Báo cáo khoa học: Role of the surface charges D72 and K8 in the function and structural stability of the cytochrome c6 from Nostoc sp. PCC 7119 potx

Role of the surface charges D72 and K8 in the function
and structural stability of the cytochrome c
6
from Nostoc
sp. PCC 7119
Christian Lange
1
, Irene Luque
2
, Manuel Herva
´
s
1
, Javier Ruiz-Sanz
2
, Pedro L. Mateo
2
and
Miguel A. De la Rosa
1
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 c
6
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 c
6
from
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 c
6
from Nostoc
(formerly Anabaena) sp. PCC 7119 (cyt c
6
) [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 c
6
from Nostoc sp.
PCC 7119 (cyt c
6
). 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 c
6
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 c
6
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 c
6
. 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 c
6
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
m
pH 7
, midpoint redox potentials at pH 7 and
at 25 °C; DG
int
, interaction Gibbs energies; k
bim
, 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.
FEBS Journal 272 (2005) 3317–3327 ª 2005 FEBS 3317
unfolding 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 c
6
, based on the known structure of cytochrome c
6
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 c
6

, the conserved
residues K8 and D72 are located near the crossing
region of the helices, and the N
x
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 c
6
with
PS I, as well as to its structural stability. For this pur-
pose, a series of mutant forms of cyt c
6
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 absorption 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 c
6
and its mutants
Wild-type cyt c
6
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 (E
m
pH 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 c
6
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 c
6
were
highly active electron donors to PS I (Fig. 1). The
determined values for the bimolecular rate constant
(k
bim

) for wild-type cyt c
6
and the mutant D72K were
in excellent agreement with the values that had been
reported previously [16]. Overall, a good correlation
(R ¼ 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 c
6
. 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 k
bim
values,
and the same was observed for all variants carrying
Table 1. Redox potentials of wild-type cyt c
6
and its mutant forms.
E
m
pH 7
(mV)
Wild-type 335
D72A 336

D72K 339
D72N 340
K8A 341
K8D 336
D72K ⁄ K8D 338
D72K ⁄ K8A 338
D72A ⁄ K8D 331
D72A ⁄ K8A 342
-2 -1 0 1 2
0
5
10
15
20
25
30
WT
K8D
D72K
D72N
D72A
KA
KD
AA
AD
k
bim
(10
7
M

-1
s
-1
)
relative nominal charge
K8A
Fig. 1. Bimolecular rate constants, k
bim
, for the reduction of PS I.
The k
bim
values 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.
Surface charge mutants of cytochrome c
6
C. Lange et al.
3318 FEBS Journal 272 (2005) 3317–3327 ª 2005 FEBS
the 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 c
6
to

PS I. K8 is located at the edge of the contact surface
of cyt c
6
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 k
bim
value compared with cyt c
6
wild-type, whereas for the mutant D72A, which has
the same net charge, no change in k
bim
was observed.
This result implies that changes in amino acid position
72 have additional specific effects on the interaction of
cyt c
6
with PS I, independent of the influence of global
net charge.
Urea-induced folding ⁄ unfolding of cyt c
6
:
equilibrium stability
Preliminary experiments had shown that cyt c
6
has
maximum stability against unfolding by heat and

denaturants around pH 5.5, in its physiological work-
ing range [27]. Oxidized cyt c
6
could not be fully unfol-
ded by urea at pH 5 and 30 °C. Therefore, we chose
to observe the urea-induced folding ⁄ unfolding of oxi-
dized cyt c
6
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 c
6
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] (DG
U0
) was significantly reduced (D[urea]
50
¼

)2.4 ± 0.2 m, DDG
U0
¼ )2.7 ± 1.6 kJÆmol
)1
). The
mutant protein D72N was similarly affected (D[urea]
50
¼
)1.9 ± 0.1 m, DDG
U0
¼ )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
[urea]
final
[urea]
final
[urea]
final
wild type
A
B

C
D
fluo. signal (V)
0
1
2
3
AD
D72K
fluo. signal (V)
0.0
0.3 0.6 0.9 1.2 1.5
1
2
3
4
fluo. signal (V)
time (s)
0246810
0
20
40
60
80
100
% unfolded
[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 c
6
(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 letter
the one at position 8.
C. Lange et al. Surface charge mutants of cytochrome c
6
FEBS Journal 272 (2005) 3317–3327 ª 2005 FEBS 3319
stability 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 c
6
. It is interesting
to note that charge neutralization with a hydrogen-
bond-forming asparagine residue leads to destabiliza-
tion of the protein, whereas neutralization 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 c
6
. The apparent stability of the mu-
tants D72K ⁄ K8A (D[urea]
50
¼ )1.7 ± 0.1 m, DDG
U0
¼
)1.4 ± 2.1 kJÆmol
)1
)andD72K⁄ K8D (KD) ( D[urea]
50
¼
)1.4 ± 0.1 m, DDG
U0
¼ +0.2 ± 2.8 kJÆmol
)1
) against
urea-induced unfolding was still reduced when com-
pared with wild-type cyt c
6
. This indicates that the elec-
trostatic interactions of the side-chain of D72 that
contribute to the stability of cyt c
6
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]
50
¼ +0.2 ± 0.1 m, DDG
U0
¼
+3.5 ± 2.4 kJÆmol
)1
and D[urea]
50
¼ +0.2 ± 0.1 m,
DDG
U0
¼ +0.5 ± 1.7 kJÆmol
)1
, respectively).
The equilibrium urea interaction parameter (m
eq
),
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 c
6
: 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 (k
U0
¼ 0.075 and 0.080 s
)1
, respectively), while
the mutant with the highest DG
U0
value, D72A⁄ K8D,
was found to show the lowest unfolding rate (k
U0
¼
0.023 s
)1

). The value for wild-type cyt c
6
lay between
these extremes (k
U0
¼ 0.035 s
)1
) (Table 2). In general,
a good correlation (R ¼ –0.73) was found between log
k
U0
and the Gibbs energy of unfolding at 0 [urea]
(DG
U0
). 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 c
6
and 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
)
m
eq
(kJÆmol
)1
ÆM
)1
)
log
(k
U0
Æs
)1
)
m
k1
(kJÆmol
)1
ÆM
)1
)
m

k2
(kJÆmol
)1
ÆM
)2
)
Wild-type 5.94 ± 0.07 21.4 ± 1.3 )3.6 ± 0.2 )1.5 ± 0.5 )1.7 ± 0.9 0.03 ± 0.06
K8A 5.8 ± 0.2 21.4 ± 0.8 )3.7 ± 0.2 )1.3 ± 0.2 )1.7 ± 0.3 0.05 ± 0.03
K8D 5.51 ± 0.02 20.6 ± 0.2 )3.74 ± 0.02 )1.26 ± 0.09 )1.6 ± 0.2 0.03 ± 0.01
D72K 3.6 ± 0.1 18.7 ± 0.3 )5.2 ± 0.2 )1.10 ± 0.05 )2.5 ± 0.2 0.11 ± 0.02
D72N 4.05 ± 0.06 18.3 ± 1.6 )4.6 ± 0.5 )1.13 ± 0.04 )2.0 ± 0.1 0.08 ± 0.01
D72A 5.41 ± 0.05 21.8 ± 1.4 )4.0 ± 0.2 )1.4 ± 0.5 )2.3 ± 0.7 0.09 ± 0.05
D72K ⁄ K8D 4.59 ± 0.02 21.6 ± 1.5 )4.7 ± 0.3 )1.3 ± 0.7 )2.8 ± 1.3 0.1 ± 0.1
D72K ⁄ K8A 4.27 ± 0.03 20.0 ± 0.8 )4.7 ± 0.2 )1.1 ± 0.1 )2.4 ± 0.4 0.11 ± 0.04
D72A ⁄ K8D 6.12 ± 0.02 24.6 ± 1.1 )4.0 ± 0.2 )1.6 ± 0.5 )2.4 ± 0.9 0.07 ± 0.07
D72A ⁄ K8A 6.07 ± 0.07 22.0 ± 0.4 )3.62 ± 0.05 )1.0 ± 0.4 )1.6 ± 0.6 0.04 ± 0.04
Surface charge mutants of cytochrome c
6
C. Lange et al.
3320 FEBS Journal 272 (2005) 3317–3327 ª 2005 FEBS
(‘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 à with increasing denaturant concentration.
For the global analysis, this was taken into account by
introducing the parameter m
k2
. 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 a ¼ 1 would indicate an all native-like trans-
ition state, a ¼ 0 would indicate an all unfolded-like
one. Wild-type cyt c
6
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 m
k2
value), and therefore signifi-
cantly higher a-values at high denaturant concentra-
tions than wild-type cyt c
6
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 c
6
and its mutants, DSC
was performed.
At pH 7, oxidized cyt c

6
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 c
6
, 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 c
6
variants. 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 c
6
,
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 c
6

is
shown in Fig. 3B. The Gibbs energy of unfolding as a
function of temperature, DG
U
(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 DG
U
(T) at 30 °C(DG
30

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
)1
at pH 3.0. It is clear from
these results that there is a pronounced pH effect on
the stability of cyt c
6
. 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 c
6
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 T
m
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

(kJ·K
-1
·mol
-1
)C
p
(kJ·K
-1
·mol
-1
)
Fig. 3. Temperature dependence of the partial molar heat capacity
of cyt c
6
. (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.
C. Lange et al. Surface charge mutants of cytochrome c
6
FEBS Journal 272 (2005) 3317–3327 ª 2005 FEBS 3321
seemed to be more complex (Fig. 4). For many pro-
teins the dependence of DH
U,m
on T
m
is linear, indica-
ting an entropic origin of the differences in stability
[30–32]. The slope of such a representation corres-

ponds to DC
p,U
. In our case, as shown in Fig. 4, this
dependence was not linear for many of the studied
forms of cyt c
6
. Moreover, in the mutants for which
a linear dependence was observed, e.g. K8D, the
obtained slope values were much greater than the
DC
p,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 kJÆ mol
)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
DDG
U
and DDH
U
are reported at 75 °C, which is at
approximately the median unfolding temperature. At
all studied pH conditions, wild-type cyt c
6
had 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 c
6
and 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. T
m
values 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 °CforT
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
) T
m
(°C)
pH 5.0 DG
30

C
U
(kJÆmol
)1
) T
m
(°C)
pH 4.0 DG
30

C
U

(kJÆmol
)1
) T
m
(°C)
pH 3.0 DG
30

C
U
(kJÆmol
)1
)
Wild-type 21.4 82.1 54.8 77.3 49.3 69.6 34.5
K8A 21.4 80.5 53.2 76.2 48.0 69.3 33.5
K8D 20.6 77.6 48.7 74.0 41.7 67.0 29.9
D72K 18.7 75.1 46.0 71.9 42.6 65.0 30.3
D72N 18.3 75.9 50.2 72.0 42.5 64.9 29.9
D72A 21.8 81.8 51.5 78.1 39.0 72.9 36.6
D72K ⁄ K8D 21.6 79.1 51.3 74.5 44.7 66.5 32.0
D72K ⁄ K8A 20.0 77.9 50.6 74.3 46.7 67.4 33.4
D72A ⁄ K8D 24.6 83.6 54.7 79.3 49.8 73.1 35.0
D72A ⁄ K8A 22.0 83.7 53.9 80.1 50.8 75.0 38.6
65 70 75 80 85
300
330
360
390
420
∆H

U,m
(kJ mol
-1
)∆H
U,m
(kJ mol
-1
)
65 70 75 80 85
300
330
360
390
420
A
B
T
m
(°C)
T
m
(°C)
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 T
m
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).
Surface charge mutants of cytochrome c
6
C. Lange et al.
3322 FEBS Journal 272 (2005) 3317–3327 ª 2005 FEBS
D72A ⁄ K8A at all three pH values (Fig. 5A). This ten-
dency was also observed when comparing the T
m
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 DDH
U
75 
C 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 c
6
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 ( DG
int
)at30°C 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,
the DG
int
values obtained when K8 was substituted by
aspartate were % 5kJÆmol
)1
higher 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 pK
a
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 DG
int
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 c
6
, 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
10 5 0 -5
∆∆G
U
75

ºC
(kJ mol
-1
) ∆∆H
U
75

ºC
(kJ mol
-1
)∆T
m
(K)
AA
AD
KA
KD
D72A
D72N

D72K
K8A
K8D
100 50 0 -50
AA
AD
KA
KD
D72A
D72N
D72K
K8A
K8D
840-4
ABC
Fig. 5. Effect of mutations on the thermodynamic parameters of unfolding. Difference of the thermodynamic parameters DDG
75

C
U
(A), T
m
(B) and DDH
U
75

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 DG
30

C
U
-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
)
pH 5.0 pH 3.0
DG
int
pH5
–
DG
int
pH3
(kJÆmol
)1
)
D72K ⁄ K8D 11.4 6.3 5.1
D72K ⁄ K8A 6.2 4.1 2.1
D72A ⁄ K8D 9.3 3.0 6.3
D72A ⁄ K8A 4.0 3.0 1.0
C. Lange et al. Surface charge mutants of cytochrome c
6
FEBS Journal 272 (2005) 3317–3327 ª 2005 FEBS 3323
along 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 c
6
(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 c
6
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 c
6
. 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 c
6
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 c
6
, as indicated by the reduced enthalpy of unfold-
ing of the corresponding mutants. Interestingly, 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 c
6
. 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 c
6
that were more stable than the wild-
type under all tested conditions. Of all studied forms
of cyt c
6
, 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 (Strata-
gene, La Jolla, CA) using pEACwt as template. Cyt c
6
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
K8D, K8A and D72A ⁄ K8D (AD). These proteins were

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].
Surface charge mutants of cytochrome c
6
C. Lange et al.
3324 FEBS Journal 272 (2005) 3317–3327 ª 2005 FEBS
purified 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
forms of cyt c
6
were applied to a Superdex 75 XK16 ⁄ 60
FPLC column. The running buffer for the gel filtration was

10 mm Tricine ⁄ KOH pH 7.5, 100 mm NaCl. After chroma-
tography, the purified proteins were washed with 10 mm
Tricine ⁄ KOH pH 7.5 in the ultrafiltration device, concen-
trated to 1–2 mm, and stored at )80 °C. The purity of all
preparations was > 95% as judged from the optical
absorbance ratio at 554 and 280 nm, and from Coomassie
Brilliant Blue-stained SDS ⁄ PAGE. Protein concentrations
were determined based on an absorptivity e
553
of
26 200 m
)1
cm
)1
[36] for fully reduced cyt c
6
.
Redox potentials
Redox potentials were determined by potentiometric titra-
tion with sodium dithionite and potassium ferricyanide as
described previously [38]. The determinations were carried
out in 50 mm sodium phosphate buffer pH 7 and at 25 °C.
Laser-flash absorption spectroscopy
Experiments were carried out as described previously [3].
The reaction mixture was buffered with 20 mm Tri-
cine ⁄ KOH at pH 7.5 and contained 10 mm MgCl
2
. The
temperature was 25 °C. For the determination of bimolecu-
lar rate constants (k

bim
), the kinetics of re-reduction of PS I
from Nostoc sp. PCC 7119 were measured at increasing
concentrations of wild-type cyt c
6
or of its mutant forms.
The k
bim
values were determined from the slope of a linear
fit of the observed rate constants (k
obs
), plotted against
donor concentration. For most forms of cyt c
6
, fast kinetic
phases were not observed, and it was possible to analyse all
kinetic according to a simple bimolecular reaction mechan-
ism without introducing significant deviations in k
obs
.
Stopped-flow experiments
Folding equilibrium relaxation experiments were carried
out as described previously [22]. In brief, equal concentra-
tions of cyt c
6
(wild-type or mutant forms) in 20 mm
sodium phosphate pH 7.0 (buffer) and in 9.8 m urea buf-
fered with 20 mm sodium phosphate pH 7.0 (buffered urea
solution), both containing 50 lm potassium ferricyanide in
order to keep the protein in its oxidized state throughout

the folding ⁄ unfolding experiment, were mixed in varying
ratios (r)inalSFM20 stopped-flow device (BioLogic SA,
Grenoble, France) with a constant total flow rate of
5.6 mL s
)1
in a constant total volume of 560 lL. Protein
folding ⁄ unfolding was monitored by tryptophan 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 depend-
ence of the Gibbs energy of unfolding (DG
U
) on the urea
concentration ([urea]) according to
DG
U
¼ DG
U0
þ m
eq
Á½ureað1Þ
with DG
U
at 0 [urea], DG
U0
, and the urea interaction
parameter m
eq
, and assuming a second-order dependence of
the logarithm of the unfolding rate constant (k

U
) on [urea],
according to
ln k
U
¼ ln k
U0
À
1
RT
Áðm
k1
Á½ureaþm
k2
Á½urea
2
Þ; ð2Þ
with k
U
at 0 [urea], k
U0
, and the first- and second-order
urea dependencies m
k1
and m
k2
. To estimate the urea-
dependent shift in transition state structure, a values were
calculated from the fit parameters according to
a ¼

m
eq
Àðm
k1
þ 2 Á m
k2
Á½ureaÞ
m
eq
: ð3Þ
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
The heat capacity of wild-type cyt c
6
and its mutant forms
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
used in the urea-induced folding ⁄ unfolding experiments.

DSC experiments were performed at a heating rate of
1.5 KÆmin
)1
between 5 and 100 °C, using protein concen-
trations from 10 to 50 lm (0.1–0.5 mgÆmL
)1
). Baseline
scans were performed with the corresponding dialysis buffer
loaded in both calorimetric cells. The partial molar heat
capacity (C
p
) was calculated assuming a value of
0.73 mLÆg
)1
for the partial specific volume of the proteins.
C. Lange et al. Surface charge mutants of cytochrome c
6
FEBS Journal 272 (2005) 3317–3327 ª 2005 FEBS 3325
After 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
origin 5.0 with user-defined fit functions based on the
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

each form of cyt c
6
, the values of the change in molar heat
capacity upon unfolding (DC
p,U
) were determined by global
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.
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