (for the double
mutants D72A⁄ K8D and D72A ⁄ K8A) at pH 3.0 Assuming a standard pKa value for the carboxyl group, the aspartate residue introduced at position 8 would be almost completely neutralized at pH 3.0 Thus, replacement of K8 with a negatively charged residue resulted in stronger destabilizing interactions than a neutral residue, when the negative charge at position 72 was removed
The influence of the nature of the introduced muta-tions and the complex pH dependence of the DGint values for the different mutant pairs strongly suggest that much more complex effects (multiple interactions and⁄ or structural rearrangements) than a direct elec-trostatic interaction between the residues at amino acid positions 8 and 72 play a role A refined model for the structure of cyt c6, incorporating NMR data, has become available during revision of this work [35] In this model (Fig 6), K8 is located relatively far from other charged residues, and its side chain is highly solvent exposed It forms backbone hydrogen bonds
AA
AD
KA
KD
D72A
D72N
D72K
K8A
K8D
∆∆G U75 ºC
(kJ mol-1)
∆T m(K)
AA AD KA KD D72A D72N D72K K8A K8D
AA AD KA KD D72A D72N D72K K8A K8D
Fig 5 Effect of mutations on the thermodynamic parameters of unfolding Difference of the thermodynamic parameters DDGU75C(A), Tm (B) and DDH U75
C
between wild-type and each mutant Black bars correspond to pH 5.0, grey bars to pH 4.0 and light grey bars to pH 3.0 Double mutants are abbreviated with the first letter representing the residue at position 72 and the second letter the one at position 8.
Table 4 Double-mutant cycle analysis Interaction Gibbs energies
at 30 C (DG int ) were calculated from the DGU30C-values reported
in Table 3 according to DG int (D72X ⁄ K8Y) ¼ DG 30 C
U (wild-type) + DG 30 C
U (D72X ⁄ K8Y) – DG 30 C
U (D72X) – DG 30 C
U (K8Y).
DG int (kJÆmol)1)
DG intpH5– DG intpH3 (kJÆmol)1)
Trang 8along the N-terminal a helix involving the amino acids
V4⁄ N5, and S11 ⁄ A12 The side chain of D72 is part of
an acidic patch, together with the residues D2 and
E68 It forms backbone hydrogen bonds along the
C-terminal a helix to E68 and Y76 Interestingly, one
of the side chain oxygens of D72 appears to be
hydro-gen-bonded to the backbone N atom of G6, bridging
the crossing between the N- and C-terminal a helices
of cyt c6 (Fig 6) Any mutation leading to charge
neutralization or inversion of a residue within this
helix-crossing region may cause rearrangements and a
reorganization of the local hydrogen-bond network It
is, for example, conceivable that upon replacement of
D72 by alanine one of the residues E68 or Q69 ‘takes
over’ in forming a stabilizing hydrogen bond to an
amino acid within a reoriented N-terminal a helix
However, any such interpretation of the experimental
data remains speculative until reliable structural
infor-mation for cyt c6 and its mutant forms becomes
avail-able
Conclusions
The results of this study stress the importance of
elec-trostatic interactions for the function, as well as for
the stability, of cyt c6 Neutralization or inversion of
the positive charge at position K8 significantly reduced
the efficiency of electron transfer to PS I This effect was not reversed when the global charge of the protein was restored by additional mutations at position 72 This implies that residue K8 plays a role in the forma-tion of the electron transfer complex between cyt c6 and PS I However, replacement of the negative charge
at position D72 by asparagine or lysine increased the efficiency of electron transfer to PS I, at least partly,
by favouring long-range electrostatic attraction between the reaction partners These mutations, however, were found to destabilize the protein significantly Again, this effect of the mutations could not be fully reversed
by restoring the global charge balance The negative charge at position D72 apparently participates in elec-trostatic interactions that stabilize the structure of cyt c6, as indicated by the reduced enthalpy of unfold-ing of the correspondunfold-ing mutants Interestunfold-ingly, the negative effect of its deletion was reduced, due to entropic contributions, when aspartate was not replaced by the isosteric asparagine, or by lysine, but
by the small amino acid alanine For the variants carrying the mutation D72A, the positive change in entropy upon unfolding was reduced with respect to other variants of cyt c6 This smaller unfolding entropy could be due to the effect of the mutation on the native state (higher flexibility and⁄ or lower exposure of hydrophobic area to the solvent in the mutant D72A) and⁄ or on the unfolded state (lower flexibility and ⁄ or higher solvation of hydrophobic surface area) Com-plementing the mutation D72A with a charge neutral-ization or inversion at position K8 led to mutant forms of cyt c6 that were more stable than the wild-type under all tested conditions Of all studied forms
of cyt c6, AD was the thermodynamically most stable
at pH 7, whereas AA was the most stable at pH 4 and
3 The price for this stabilization, however, was a reduction in catalytic efficiency by > 50%
Experimental procedures
Protein expression and purification Expression plasmids pEACwt and pEACD72K were kindly provided by F.P Molina-Heredia [16,36] Expression plas-mids for the other mutants were generated by site-directed mutagenesis according to the QuikChange method
wild-type and its mutant forms were expressed under aero-bic conditions in E coli GM119 cells that had been cotransfected with the plasmid pEC86 [37] (kindly provided
by L Tho¨ny-Meyer, ETH Zu¨rich) and were purified as des-cribed previously [16], with the exception of the mutants
Fig 6 Structural model of cyt c 6 , showing secondary structure
ele-ments The side chains of amino acids K8 (blue), E68 (light red),
Q69 (light green) and D72 (red), as well as the heme moiety
(orange), are depicted as stick models The H-bond from the
back-bone amide group of G6 to the carboxyl oxygen of D72 is shown
as dotted green line The image was generated with PYMOL 0.97
(DeLano Scientific, San Carlos, CA, USA) and is based on the
struc-tural model from [35].
Trang 9purified by a combination of anion-exchange
chromatogra-phy and gel filtration In brief, the periplasmic extracts
con-taining the mutants K8D, K8A or AD were dialysed
against 0.5 mm sodium phosphate buffer pH 7, oxidized by
the addition of 50 lm potassium ferricyanide, and loaded
onto a DEAE cellulose column (50 mL) that had been
equilibrated with a 1 mm sodium phosphate buffer, pH 7
The mutant proteins were found in the flow-through After
concentration under nitrogen pressure in an Amicon 8050
ultrafiltration device fitted with YM 3 membranes
(Milli-pore, Bedford, MA), the fractions containing the mutant
FPLC column The running buffer for the gel filtration was
chroma-tography, the purified proteins were washed with 10 mm
preparations was > 95% as judged from the optical
absorbance ratio at 554 and 280 nm, and from Coomassie
Redox potentials
Redox potentials were determined by potentiometric
titra-tion with sodium dithionite and potassium ferricyanide as
described previously [38] The determinations were carried
Laser-flash absorption spectroscopy
Experiments were carried out as described previously [3]
The reaction mixture was buffered with 20 mm
from Nostoc sp PCC 7119 were measured at increasing
phases were not observed, and it was possible to analyse all
kinetic according to a simple bimolecular reaction
Stopped-flow experiments
Folding equilibrium relaxation experiments were carried
out as described previously [22] In brief, equal
sodium phosphate pH 7.0 (buffer) and in 9.8 m urea
buf-fered with 20 mm sodium phosphate pH 7.0 (bufbuf-fered urea
solution), both containing 50 lm potassium ferricyanide in
order to keep the protein in its oxidized state throughout
ratios (r) in a lSFM20 stopped-flow device (BioLogic SA, Grenoble, France) with a constant total flow rate of
fluores-cence Kinetic traces were recorded in duplicate or triplicate and averaged for analysis The data for all mutants were fitted globally to a two-state model assuming linear
concentration ([urea]) according to
according to
urea-dependent shift in transition state structure, a values were calculated from the fit parameters according to
The global fitting of the data was carried out with the non-linear analysis module of origin 5.0 (Microcal, Northamp-ton, MA, USA)
Microcalorimetry
was measured as a function of temperature with a VP-DSC differential scanning microcalorimeter (Microcal) Samples were dialysed overnight against the desired buffer and sub-sequently oxidized by addition of potassium ferricyanide to
a final concentration of 2.5 mm Immediately before load-ing the samples into the calorimetric cell, ferricyanide was removed by gel filtration on a PD-10 column (Amersham Biosciences, Little Chalfont, UK) The buffers used in the experiments were 60 mm sodium acetate, pH 5.0, 250 mm sodium acetate, pH 4.0, or 45 mm sodium phosphate,
pH 3.0, all giving rise to the same ionic strength (41 mm)
as the 20 mm sodium phosphate buffer, pH 7, which was
DSC experiments were performed at a heating rate of
scans were performed with the corresponding dialysis buffer loaded in both calorimetric cells The partial molar heat
Trang 10After transforming the DSC traces into partial molar heat
capacity curves, they were subjected to individual and
glo-bal curve fitting using the nonlinear analysis module of
equations corresponding to a two state model (NfiU) The
temperature dependence of the heat capacity was described
as a linear and quadratic function for the native and
unfol-ded species, respectively [39] The corresponding parameters
were left to float during the curve fitting, with the exception
of the first- and second-order coefficients of the unfolded
heat capacity, which were fixed and evaluated from the
amino acid content as described previously [40,41] For
fitting of the DSC traces recorded at all three studied pH
values
Chemicals
Urea was SigmaUltra grade All other chemicals were
at least analytical grade All solutions were prepared with
MilliQ water
Acknowledgements
This work was supported by the Spanish Ministry of
Science and Technology (MCYT grants BMC
2000-444 and BIO2003-04274) and the Junta de Andalucı´a
(PAI, CVI-198) CL received a fellowship from the
European Union’s Research Training Network
pro-gram (HRPN-CT-1999-00095) IL was supported by a
research contract from the University of Granada and
is the recipient of a Ramo´n y Cajal research contract
from the Spanish Ministry of Science and Technology
References
1 Wood PM (1978) Interchangeable cooper and iron
pro-teins in algal photosynthesis: studies on plastocyanin
87, 9–19
2 Ho KK & Krogmann DW (1984) Electron donors to
P700 in cyanobacteriae and algae: an instance of
un-usual genetic variability Biochim Biophys Acta 766,
310–316
3 Herva´s M, Navarro JA, Dı´az-Quintana A, Bottin H &
De la Rosa MA (1995) Laser-flash kinetic analysis of
the fast electron transfer from plastocyanin and
the evolution of the reaction mechanism Biochemistry
34, 11321–11326
4 Herva´s M, Navarro JA, Dı´az-Quintana A & De la Rosa
MA (1996) A comparative thermodynamic analysis by
laser-flash absorption spectroscopy of photosystem I
Biochemistry 35, 2693–2698
5 Ullman GM, Hauswald M, Jensen A, Kostic NM & Knapp EW (1997) Comparison of the physiologically
the basis of their electrostatic potentials Tryptophan 63
in plastocyanin Biochemistry 36, 16187–16196
6 Crowley PB, Diaz-Quintana A, Molina-Heredia FP, Nieto P, Sutter M, Haehnel W, De La Rosa MA & Ubbink M (2002) The interactions of cyanobacterial
NMR J Biol Chem 277, 48685–48689
7 Dikiy A, Carpentier W, Vandenberghe I, Borsari M, Safarov N, Dikaya E, Van Beeumen J & Ciurli S (2002) Structural basis of the molecular properties of
8 Fraza˜o C, Soares CM, Carrondo MA, Pohl E, Dauter Z, Wilson KS, Herva´s M, Navarro JA, De la Rosa MA & Sheldrick GM (1995) Ab initio determination of the
plastocyanin Structure 3, 1159–1170
9 Kerfeld CA, Anwar HP, Interrante R, Merchant S & Yeates TO (1995) The structure of chloroplast
oligomerization J Mol Biol 250, 627–647
10 Banci L, Bertini I, De la Rosa MA, Koulougliotis D, Navarro JA & Walter O (1998) Solution structure of
Monoraphi-dium braunii Biochemistry 37, 4831–4843
11 Beißinger M, Sticht H, Sutter M, Ejchart A, Haehnel W
from the thermophilic cyanobacterium Synechococcus elongatus EMBO J 17, 27–36
12 Schnackenberg J, Than ME, Mann K, Wiegand G, Huber R & Reuter W (1999) Amino acid sequence, crystallization and structure determination of reduced
Scenedesmus obliquus J Mol Biol 290, 1019–1030
13 Yamada S, Park S-Y, Shimizu H, Koshizuka Y, Kadok-ura K, Satoh T, Suruga K, Ogawa M, Isogai Y, Nishio
alga Porphyra yezoensis at 1.57 A˚ resolution Acta Crys-tallogr D56, 1577–1582
14 Sawaya MR, Krogmann DW, Serag A, Ho KK, Yeates
TO & Kerfeld CA (2001) Structures of cytochrome
Arthrospira maxima Biochemistry 40, 9215–9225
15 De la Cerda B, Dı´az-Quintana A, Navarro JA, Herva´s M
& De la Rosa MA (1999) Site-directed mutagenesis of
Chem 274, 13292–13297
16 Molina-Heredia FP, Dı´az-Quintana A, Herva´s M, Navarro JA & De la Rosa MA (1999) Site-directed