Dynamics in the transient complex of plastocyanin-cytochrome f from Prochlorothrix hollandica.

Abstract

The nature of transient protein complexes can range from a highly dynamic ensemble of orientations to a single well-defined state. This represents variation in the equilibrium between the encounter and final, functional state. The transient complex between plastocyanin (Pc) and cytochrome f (cytf) of the cyanobacterium Prochlorothrix hollandica was characterized by NMR spectroscopy. Intermolecular pseudocontact shifts and chemical shift perturbations were used as restraints in docking calculations to determine the structure of the wild-type Pc-cytf complex. The orientation of Pc is similar to orientations found in Pc-cytf complexes from other sources. Electrostatics seems to play a modest role in complex formation. A large variability in the ensemble of lowest energy structures indicates a dynamic nature of the complex. Two unusual hydrophobic patch residues in Pc have been mutated to the residues found in other plastocyanins (Y12G/P14L). The binding constants are similar for the complexes of cytf with wild-type Pc and mutant Pc, but the chemical shift perturbations are smaller for the complex with mutant Pc. Docking calculations for the Y12G/P14L Pc-cytf complex did not produce a converged ensemble of structures. Simulations of the dynamics were performed using the observed averaged NMR parameters as input. The results indicate a surprisingly large amplitude of mobility of Y12G/P14L Pc within the complex. It is concluded that the double mutation shifts the complex further from the well-defined toward the encounter state.

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Dynamics in the Transient Complex of
Plastocyanin-Cytochrome f from Prochlorothrix hollandica
Rinske Hulsker,†Maria V. Baranova,‡George S. Bullerjahn,‡and
Marcellus Ubbink*,†
Leiden Institute of Chemistry, Leiden UniVersity, Gorlaeus Laboratories, P.O. Box 9502, 2300
RA Leiden, The Netherlands, Department of Biological Sciences, Bowling Green State
UniVersity, Bowling Green, Ohio 43403
Received September 27, 2007; E-mail: m.ubbink@chem.leidenuniv.nl
Abstract: The nature of transient protein complexes can range from a highly dynamic ensemble of
orientations to a single well-defined state. This represents variation in the equilibrium between the encounter
and final, functional state. The transient complex between plastocyanin (Pc) and cytochrome f (cytf) of the
cyanobacterium Prochlorothrix hollandica was characterized by NMR spectroscopy. Intermolecular
pseudocontact shifts and chemical shift perturbations were used as restraints in docking calculations to
determine the structure of the wild-type Pc-cytf complex. The orientation of Pc is similar to orientations
found in Pc-cytf complexes from other sources. Electrostatics seems to play a modest role in complex
formation. A large variability in the ensemble of lowest energy structures indicates a dynamic nature of the
complex. Two unusual hydrophobic patch residues in Pc have been mutated to the residues found in other
plastocyanins (Y12G/P14L). The binding constants are similar for the complexes of cytf with wild-type Pc
and mutant Pc, but the chemical shift perturbations are smaller for the complex with mutant Pc. Docking
calculations for the Y12G/P14L Pc-cytf complex did not produce a converged ensemble of structures.
Simulations of the dynamics were performed using the observed averaged NMR parameters as input. The
results indicate a surprisingly large amplitude of mobility of Y12G/P14L Pc within the complex. It is concluded
that the double mutation shifts the complex further from the well-defined toward the encounter state.
Introduction
Recent studies have shown the existence of dynamic encoun-
ter complexes in transient protein-protein and protein-DNA
interactions.1-3The encounter state (or encounter complex) is
thought to precede the well-defined (or single-orientation)
complex as illustrated in a two-step model for protein complex
formation (Figure 1). In earlier studies, transient protein
complexes were found to range from entirely dynamic to mostly
well-defined.4-12The complex of plastocyanin (Pc) and cyto-
chrome f (cytf) is an interesting example in this respect, because
comparative studies have shown that the degree of dynamics
within the complex varies strongly between species.
The soluble plastocyanin transfers electrons in oxygenic
photosynthesis between the membrane-bound cytochrome b6f
and photosystem I complexes.13,14Pc is a small (∼11 kDa) blue
copper protein, which contains a type I copper site for electron
transfer.15,16Cytf is a c-type heme containing cytochrome, of
which the truncated N-terminal soluble part (∼28 kDa) is used
for in vitro experiments (refs 17-19 and references therein).
Because of the transient nature of the complex all structures of
†Leiden University.
‡Bowling Green State University.
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Figure 1. Model for the formation of a protein complex.2Free proteins
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Published on Web 01/18/2008
10.1021/ja077453p CCC: $40.75 © 2008 American Chemical Society
J. AM. CHEM. SOC. 2008, 130, 1985-1991 9 1985

Page 2

Pc-cytf complexes determined so far have been determined by
NMR spectroscopy.4,8,20,21The first structure of the plant
complex revealed an electron-transfer pathway between the
hydrophobic patch surrounding the copper site of Pc and
heme ligand Tyr1 in cytf.8Kinetic studies in vitro13,22-27have
shown that electrostatics play an important role in complex
formation. In vivo studies suggest, however, that charged
residues are not relevant for fast electron-transfer reactions.28,29
Nonetheless, the structure of the plant complex showed that the
acidic “eastern” patch on Pc interacts with a set of basic residues
in the small domain of cytf. The results suggested that this
complex is mostly in a well-defined state. The structure of the
complex from cyanobacterium Nostoc sp. PCC 7119 (former
A. Variabilis) is similar to that from plants, yet the interaction
charges between Pc and cytf are interchanged.21The complex
of Phormidium laminosum Pc and cytf demonstrated that the
charge interactions are not absolutely necessary for a functional
complex. In this case, the proteins mostly interact through
hydrophobic contacts. The structure showed poorer convergence
to a well-defined state, suggesting more dynamics within the
complex.4
Plastocyanin from cyanobacterium Prochlorothrix hollandica
contains two unusual residues located in the hydrophobic patch,
which have been mutated to the residues normally found in these
positions (Tyr12Gly/Pro14Leu). The mutant Pc has been shown
to react differently with photosystem I30and was suggested to
be more dynamic in complex with cytf from Ph. laminosum.31
Here, we report the structure of the physiological complex
between P. hollandica Pc and cytf and show that the existing
equilibrium between encounter and well-defined state in the
complex is shifted toward the encounter state by the mutation
of two unusual hydrophobic patch residues (Y12G/P14L). It is
concluded that this complex is on the border between dynamic
and well-defined states.
Materials and Methods
Protein Expression and Purification. Mutant and wild-type15N-
labeled P. hollandica plastocyanin were expressed and purified as
described before.31Cytf was expressed and purified essentially as
described,32in which the coding sequence of the P. hollandica cytf
soluble domain (GenBank AF486288) was ligated in-frame to the Ph.
laminosum Pc leader coding sequence and expressed in Escherichia
coli. The purification procedure followed the protocol for the Ph.
laminosum protein.32Yields of pure cytf were typically 3 mg protein
per liter of culture.
Cd-Substitution of Pc. Cd-substitution of plastocyanin was es-
sentially done as described33with the following modifications. Of a
200 mM KCN, 500 mM Tris/HCl, pH 7.0 solution, 0.5 mL was added
to 0.5 mL of 1 mM oxidized Pc. The sample was then loaded on a
G25 Sephadex column pre-equilibrated with 1 mM CdCl2, 50 mM
HEPES, pH 7.0. The buffer was exchanged to water and then to 10
mM sodium phosphate, pH 6.0.
NMR Samples. All protein samples contained 10 mM sodium
phosphate, pH 6.0, 6% D2O. Protein concentrations were determined
by optical spectroscopy using ?602of 4.9 mM-1cm-1for oxidized Cu-
plastocyanin (PCu), ?554of 24.9 mM-1cm-1for reduced cytf, and ?278
of 7.6 mM-1cm-1for Cd-plastocyanin (PCd) (?278based on atomic
absorption measurements). The pH was adjusted with microliter aliquots
of 0.1 or 0.5 M HCl. Samples for assignment contained 1.0 mM15N-
labeled PCd. For titrations, both Cu(II) and Cd-substituted15N-labeled
Pc were concentrated to 0.2 mM. Wild-type PCd could not be used for
titrations because of unfolding in the presence of high amounts of cytf.
This was not the case for the mutant PCd, which is more stable in
both the Cu- and Cd-substituted form. The copper proteins were reduced
by addition of 2.0 mM ascorbate and flushed with argon to prevent
reoxidation. Aliquots of a 1.33 mM cytf stock solution were added to
the Pc containing samples. Samples for determination of PCS contained
85 µM15N-labeled wild type or mutant PCd and 50 or 100 µM oxidized
cytf, respectively. Cytf was subsequently reduced in the sample by
addition of 20 equiv of sodium ascorbate. The pH was measured before
and after each experiment.
NMR Spectroscopy. All NMR spectra were recorded at 300 K on
a Bruker DMX600 spectrometer equipped with a triple-resonance TCI-
ZGRAD ATM Cryoprobe (Bruker, Karlsruhe, Germany). Chemical-
shift perturbation and pseudocontact shift(s) (PCS) studies were
performed by acquiring15N,1H HSQC (heteronuclear single quantum
coherence spectra). Spectral widths of 40 ppm (15N) and 13.5 ppm (1H)
were used, and 1024 and 256 complex points were acquired in the
direct and indirect dimension, respectively. Cd-substituted wild-type
and mutant Pc resonances were assigned using 3D NOESY-HSQC and
3D TOCSY-HSQC experiments. Data were processed with AZARA
2.734and analyzed in ANSIG for Windows.35
Binding Curves and Chemical Shift Mapping. Averaged chemical
shift perturbations (∆δavg) were derived from the following equation:
2(
where ∆δNand ∆δHare the chemical shift perturbation after extrapola-
tion to the 100% bound state of the amide nitrogen and proton,
respectively.36Chemical shift titration curves were analyzed with a two-
parameter nonlinear least-squares global fit to a 1:1 binding model,
which corrects for dilution effects:11,23
where R is the [cytf]:[15N-Pc] ratio, ∆δbind is the chemical shift
perturbation at a given R, ∆δmaxis the chemical shift perturbation at
100% bound15N-Pc, P is the initial [15N-Pc], C is the stock concentra-
tion of cytf and Kais the association constant of the complex.
(20) Lange, C.; Cornvik, T.; Dı ´az-Moreno, I.; Ubbink, M. Biochim. Biophys.
Acta 2005, 1707, 179-188.
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Biol. Chem. 2005, 280, 18908-18915.
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1183.
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1996, 35, 14590-14598.
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∆δavg)?
1
∆δN
25
2
+ ∆δH
2)
(1)
∆δbind)1
2∆δmax(A - ?(A2- 4R)
A ) 1 + R +PR + C
(2a)
PCKa
(2b)
A R T I C L E S
Hulsker et al.
1986 J. AM. CHEM. SOC.9VOL. 130, NO. 6, 2008

Page 3

Structure Determination. The coordinates for P. hollandica Pc were
taken from the solution structure (PDB entry 1B3I37). Mutations Y12G
and P14L were introduced in silico, using DeepView/Swiss-PdbViewer
version 3.7.38A model of P. hollandica cytf based on Ph. laminosum
cytf (PDB entry 1CI339) was created with MODELLER 6v2.40Docking
of Pc onto cytf was done using restrained rigid body molecular dynamics
in XPLOR-NIH 2.9.9.41The coordinates of cytf were fixed, while Pc
was placed at a random position and allowed to move under the forces
of restraints and a van der Waals repel function. Only the interactions
between the backbone and C?atoms of Pc and all atoms of cytf were
considered at this stage. The restraints were divided into three classes.
Chemical shift perturbations in the presence of reduced cytf were
attributed to the proximity of the protein. The average relative solvent-
accessible surface area of each Pc residue was calculated with
NACCESS 2.1.1.42Residues with a surface accessible surface area of
more than 50% and ∆δbindg 0.1 (15N) or 0.02 (1H) were included in
the class for interface restraints. Pseudocontact and angle restraints based
on PCS were defined as described.8Residues that do not experience a
PCS were included in a minimal distance restraint class. A summary
of the restraints is given in Table 1. The product of the number of
restraints and the scale used in the calculations indicates the weight of
each group. The rigid-body molecular dynamics was essentially done
as described before.4,8,20,21A run comprised 3000 cycles, each of 1000
steps. Structures below an energy threshold were saved, yielding ∼200
structures per run. To obtain multiple independent dockings during a
run Pc was randomly displaced after having reached an energy
minimum, with energies changing less than 2-fold during 10 cycles.
Approximately 130 displacements occurred per run. The resulting
structures were ranked according to total restraint energy, and the lowest
energy structures were subjected to energy minimization of the side
chains in the interface. This ensemble of 20 lowest energy structures
has been deposited in the Protein Data Bank (entry 2P80). Figures 3,
5, and 8 were made in PyMOL v0.98.43The buried interface area was
calculated using NACCESS.
Pseudocontact Simulations. The PCS simulations were done using
XPLOR-NIH. The lowest energy structure of wild-type Pc-cytf was
used as the initial orientation of the Y12G/P14L Pc-cytf complex. The
relative diffusional movement in the mutant complex of Pc-cytf was
decomposed into two types of rotations. Pc was rotated around its center
of mass (wobble) and around the origin of the magnetic susceptibility
tensor frame; the heme iron in cytf (rotation). For this purpose three
pseudoatoms, representing the magnetic susceptibility tensor were
placed at 2 Å from the heme metal center in cytf. They were used as
a reference frame with the ?zz component of the tensor placed along
the Fe-Tyr1N vector. Each rotation was again decomposed in three
directions (X, Y, and Z), resulting in sets of six variables for the complete
movement. For a given set (for example, 40°, 80°, 35°/60°, 60°, 60°)
an ensemble of 50 structures was created by six rotations over random
angles within the range given for each of the six variables (i.e., between
0° and 40° for the first angle, and so on), and this procedure was
repeated 50 times. For such an ensemble the average PCS was
calculated for each Pc nucleus. The equation used to calculate PCS,
assuming an axial magnetic susceptibilty tensor, oriented along the Fe-
Tyr1N vector8is
where ∆δPCis the size of the PCS in ppm, r is the distance (Å) from
the Pc nucleus to the iron, and θ is the angle between the nucleus, the
iron, and the nitrogen of the N-terminal amino group of cytf. F reflects
the fraction Pc in complex with cytf; the value used in these simulations
is 0.7. NA is Avogadro’s number and ?ax the size of the magnetic
susceptibility tensor, taken to be 2.0 × 10-8m3mol-1, on the basis of
other c-type cytochromes.44
If the rotations caused the structures to either clash or not touch,
the distance was increased or decreased in steps of 1 Å, respectively,
until both proteins were just in contact. To determine the correlation
between the observed and simulated PCS the Q-factor was calculated
with the following equation:
where ∆δPC
size of the average simulated PCS for residue i.
obsis the size of the observed PCS in ppm and ∆δPC
simis the
Results and Discussion
Complex of P. hollandica PCu and cytf: Wild-Type versus
Y12G/P14L Pc. To compare the effects of binding, chemical
shift perturbations were analyzed for wild-type and Y12G/P14L
P. hollandica Pc upon titration with cytf. The presence of
reduced cytf gives rise to distinct changes in the1H-15N HSQC
spectrum of15N-PCu(I). A single averaged resonance was
observed for each amide indicating that exchange between free
and bound Pc is fast on the NMR-time scale. The observed
chemical shift changes (∆δbind) of the most affected residues
were plotted against the molar ratio of cytf:
2) and fitted to a 1:1 binding model.23This yields a Kaof 25
((2) × 103M-1for wild-type PCu and 20 ((1) × 103M-1for
Y12G/P14L PCu. Although the binding constants are very
similar, there is a significant difference in the size of the
chemical shift perturbations. This becomes apparent when the
average chemical shift perturbations (∆δavg) per residue are
considered (Figure 3). From the figure, it is clear that the
hydrophobic patch surrounding the copper site is the main site
involved in complex formation, as seen in Pc-cytf complexes
from other organisms.4,8,20,21,45The residues that are affected
in the wild-type complex are generally also affected in the
mutant complex, but the size of the chemical shift changes is
clearly smaller, by about 40%. This is comparable to the results
obtained for the complex of P. hollandica Pc and cytf from Ph.
laminosum,31and suggests increased dynamics in the complex
of Y12G/P14L Pc and cytf. A comparison of the size of the
PCS found for the complex with wild type and mutant Pc (see
later, Figure 7A) supports this notion.
15N-PCu (Figure
(37) Babu, C. R.; Volkman, B. F.; Bullerjahn, G. S. Biochemistry 1999, 38,
4988-4995.
(38) Guex, N.; Peitsch, M. C. Electrophoresis 1997, 18, 2714-2723.
(39) Carrell, C. J.; Schlarb, B. G.; Bendall, D. S.; Howe, C. J.; Cramer, W. A.;
Smith, J. L. Biochemistry 1999, 38, 9590-9599.
(40) Sali, A.; Blundell, T. L. J. Mol. Biol. 1993, 234, 779-815.
(41) Schwieters, C. D.; Kuszewski, J. J.; Tjandra, N.; Clore, G. M. J. Magn.
Reson. 2003, 160, 65-73.
(42) Hubbard, S. J. and Thornton, J. M. NACCESS; Department of Biochemistry
and Molecular Biology, University College London: London, 1993.
(43) DeLano, W. L. The PyMOL Molecular Graphics System 2002; DeLano
Scientific LLC: Palo Alto, CA, 2002.
(44) Worrall, J. A. R.; Kolczak, U.; Canters, G. W.; Ubbink, M. Biochemistry
2001, 40, 7069-7076.
(45) Crowley, P. B.; Ubbink, M. Acc. Chem. Res. 2003, 36, 723-730.
Table 1. Restraint Groups
type numberscale number × scale
interface
pseudocontact
minimal distance
angle
22
36
94
36
10
10
220
360
2823
aa
aScaling of the angle restraints is not comparable to the other (distance)
restraints.
∆δPC)
1036F
12πNAr3?ax(3 cos2θ - 1) (3)
Q ) [∑
i
{∆δPC
obs(i) - ∆δPC
sim(i)}2/∑
i
∆δPC
obs(i)2]1/2
(4)
Dynamics in the complex plastocyanin−cytochrome f. A R T I C L E S
J. AM. CHEM. SOC. 9 VOL. 130, NO. 6, 2008 1987

Page 4

The affinity between Pc and cytf decreases with increasing
ionic strength (Figure 4). A reduction of ∆δbindof 46% at 200
mM NaCl is observed, indicating electrostatics play a role in
complex formation. The Pc Y12G/P14L complex is similarly
affected by ionic strength, with ∆δbindreduced by 42%. This
demonstrates that the effect of ionic strength on complex
formation is similar in the wild-type and mutant complexes.
Structure of the Complex of P. hollandica Pc-cytf. The
structure of P. hollandica Pc-cytf complex has been determined
by rigid-body structure calculations using restraints obtained
by NMR spectroscopy. Two types of NMR data were used. One
is chemical shift perturbations of solvent exposed Pc residues,
which give information on the proximity of these residues to
cytf. The other is intermolecular pseudocontact shifts, which
are observed in the presence of paramagnetic, oxidized cytf and
give both distance and angular information on the proximity of
Pc residues to the Fe(III).8To be able to study the interaction
of Pc with both oxidized and reduced cytf without interference
from electron-transfer reactions, the Cu in Pc was substituted
with Cd (PCd).
A titration followed by1H-15N HSQC spectra showed that
the complex of PCd Y12G/P14L has a Kaof 26 ((3) × 103
M-1, similar to that of the complex with PCu Y12G/P14L. The
observed ∆δavg in the mutant PCd complex are identical for
forty percent of the perturbed residues, while for residues in
the vicinity of the metal site and the “eastern patch” ∆δavgvalues
differ between Pc containing Cu(I) and Cd(II). Similar differ-
ences have been observed before for Ph. laminosum Cd-
substituted Pc in complex with cytf46and are most likely caused
by the charge difference between the metals. Comparison of
∆δavgor Kabetween the wild type PCu and PCd complexes
was not possible because of experimental limitations (see
Materials and Methods).
The rigid body calculations with the restraints summarized
in Table 1 converge to an ensemble of complexes, which is
depicted by an overlay of the twenty lowest energy structures
in Figure 5A. The ensemble is characterized by a Cu-Fe
(46) Crowley, P. B. Transient protein interactions of photosynthetic redox
partners. Ph. D. Thesis. Leiden University, Leiden, the Netherlands, 2002.
Figure 2. Binding curves for complex formation between P. hollandica
Pc and cytf. The |∆δbind| of individual residues is plotted as a function of
the cytf:Pc ratio. Global nonlinear least-squares fits (solid lines) to a 1:1
binding model23yielded a Kaof 25 ((2) × 103M-1for wild-type PCu (A)
and 20 ((1) × 103M-1for Y12G/P14L Pc (B).
Figure 3. Surface representations of (A) P. hollandica Pc (PDB file 1BI3)
and (B) a model of P. hollandica Pc Y 12G/P14L. Average chemical shift
perturbations (∆δavg) for PCu are color coded as follows: blue, ∆δavge
0.025 ppm; yellow, ∆δavgg 0.025 ppm; orange, ∆δavgg 0.10 ppm; and
red, ∆δavgg 0.175 ppm.
Figure 4. Salt dependence of the |∆δbind| of the most shifted residues of
wild-type PCu (A) and Y12G/P14L PCu (B) in complex with cytf (the ratio
cytf:PCu is 3:1). Symbols indicate the presence of 0 mM NaCl (9), 50
mM NaCl (b), 100 mM NaCl (1), and 200 mM NaCl (2).
A R T I C L E S
Hulsker et al.
1988 J. AM. CHEM. SOC.9VOL. 130, NO. 6, 2008

Page 5

distance of 13.4 ( 1.4 Å. The average positional rmsd from
the mean structure of the Pc backbone atoms in the 20 lowest
energy structures is 4.6 ( 2.7 Å. This variability is mainly due
to a relative translational displacement of the Pc on the cytf
surface. Violation analysis of both angles and PCS (Figure 6)
shows that there is a large degree of variation between the
structures. In structure calculations of other Pc-cytf complexes
better convergence was observed using similar input.8,20,21
Therefore, we believe the limited convergence is an indication
of real dynamics rather than a lack of sufficient restraints. The
observed restraints in this case represent an average that can be
approximated by an ensemble of structures. For residues in the
loop regions 35-41 and 46-51 of Pc, negative PCS are
predicted in part of the structures. This is related to the angle
between the nucleus-iron vector and the Fe-Tyr1 N bond, which
exceeds 54.7°, resulting in a sign change of the PCS.47Both
the rmsd and violations for the ensemble indicate that the P.
hollandica Pc-cytf complex is much more dynamic than the
plant and Nostoc sp. PCC 7119 complexes8,21and more closely
resembles the highly dynamic P. laminosum complex.4
The orientation of wild-type Pc in the lowest energy P.
hollandica Pc-cytf complex (Figure 5B) is reminiscent of the
orientation found for Nostoc sp. PCC 7119.21The binding
interface comprises 15 Pc residues, all located in the hydro-
phobic patch, including Tyr12 and Pro14. The buried interface
area for Pc is calculated to be ∼860 Å2. Similarly, 15 cytf
residues and the propionate side chains of the heme contribute
to a buried interface area of ∼725 Å2. Although the chemical
shift changes in the complex are salt dependent (see earlier)
the only charged residues in the interface are Asp63 in cytf,
which interacts with Thr58 in Pc and Arg86 in Pc, which could
interact with Tyr162 in cytf. Some polar residues are present in
the interface, mainly on the cytf side, but clear electrostatic
contributions as seen in the plant and Nostoc Pc-cytf complexes
are not found. The lack of interaction between the eastern patch
on Pc and the small domain of cytf as found in the plant and
Nostoc sp. PCC 7119 complex could account for the more
dynamic nature of the P. hollandica complex. Such interactions
are lacking in the Ph. laminosum Pc-cytf complex, which is
also very dynamic. In the lowest energy structure the coupling
pathway for electron transfer comprises the heme ligand Tyr1
and the solvent-exposed copper ligand His85. This pathway has
also been found in plant Pc-cytf complexes.8,20It has to be
(47) Arnesano, F.; Banci, L.; Piccioli, M. Q. ReV. Biophys. 2005, 38, 167-
219.
Figure 5. Structure of P. hollandica Pc-cytf complex: (A) Superposition of the 20 lowest energy conformers. Cytf is shown as a red ribbon, the heme as
sticks, and the Fe ion as a sphere; the backbone of Pc is shown as a black CRtrace, with the copper ion as a gray sphere. (B) Lowest energy representation,
Pc is shown as a blue ribbon, with the Cu ion in magenta. Copper ligand His85 and heme ligand Tyr1 are shown as sticks.
Figure 6. Violation analysis for the wild-type Pc-cytf complex. The top
panel shows the observed PCS (b) and the back-calculated PCS (O) for
the backbone amide atoms in the 20 lowest energy structures. For every
residue the15N value is shown (major tick), followed by the1H value (minor
tick). The bottom panel shows the back-calculated angles (0) between the
nucleus, the heme iron, and the Tyr1 N atom. Positive PCS are expected to
give an angle <54.7° (solid line).
Dynamics in the complex plastocyanin−cytochrome f.
A R T I C L E S
J. AM. CHEM. SOC. 9 VOL. 130, NO. 6, 2008 1989

Page 6

noted that because of the variation in the ensemble a detailed
analysis of electron-transfer pathways is not possible.
Dynamics in P. hollandica Y12G/P14L Pc-cytf Complex.
When the PCS in the wild type and Y12G/P14L Pc-cytf
complexes are compared a clear decrease in their size for the
mutant complex can be observed (Figure 7A), except for the
last 10 residues. The decrease in size of chemical shift
perturbations (see earlier) and PCS in the mutant complex leads
to less and weaker restraints, which in turn cause more possible
orientations with similar energies. As a result, rigid-body
structure calculations for the P. hollandica Y12G/P14L Pc-
cytf complex did not lead to any converged ensemble of
structures, contrary to the case of the wild-type complex. The
inhomogeneous decrease in PCS and lack of convergence of
the calculations can be attributed to a more dynamic nature of
the Y12G/P14L Pc-cytf complex.
Simulations were done to determine the degree of movement
in the mutant complex sufficient to result in the observed
averaged PCS (Figure 7A). The orientation of the lowest energy
complex between wild-type Pc and cytf was used as a starting
point. Rotation of Pc around the Fe axis in each direction was
analyzed, an example for 60° is shown in Figure S1. It was
concluded that rotation in a single direction does not result in
an ensemble of orientations that closely matches the observed
and simulated PCS. The effect of rotation around the center of
mass of Pc Y12G/P14L (wobble) of various degrees was
analyzed as well (Figure S2). This movement clearly affects
the overall size of the average PCS. It has to be noted though,
that the chemical shift perturbation map indicates that the
binding site is localized mostly at the hydrophobic patch. Thus,
an ensemble of orientations that results from this movement
over for example 180° is unrealistic. By trying systematicly
combinations of rotation and wobbling, it was found that rotation
of Pc around the Fe with amplitudes of 40°, 80°, and 35° in the
x-, y-, and z-direction, respectively, combined with rotation
around the center of mass of Pc Y12G/P14L (wobble) of 60°
in all directions results in an ensemble of orientations with
average PCS values that closely resemble those observed (Figure
7B). This solution is not unique; there are more combinations
of variables that lead to similar results. For example rotation of
60°, 90°, and 45° in the x-, y- and z-direction, respectively,
combined with rotation around the center of mass of Pc Y12G/
P14L (wobble) of 60° in all directions was found to result in
very similar average PCS and a similar spread in Pc positions
Figure 7. (A)1H PCS in wild type and mutant Pc in complex with cytf; (B) observed and simulated1H PCS in mutant Pc in complex with cytf.
Figure 8. Representation of the dynamics in the Pc Y12G/P14L-cytf complex. Cytf is shown as a red ribbon, the heme as sticks, and the Fe ion as a sphere.
The Cu ion in a set of 50 Pc Y12G/P14L molecules is shown as magenta spheres. The two most extreme orientations of Pc Y12G/P14L are shown as blue
ribbons.
A R T I C L E S
Hulsker et al.
1990 J. AM. CHEM. SOC.9VOL. 130, NO. 6, 2008

Page 7

(Figure S3). Some Pc Y12G/P14L residues have deviating PCS
values in all simulations. These residues (33-37 and 57-59)
are found in the interface and are also the most violated in the
wild-type structure (Figure 6), which suggests that these
deviations are not specific for the Y12G/P14L Pc-cytf complex.
It must be noted, however, that residues 33-37 are found to be
the most affected by the mutations Y12G/P14L apart from
residues neighboring the mutations,31perhaps indicating a
structural difference in this part of the mutant Pc, as compared
to the wild type.
The 50 orientations of Pc Y12G/P14L that result from the
simulation mentioned above are visualized in Figure 8. From
these simulations, it is clear that a considerable range of
orientations is sampled in the mutant complex. It also demon-
strates that merely the observation of PCS cannot be used as
evidence for a well-defined complex. The Cu-Fe distances are
mostly too large for electron transfer, so only a subset of states
will be suitable for electron transfer. Since the wild-type and
mutant complex have similar binding constants and the electron-
transfer rates to Pc are similar (k2 ) 2-3 × 108M-1s-1;
Baranova and Bullerjahn, manuscript in preparation), it provides
an example of two complexes that differ mainly in their
dynamics.
It can be concluded that the mutation of the two hydrophobic
patch residues Tyr12 and Pro14 to the residues found in other
plastocyanins, results in a more dynamic complex of Pc and
cytf. These mutations provide a way to compare the reasonably
well-defined wild-type Pc-cytf complex with one that is more
dynamic, presenting an opportunity to examine the movements
and dynamics in the encounter state of a transient protein-
protein complex.
Acknowledgment. C. Erkelens and F. Lefeber are gratefully
acknowledged for assistance with the NMR experiments. R.H.
and M.U. acknowledge financial support from The Netherlands
Organisation for Scientific Research, VIDI-Grant 700.52.425.
G.S.B. was supported under award 0070334 from the National
Science Foundation.
Supporting Information Available: Three figures showing
results of PCS simulations for various sets of rotation angles.
This material is available free of charge via the Internet at
http://pubs.acs.org.
JA077453P
Dynamics in the complex plastocyanin−cytochrome f.
A R T I C L E S
J. AM. CHEM. SOC. 9 VOL. 130, NO. 6, 2008 1991