S uc u e o he Complex be ween Plas ocyanin and Cy och ome
om he Cyanobac e ium Nos oc sp. PCC 7119 as De e mined by
Pa amagne ic NMR
THE BALANCE BETWEEN ELECTROSTATIC AND HYDROPHOBIC INTERACTIONS WITHIN THE TRANSIENT
COMPLEX DETERMINES THE RELATIVE ORIENTATION OF THE TWO PROTEINS*□S
Recei ed o publica ion, No embe 25, 2004, and in e ised o m, Feb ua y 3, 2005
Published, JBC Pape s in P ess, Feb ua y 10, 2005, DOI 10.1074/jbc.M413298200
I ene Dı´az-Mo eno‡§, An onio Dı´az-Quin ana‡, Miguel A. De la Rosa‡, and Ma cellus Ubbink§¶
F om he ‡Ins i u o de Bioquı´mica Vege al y Fo osı´n esis, Uni e sidad de Se illa y Consejo Supe io de In es igaciones
Cien ı´ icas, A da. Ame´ ico Vespucio 49, 41092 Se illa, Spain, and §Leiden Ins i u e o Chemis y, Leiden Uni e si y,
Go laeus Labo a o ies, P. O. Box 9502, 2300 RA Leiden, The Ne he lands
The complex be ween cy och ome and plas ocyanin
om he cyanobac e ium Nos oc has been cha ac e ized
by NMR spec oscopy. The binding cons an is 16 mM
ⴚ1
,
and he li e ime o he complex is much less han 10 ms.
In e molecula pseudo-con ac shi s obse ed o he
plas ocyanin amide nuclei, caused by he heme i on, as
well as he chemical-shi pe u ba ion da a we e used
as he sole expe imen al es ain s o de e mine he o i-
en a ion o plas ocyanin ela i e o cy och ome wi h a
p ecision o 1.3 Å. The da a show ha he hyd ophobic
pa ch su ounding y osine 1 in cy och ome docks he
hyd ophobic pa ch o plas ocyanin. Cha ge complemen-
a i ies a e ound be ween he ims o he espec i e
ecogni ion si es o cy och ome and plas ocyanin. Sig-
ni ican di e ences in he ela i e o ien a ion o bo h
p o eins a e ound be ween his complex and hose p e-
iously epo ed o plan s and Pho midium, indica ing
ha elec os a ic and hyd ophobic in e ac ions a e bal-
anced di e en ly in hese complexes.
In oxygen-e ol ing pho osyn he ic o ganisms, ligh -d i en
ATP syn hesis equi es he pa icipa ion o cy och ome b
6
complex (1, 2), which couples p o on ansloca ion ac oss he
hylakoid memb ane o he elec on anspo be ween PSI
1
and PSII (3). In he cy och ome b
6
complex, cy och ome (C )
ans e s elec ons om he Rieske i on sul u clus e o a
soluble me allop o ein ha ac s as he elec on dono o he
P700 co ac o o PSI. The C subuni consis s o a ⬃28-kDa
N- e minal soluble pa ancho ed o he memb ane by a C-
e minal helix (4). I ep esen s an a ypical c- ype cy och ome
because o bo h i s
␤
-shee seconda y s uc u e and he un-
usual heme axial coo dina ion (5). The long axis o he soluble
pa is il ed ela i e o he memb ane no mal, and he heme is
o ien ed app op ia ely o app oach o he Rieske p o ein om
he memb ane side and o plas ocyanin (Pc) om he luminal
side (6, 7).
Pc is he mos ubiqui ous elec on ca ie be ween C and
P700 (8). I is a ype I cup edoxin (9) ha consis s o an
an i-pa allel
␤
-sandwich s uc u e wi h a single coppe a om
(10–12) ha is coo dina ed by wo ni ogen a oms and wo
sul u a oms om highly conse ed esidues.
In addi ion o i s physiological ele ance, he elec on ans-
e eac ion be ween C and Pc ep esen s an excellen case o
s udy he ansien na u e o p o ein in e ac ions in elec on
ans e chains (13). The li e ime o his kind o complexes is on
he o de o 1 ms o less. Due o he la ge amoun o unc ional
da a a ailable, his eac ion has become a e y use ul model o
es heo e ical app oaches o he p edic ion o s uc u es o
p o ein-p o ein complexes (14–20).
In plan s his eac ion shows as kine ics a 100 mMionic
s eng h (⬎10
8
M
⫺1
s
⫺1
) despi e bo h i s modes binding con-
s an (⬃7m
M
⫺1
) unde hese condi ions and he small di e -
ence in edox po en ial (20 mV) be ween dono and accep o
(21, 22). The mechanism o his elec on ans e eac ion has
been s udied wi h se e al echniques (22). Such s udies sup-
po he impo ance o elec os a ic in e ac ions in ol ing he
acidic pa ches (“si e 2”) on Pc and he basic esidues o C o
binding unde in i o condi ions (23–26) and he essen ial ole
o speci ic esidues in he hyd ophobic pa ches o bo h C (27)
and Pc (26). No ably, he ele ance o he elec os a ic in e ac-
ions could no be con i med in i o (15). In he sys em om
Pho midium laminosum, he only cyanobac e ium o which
he kine ics o he educ ion eac ion ha e been analyzed so a ,
he elec os a ic e ec s appea o be weake and less op imized
compa ed wi h plan s (28, 29).
The solu ion s uc u es o wo Pc-C complexes ha e been
ob ained by dissec ing he diamagne ic and pa amagne ic con-
ibu ions o he chemical-shi pe u ba ions o Pc esonances
upon C binding. The i s one (PDB en y 2PCF) co esponds
o he complex be ween spinach Pc and u nip C (30), and he
second co esponds o he p o eins om P. laminosum (31).
Bo h s uc u es show modes in e ace a eas (600–850 Å
2
pe
p o ein). Mo eo e , in bo h cases he hyd ophobic pa ch o Pc
* This wo k was suppo ed by P og am Human Po en ial and Mobil-
i y o Resea che s o he Eu opean Commission Con ac HPRN-CT-
1999-00095 “T ansien Ne wo k,” Spanish Minis y o Educa ion, Cul-
u e, and Spo G an AP2000-2937, Spanish Minis y o Science and
Technology G an BMC2003-00458, he Andalusian Go e nmen G an
CVI-0198, and Ne he lands O ganiza ion o Scien i ic Resea ch G an
700.52.425. The cos s o publica ion o his a icle we e de ayed in pa
by he paymen o page cha ges. This a icle mus he e o e be he eby
ma ked “ad e isemen ” in acco dance wi h 18 U.S.C. Sec ion 1734
solely o indica e his ac .
□SThe on-line e sion o his a icle (a ailable a h p://www.jbc.o g)
con ains supplemen al ma e ial.
The a omic coo dina es and s uc u e ac o s (code 1TU2) ha e been
deposi ed in he P o ein Da a Bank, Resea ch Collabo a o y o S uc-
u al Bioin o ma ics, Ru ge s Uni e si y, New B unswick, NJ
(h p://www. csb.o g/).
¶To whom co espondence should be add essed. Tel.: 31-71-527-
4628; Fax: 31-71-527-4349; E-mail: [email p o ec ed].
1
The abb e ia ions used a e: PSI, pho osys em I; PSII, pho osys em
II; C , wa e -soluble agmen o cy och ome ; HSQC, he e onuclea
single-quan um cohe ence; Pc, plas ocyanin; PCd, cadmium plas ocya-
nin; PCS, pseudo-con ac shi s; .m.s.d., oo mean squa e de ia ion;
WT, wild ype.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 19, Issue o May 13, pp. 18908–18915, 2005
© 2005 by The Ame ican Socie y o Biochemis y and Molecula Biology, Inc. P in ed in U.S.A.
This pape is a ailable on line a h p://www.jbc.o g18908
This is an Open Access a icle unde he CC BY license.
(“si e 1”) lies nea Ty -1 o C , hus p o iding an app op ia e
en i onmen o e icien elec on ans e owa d he coppe
a om h ough he exposed coppe -coo dina ing His esidue. In
addi ion o his, chemical-shi pe u ba ion da a ha e been
epo ed o se e al he e ologous plan and cyanobac e ial sys-
ems (32–34). Despi e hei simila i ies, signi ican di e ences
a e ound be ween he cyanobac e ial and he plan complexes.
In Pho midium, Pc binds C in a “head-on” con o ma ion in
which he hyd ophobic pa ch accoun s o he whole ecogni ion
in e ace in Pc, con a y o he “side-on” in e ace ha also
in ol es he acidic pa ches, which is ound in he plan com-
plex. Bo h kinds o complexes ha e been p edic ed in heo e -
ical s udies using he co-o dina es om plan p o eins (15–17).
The ionic s eng h dependences o he s uc u es sugges ha
in he plan complex elec os a ics play a dominan ole,
whe eas in Pho midium complex o ma ion is go e ned by he
hyd ophobic e ec .
I is known ha Pho midium is a he mophilic o ganism
(35). A highe ambien empe a u e could in luence he balance
be ween elec os a ic o ces and hyd ophobic e ec s, making
his complex unusual and di e en om ha in o he cya-
nobac e ia. Hence, i is unknown i di e ences be ween he
epo ed plan and Pho midium complexes a e applicable o all
cyanobac e ia. He e, he s uc u e o he complex be ween Pc
and C om ano he cyanobac e ium, Nos oc ( o me ly
Anabaena), has been de e mined. In e es ingly, he esul s
he ein p esen ed a e consis en wi h a single con o ma ion in
he ansien complex be ween Pc and C ha esembles he
cha ac e is ic side-on binding mode p esen in plan s ye has
an in e ace simila o ha ound in he Pho midium complex.
EXPERIMENTAL PROCEDURES
P o ein P epa a ion—Uni o mly (99%)
15
N-labeled Nos oc sp. PCC
7119 Pc was p oduced in Esche ichia coli JM109 ans o med wi h
pEAP-WT (36). The cul u e condi ions and pu i ica ion me hods will be
published elsewhe e (64). Cadmium subs i u ion o he coppe in plas-
ocyanin was pe o med as published (37) excep ha a PD-10 column
(Ame sham Biosciences) p e-equilib a ed wi h a solu ion o 50 mM
HEPES, pH 7.0, con aining 1 mMCdCl
2
was used ins ead o a Sephadex
G25 gel il a ion column.
The soluble pa o Nos oc sp. PCC 7119 C was p oduced in E. coli
DH5
␣
ans o med wi h bo h pEC86, con aining he c- ype cy och ome
ma u a ion casse e (38), and an exp ession ec o o C , pEAF-WT,
ob ained by inse ion o a chime ic pe A gene in pBluesc ip II (S a -
agene). This chime ic gene coded o a usion p o ein wi h C unca ed
a he C e minus (a posi ion 253) and he signal pep ide o cy och ome
c
6
(36). Cells we e g own in LB medium wi h 100
␮
g/ml ampicillin, 12
␮
g/ml chlo amphenicol, and 6 mg/ml Fe(NH
4
)
3
ci a e unde semi-
anae obic condi ions (39) a 35.5 °C, 150 pm o 32 h up o an A
600
o
1.3. P o ein yields up o 1.5 mg/li e we e ob ained in his manne . The
pu i ica ion p ocedu e used o C exp essed om pEAF-WT will be
desc ibed elsewhe e.
2
NMR Sample P epa a ion—Pc and PCd p o ein solu ions we e con-
cen a ed o he equi ed olume by ul a il a ion me hods (Amicon,
YM3 memb ane) and exchanged in o 10 mMsodium phospha e, pH 6.0,
H
2
O/D
2
O 95:5 solu ions. P o ein concen a ions we e de e mined by
abso p ion spec opho ome y using a
⑀
598
o 4.5 mM
⫺1
cm
⫺1
o he
oxidized o m o Pc and a
⑀
278
o 5.5 mM
⫺1
cm
⫺1
o PCd. The PCd
⑀
278
was es ima ed using p o ein concen a ion alues om B ad o d as-
says. A A
278
/A
598
a io o 1.0 o he oxidized Pc indica ed su icien
pu i y o cha ac e iza ion by NMR. The s ock concen a ions we e 2.0
mM
15
N-labeled Pc and 2.7 mM
15
N-labeled PCd.
The soluble domain o C was concen a ed using Amicon YM10
memb ane and exchanged in o 10 mMsodium phospha e, pH 6.0, 3 mM
sodium asco ba e, H
2
O/D
2
O 95:5 solu ions. The concen a ion de e mi-
na ion was based on op ical spec oscopy using an
⑀
556
o 31.5 mM
⫺1
cm
⫺1
o he educed C (30). A 3.7 mM e ous C s ock solu ion wi h a
A
278
/A
598
a io o 0.9 was used. C was kep in a educed o m wi h a ew
equi alen s o sodium asco ba e and was s able in his o m o days.
The e ic o m was p epa ed by he addi ion o a 5- old excess o
po assium e icyanide (K
3
[Fe(CN)
6
]) ollowed by gel il a ion (Ame -
sham Biosciences Supe dex G75) o emo e e ocyanide. Comple e
oxida ion was e i ied by he disappea ance o he abso p ion band a
556 nm. Then, a 2.0 mM e ic C s ock solu ion was p epa ed.
NMR Spec oscopy—All NMR expe imen s we e pe o med on a
B uke DMX 600 NMR spec ome e ope a ing a 298 K. The
1
H and
15
N assignmen s o educed Nos oc Pc assignmen s we e aken om
Badsbe g e al. (40). Fo sequence-speci ic assignmen o he backbone
amide esonances o PCd (Supplemen al Table S2), a wo-dimensional
1
H,
15
N HSQC (41), wo-dimensional
1
H,
15
N HSQC nuclea O e hause
enhancemen spec oscopy wi h 150 ms mixing ime, and wo-dimen-
sional
1
H,
15
N HSQC o al co ela ion spec oscopy wi h 80-ms mixing
ime spec a we e eco ded.
The e ec s o complex o ma ion on PCd we e ollowed by acqui ing
wo-dimensional
1
H,
15
N HSQC spec a du ing i a ions o aliquo s o a
3.7 mM e ous o 2.0 mM e ic C solu ion in o a solu ion o 0.2 mM
15
N-labeled PCd. The spec al wid hs we e 32.0 ppm (
15
N) and 12.0 ppm
(
1
H), and 256 and 1024 complex poin s we e acqui ed in he indi ec and
di ec dimensions, espec i ely. Fo measu emen s o he pseudo-con-
ac shi s (PCS)
1
H,
15
N HSQC spec a o ee Pc, he oxidized complex
and he educed complex we e acqui ed, always on he same sample.
Fe ic C om a s ock solu ion was added o a
15
N-labeled PCd sample
wi h inal concen a ions o 0.35 and 0.50 mM, espec i ely. C was
educed wi h 10 mol eq o a concen a ed sodium asco ba e solu ion.
Gi en he inal C concen a ion and he binding cons an , he pe cen -
age o Pc bound was calcula ed o be 55%.
All da a p ocessing was pe o med wi h AZARA (www.bio.cam.ac.uk/
aza a), and analysis o he chemical-shi pe u ba ions (⌬
␦
Bind
) wi h
espec o he ee p o ein was pe o med in Ansig (42–44). The spec a
we e calib a ed agains he in e nal s anda d [
15
N]ace amide (0.5 mM).
Binding Cu es—Ti a ion cu es we e ob ained by plo ing ⌬
␦
Bind
agains he mola a io o C
II/III
:PCd o he mos s ongly a ec ed
signals. Non-linea leas squa es i s o a 1:1 binding model (21) we e
pe o med in O igin 6.0 (Mic ocal Inc.). This model accoun s o he
dilu ion e ec o bo h p o eins du ing he i a ion, wi h he a io o C
and PCd and ⌬
␦
Bind
as he independen and dependen a iables,
espec i ely. The binding cons an (K
a
) and he maximum chemical
shi change (⌬
␦
max
) we e he i ed pa ame e s. A global i o he da a
was pe o med in which he cu es we e i ed simul aneously o a
single K
a
alue, whe eas he ⌬
␦
max
o each esonance was allowed o
a y.
Chemical Shi Mapping—The shi s obse ed in he complex PCd-C
II
wi h3eqo C we e ex apola ed o 100% bound o all esidues using
he K
a
ob ained om he i s. The a e age chemical-shi pe u ba ion
(⌬
␦
a g
) o each amide was calcula ed using he ollowing equa ion (45),
⌬
␦
a g ⫽
冑
(⌬
␦
N/5)2⫹⌬
␦
H2
2(Eq. 1)
in which ⌬
␦
N
is he change in he
15
N chemical shi , and ⌬
␦
N
is he
change in he
1
H chemical shi when he p o ein is 100% bound o C .
Res ain s Classes—De ails o he es ain s de ini ions a e p o ided
in he supplemen al ma e ial. B ie ly, ou g oups o es ain s we e
de ined. The in e ace es ain s ep esen he chemical-shi pe u ba-
ion da a o Pc nuclei (Supplemen al Table S1). These a e sa is ied
when he nuclei a e close he C su ace. Addi ional in e ace es ain s
we e de ined o se e as a weak an de Waals epel unc ion. PCS we e
used o de ine pseudo-con ac es ain s and angle es ain s acco ding
o he p ocedu e desc ibed in Ubbink e al. (30), and minimal dis ance
es ain s we e de ined o amide g oups ha did no expe ience a PCS.
Elec os a ic es ain s based on kine ic a he han NMR da a we e
used p e iously (30) o ep esen he elec os a ic a ac ion be ween
PCd and C . In he Nos oc complex, hese we e no used because he
NMR expe imen al da a we e su icien o ob ain a well de ined
s uc u e.
A summa y o he es ain g oups is lis ed in Table I. The p oduc
be ween he numbe o es ain s and he scaling ac o used in he
calcula ions indica es he impo ance o each es ain g oup. No e ha
he pseudo-con ac es ain s, which gi e quan i a i e in o ma ion, a e
dominan .
Nos oc C Homology Model—A homology model o C was buil using
he COMPOSER (46) module o SYBYL 6.5 (T ipos Inc.) using X- ay
di ac ion da a om B assica apa, PDB en y 1CTM ( esolu ion 2.30
Å (5)) and PDB en y 1HCZ ( esolu ion 1.96 Å (47)) as empla es.
Including he s uc u e o Pho midium C (PDB en y 1CI3 (48)) as he
empla e did no imp o e he model. Th ee sequence s e ches ( esidues
1–9, 13–183, and 198–254) we e conside ed as conse ed. These egions
2
C. Alba a´n, J. A. Na a o, F. P. Molina-He edia, P. del S. Mu doch,
M. A. De la Rosa, and M. He a´s, submi ed o publica ion.
Plas ocyanin-Cy och ome In e ac ions 18909
showed iden i ies o 66.7, 64.9, and 54.4%, espec i ely. Two loops
co esponding o esidues T p-4—Gln-6 and Ala-184—Val-197 we e
simula ed o alloca e 1- and 3- esidue inse ions, espec i ely, using he
TWEAK op ion. On a e age, he .m.s.d. o backbone a oms o he model
o C wi h espec o he abo e s uc u es was 0.56 Å. The s uc u e o
C om Mas igocladus (made a ailable only a e comple ion o ou
calcula ions, PDB en y 1VF5 (6)) shows a simila ex ension o he
small domain o C . The Nos oc C model shows a 1.16-Å .m.s.d. wi h
his s uc u e. The la ges di e ences co espond o esidues 184–197,
which show high B- ac o s in he Mas igocladus C c ys al s uc u e,
sugges ing ha his loop may be lexible.
S uc u e Calcula ions—S uc u e calcula ions we e pe o med us-
ing XPLOR-NIH Ve sion 2.9.1 (49, 50). The s uc u es o Nos oc Pc
(PDB en y 1NIN (40)) and he homology model o C we e ea ed as
igid bodies, and he co-o dina es o C we e ixed. PCd was placed a a
andom posi ion and allowed o mo e in a es ained igid-body molec-
ula dynamics calcula ion. None o he s anda d ene gy e ms was
used. Only he g oups o expe imen al es ain s desc ibed abo e we e
applied o dock he p o eins. Fi e housand cycles (see supplemen al
ma e ial) o calcula ions we e pe o med (9 h on a dual p ocesso
Pen ium IV PC unning unde LINUX). Only s uc u es wi h a o al
es ain s “ene gy” (E
o
) below a h eshold we e sa ed, yielding ⬃90
s uc u es. To assu e su icien sampling o he o ien a ion space, a
la ge andom displacemen o Pc occu ed when a (local) minimum had
been ound, as judged om a o al es ained ene gy ha had no
changed o mo e han 50% du ing 10 cycles. Abou 200 o such dis-
placemen s occu ed in a ep esen a i e un. As an illus a ion, E
o
has
been plo ed agains he cycle numbe in Fig. S2 o a sec o o one
ajec o y, co esponding o 200 cycles.
The esul ing s uc u es we e anked acco ding o o al es ain
ene gy and he op en s uc u es, wi h o al es ain ene gy alues
om 28 o 29 a bi a y uni s subjec ed o es ained ene gy minimiza-
ion o he side chains ollowed by a sho es ained igid body ene gy
minimiza ion, bo h using he XPLOR-NIH epulsi e an de Waals
e m wi h educed scaling. This la gely emo ed he collisions be ween
Pc and C a oms while main aining he low o al es ain ene gy alue.
The en bes s uc u es ha e been deposi ed in he P o ein Da a Bank
unde en y 1TU2. Bu ied su ace a eas ha e been calcula ed using
NACESS (51).
Elec on T ans e Pa hways—To de e mine he esidues ha could
be in ol ed in he elec on anspo , he bes elec on ans e pa hway
o each o he ene gy-minimized complex s uc u es was calcula ed
using G eenpa h Ve sion 0.971 (52). This p og am pe o ms a G een
unc ion analysis based on wo-s a e supe -exchange model (53). No
enhanced coupling was used o a oma ic ings. Fo ep esen a ion
pu poses, we selec ed all he coo dina es o he esidues ha appea in
any o hese pa hs ins ead o jus ep esen ing he bonds and jumps
in ol ed.
RESULTS AND DISCUSSION
Binding A ini y—To cha ac e ize he complex o Pc and C
om Nos oc,C was i a ed in o a solu ion o
15
N-labeled
Cd-subs i u ed Pc, PCd. The coppe in Pc was eplaced by he
edox inac i e subs i u e cadmium o allow o s udies wi h
bo h oxidized and educed C wi hou in e e ence om elec-
on ans e eac ions. The e ec s o he i a ions we e ol-
lowed wi h wo-dimensional HSQC expe imen s. Shi ing o
esonances du ing he i a ion indica ed ha binding and
dissocia ion we e as on he NMR imescale (⬎100 s
⫺1
). In Fig.
1, he chemical-shi pe u ba ions due o binding o educed C
(C
II
) a e plo ed o se e al esidues o PCd. The cu es clea ly
illus a e ha he chemical-shi pe u ba ions inc ease as a
unc ion o he C
II
concen a ion. A global i o he da a o a
1:1 binding model (21) yielded a binding cons an o 16 ⫾1⫻10
3
M
⫺1
, and he same a ini y was ob ained wi h oxidized C (K
A
⫽
16 ⫾2⫻10
3
M
⫺1
; da a no shown). This alue is sligh ly lowe
han ha ob ained o na i e (Cu(I)) Nos oc Pc (26 ⫾1⫻10
3
M
⫺1
(64)). Hence, i can be concluded ha he binding a ini y is
independen o he oxida ion s a e o C bu appea s o a y
sligh ly be ween Pc wi h a singly cha ged me al and a doubly
cha ged one.
In e ace Map—In Fig. 2, he size o he chemical-shi pe -
u ba ions o esidues a ec ed upon i a ion wi h C
II
a e
colo -coded on o he su ace o Pc. Pe u bed esidues map in
sequence s e ches 7–17, 32–44, 63–72, and 88–100. These
s e ches o m a la ge a ea comp ising esidues om bo h
classical binding si es (10, 25). Th ee p oline esidues, a posi-
ions 37, 38, and 91 (g ay), a e loca ed in he middle o he
in e ace, close o he coppe ligand His-92. In addi ion o he
main in e ac ion a ea, h ee isola ed esidues (Lys-51, Asp-54,
and Leu-55) unde go a signi ican pe u ba ion. These esidues
a e loca ed in a egion below si e 2, comp ising mainly cha ged
esidues ha ha e an impo an ole in he Nos oc complex
s uc u e, as is explained below. The pe u ba ion map o PCd
is simila o ha ound o Pc(Cu(I)) (64).
The o e all sizes o he pe u ba ions and he localized na-
u e o he binding map indica e ha he complex be ween PCd
and C is well de ined acco ding o he classi ica ion o well
de ined e sus dynamic complexes, sugges ed by Wo all e al.
(55) and P udeˆncio and Ubbink (56). Tha is, PCd adop s a
single p edominan con o ma ion du ing mos o he li e ime o
he complex. This is also suppo ed by he obse a ion o in e -
molecula pseudo-con ac e ec s, as explained below.
Expe imen al Res ain s and S uc u e Calcula ion—When
compa ing he pe u ba ions o Pc esonances obse ed wi h
educed and oxidized C , i is s iking ha in he la e case
many Pc nuclei expe ience an addi ional shi ha is o simila
size o
1
H and
15
N. This is illus a ed in Fig. 3. The op panel
shows he sizes o he pe u ba ions o
1
H and
15
N o each Pc
esidue in he p esence o educed C . I is clea ha nei he
size no sign co ela e be ween he wo ypes o nuclei. The
bo om panel shows he addi ional shi s ob ained when com-
FIG.1.Binding cu es o he in e ac ion o Nos oc PCd in he
p esence o educed C .⌬
␦
Bind
is plo ed agains he a io o C and
PCd. The da a we e i ed globally (non-linea , leas squa es) o a 1:1
binding model, yielding a binding cons an o 16 ⫾2⫻10
3
M
⫺1
.
TABLE I
Res ain s g oups
Res ain g oup Type Numbe o
es ain s Scaling Numbe ⫻scaling
In e ace Dis ance 41 5 205
Pseudo-con ac Dis ance 81 20 1620
Minimal dis ance Dis ance 90 10 900
Angle Angle 81
a
a
Scaling o he angle es ain s is no compa able wi h ha o dis ance es ain s.
Plas ocyanin-Cy och ome In e ac ions18910
pa ing Pc in he p esence o oxidized and educed C (⌬
␦
oxidized
-
⌬
␦
educed
). In his case he e is a clea co ela ion o he shi s,
sugges ing ha he addi ional shi s a e caused by in e molec-
ula PCS om he e ic heme i on on o Pc nuclei because hese
should be simila (in ppm) o he p o on and ni ogen nuclei o
a gi en amide. The s onges PCS a e ound in he hyd ophobic
pa ch egion, sugges ing ha his egion comes closes o he
heme i on in he complex.
The binding e ec s and he PCS can be used o de e mine he
o ien a ion o Pc ela i e o C in he complex. Bo h ypes o
shi s we e ansla ed in o es ain s o a igid-body docking
calcula ion. The binding shi s we e used in a quali a i e man-
ne ; any shi la ge han he h eshold yields a es ain ha
equi es ha he amide is b ough close o he su ace o C
(in e ace es ain s, Supplemen al Table S1). The PCS a e
used quan i a i ely; du ing he calcula ions, he PCS a e cal-
cula ed o he gi en posi ion o Pc and compa ed wi h he
expe imen al alues. I he di e ence is la ge han he e o
ma gins, he es ain is iola ed. The posi ion o Pc is changed,
and he PCS a e calcula ed again. Thus, in an i e a i e ashion,
he op imal posi ion, wi h minimal es ain iola ions is
ound. These a e called he pseudo-con ac es ain s (Supple-
men al Table S1). The sign o he PCS gi es in o ma ion abou
he angle be ween i on-nucleus ec o and he o ien a ion o
he magne ic suscep ibili y enso , hus p o iding angle e-
s ain s. The absence o a PCS can be used o de ine a minimal-
dis ance es ain , which is iola ed when he nucleus ge s oo
close o he heme i on. Also, hese es ain s a e e alua ed
i e a i ely du ing he calcula ions. All hese es ain s we e
used in a igid-body calcula ion o ob ain he o ien a ion o Pc
ela i e o C wi h minimal iola ions o he expe imen al
es ain s. Fo his pu pose he solu ion s uc u e o Pc (40)
and a homology model o C , based on i s conse ed amino acid
sequence, we e used. Apa om he expe imen al es ain
e ms, no o he o ces, such as elec os a ics, we e used, excep
o a weak an de Waals epel unc ion o a oid ex ensi e
collisions be ween he a oms o bo h p o eins. Fu he de ails
abou he de ini ions and numbe s o es ain s as well as he
calcula ions a e gi en unde “Expe imen al P ocedu es” and
he Supplemen al Ma e ial.
Resul s o he S uc u e Calcula ion—Two ypes o complexes
we e ob ained om he abo e es ained igid body calcula-
ions. All he s uc u es wi h he lowes sum o iola ions
(28.5 ⫾0.5 a bi a y uni s) clus e in one o ien a ion, wi h an
a e age posi ional oo mean squa e de ia ion o 1.3 ⫾0.6 Å
(“posi ional .m.s.d.”; see below), wi h espec o hei a e age
s uc u e (Fig. 4). The i on-cadmium dis ance was 16.2 ⫾0.1 Å.
In addi ion, an al e na i e se o s uc u es wi h ano he con-
o ma ion was ound (Supplemen al Fig. S3). These ha e a
highe sum o iola ions (38.9 ⫾0.3 a bi a y uni s) and, hus,
a much wo se i o he NMR da a, in pa icula o he in e -
ace es ain s, because he in e ac ion su ace is small and
does no in ol e he hyd ophobic pa ch. These s uc u es ha e
a signi ican ly la ge deg ee o a iabili y wi h a posi ional
.m.s.d. o 3.2 Å and la ge i on-cadmium dis ance (17.4 ⫾0.1
Å). Consequen ly, hese al e na i e complexes wi h la ge io-
la ions, a small in e ace, and lowe p edic ed e iciency in he
elec on ans e eac ion a e conside ed o be a non-physical
solu ion o he calcula ions and a e no conside ed u he .
Quali y o he S uc u e; Accu acy and P ecision—The qual-
i y o he 10 bes s uc u es was e alua ed on he basis o he
iola ions be ween he calcula ed and he expe imen ally ob-
se ed alues o each g oup o es ain s (see “Expe imen al
P ocedu es”). Wi h ega d o in e ace es ain s, sys ema ic
iola ions only appea o a ew amide nuclei, wi h he la ges
ound o Lys-20. The amides o hese esidues expe ience
small chemical-shi pe u ba ions upon binding ha may be
a ibu able o indi ec e ec s (30). Fo he minimal dis ance
es ain s, he e a e a ew small iola ions ha co espond o
Ile-101 in mos o he 10 s uc u es. This amino acid is a he
bounda y o esidues ha expe ience PCS.
The iola ions o he PCS es ain s can be e alua ed om
Fig. 5A. The obse ed (open symbols) and p edic ed (dashes o
he 10 bes s uc u es) PCS a e plo ed. Fo mos esidues,
obse ed and p edic ed PCS ag ee wi hin he e o ma gins.
Howe e , some iola ions a e obse ed, o Leu-13 (
15
N),
His-39 (
15
N and
1
H), Asn-40 side chain (
15
N and
1
H), Leu-65
(
15
N), Glu-90 (
1
H), His-92 (
15
N and
1
H), and A g-93 (
1
H), wi h
FIG.3.Chemical shi changes caused by binding e ec s (up-
pe panel) and PCS (lowe panel) a e plo ed o all esidues o
PCd. The PCS we e ob ained as desc ibed in he supplemen al ma e-
ial. The e o ba s ep esen he es ima ed expe imen al a ia ion in
chemical shi de e mina ion.
FIG.2.Chemical-shi pe u ba ion map o Nos oc PCd in he p esence o educed C .Residues a e colo ed acco ding o hei la ges
⌬
␦
a g
(ppm): blue o ⬍0.025, whi e o ⱕ0.050, o ange o ⱕ0.100, ed o ⱕ0.225. P olines a e indica ed in g ay. Residues a e iden i ied wi h he
single-le e amino acid code, and he su aces ha e been o a ed 90° a ound he e ical axis o each pic u e, wi h espec o he one on he le .
Su ace ep esen a ions we e gene a ed o he s uc u e o Nos oc Pc (PDB en y 1NIN, model 1; 40) using Swiss-PdbViewe e sion 3.7 (54).
Plas ocyanin-Cy och ome In e ac ions 18911
he la ges de ia ions o he wo His esidues, which a e e y
close o he i on. I is likely ha he necessa y assump ions on
he size, axiali y, and o ien a ion o he magne ic suscep ibili y
enso (see supplemen al ma e ial) a e he limi ing ac o s in
he accu acy o he s uc u es. The angles be ween he as-
sumed
␹
zz
di ec ion and he i on-nucleus ec o , as calcula ed
om he en bes s uc u es, a e plo ed in Fig. 5B. Posi i e
PCS co espond o an angle smalle han 54°, and nega i e
PCS ha e an angle la ge han ha . The angles obse ed in he
s uc u es a e all in ag eemen wi h he posi i e sign o he
PCS. The PCd posi ion ela i e o ha C is he mos ele an
ea u e in he p ecision o he complex s uc u e. A e igid-
body and side-chain ene gy minimiza ions o he 10 s uc u es,
he posi ional .m.s.d. is de e mined by aligning he C mole-
cules in each s uc u e and calcula ing he .m.s.d. o backbone
hea y a oms o Pc compa ed wi h he a e age s uc u e. The
a e age backbone posi ional .m.s.d. o PCd is 1.3 ⫾0.6 Å o
he 10 bes s uc u es. This p ecision is achie ed wi hou he
inpu o elec os a ic es ain s in s uc u e calcula ions as was
done in he case o he complex o plan p o eins (30) in which
he posi ional .m.s.d. was 1.05 Å. In he p esen case i can be
concluded ha he numbe o expe imen al es ain s, which is
la ge han in p e ious s udies, is su icien o esol e a well
de ined s uc u e.
Desc ip ion o he S uc u e—The complex is shown in Fig. 4
in ibbon and space- illing ep esen a ions. The binding in e -
ace in ol es he hyd ophobic a eas close o he me al cen e s
in bo h p o eins. In Pc i comp ises 14 esidues om he hy-
d ophobic pa ch (Leu-14, Val-36, P o-37, P o-38, Leu-64, Me -
66, P o-68, P o-91, His-92, and Ala-95), he neighbo ing Lys-
35, and om he adjacen egion o si e 2 (Lys-62, Gln-63, and
Glu-90). The bu ied a ea in his ecogni ion pa ch on Pc is 572
Å
2
. A second, mino ecogni ion si e on Pc co esponds o es-
idues Asp-54 and Lys-57. I accoun s o 41 Å
2
o he bu ied
su ace. This pa ch is clea ly in ol ed in elec os a ic in e ac-
ions wi h cha ged esidues a he small domain egion o C .
On C , he ecogni ion si e measu es 525 Å
2
, comp ising 20
esidues ha a e a leas pa ially bu ied du ing binding.
Th ee o he i e a oma ic esidues in his pa ch, namely Ty -1,
Phe-3, and Ty -102, ep esen ⬃31% o he ecogni ion su ace
o C . Mos esidues a he ecogni ion si e a e hyd ophobic o
pola , wi h cha ged esidues lying a he im o he in e ace.
FIG.4.S uc u e o he Nos oc sp. PCC 7119 PCd-C complex. On he le he posi ions o he PCd backbone (C
␣
ace, in blue) ela i e o
he C (shown in ibbons) a e shown o he 10 complexes wi h he lowes o al o iola ions o he NMR-de i ed es ain s. The a e age posi ional
.m.s.d. o he PCd s uc u es compa ed wi h he mean is 1.3 ⫾0.6 Å. The side chains o acidic amino acids a e shown in ed, whe eas he basic
esidues a e in blue. The heme g oup ( ep esen ed in s icks) is colo ed in g een, and he cadmium a om (shown as a sphe e)isinda k blue.On he
igh , a space- illing ep esen a ion is shown in he same o ien a ion as on he le . No e he p oximi y o he hyd ophobic and elec os a ic su aces
on bo h p o eins in he complex.
FIG.5.Viola ions o he Pc-C complex. The obse ed PCS a e
shown in he op panel wi h open symbols (squa es,
15
N; ci cles,
1
H;
e o ba s indica e es ima ed expe imen al e o s in chemical shi
de e mina ion). The dashes gi e he back-calcula ed alues o he 10
bes s uc u es. Residue numbe s a e indica ed; sc, side chain. In he
bo om panel, he back-calcula ed angles be ween he nucleus ha
expe iences a PCS, he heme i on, and Ty -1 amino ni ogen a om a e
indica ed wi h a dash o he same nuclei as in he op panel. Posi i e
PCS should ha e angles ⬍54° (below he ho izon al line), and o neg-
a i e PCS he angles should be ⬎54°. I can be concluded ha all angles
in he calcula ed s uc u es ag ee wi h he obse ed sign o he PCS.
Plas ocyanin-Cy och ome In e ac ions18912

Se e al aspec s o he amino acid composi ion o he in e ace
can be ela ed o he ansien na u e o he complex. Cha ge
in e ac ions a e obse ed a he edge o he in e ace, in acco d
wi h he gene al indings o ansien complexes (33, 56).
Residues Lys-57 and Lys-62 o Pc a e close o Glu-189 and
Asp-64 om C , espec i ely. Se e al o he s (Asp-10, Lys-11,
Lys-35, Asp-44, Lys-51, Asp-54, Asp-90, and A g-93 om Pc
and Asp-100, Glu-108, Glu-165, Asp-190 and he heme p opi-
ona es in C ) a e u he away om he o he p o ein bu may
be close enough o con ibu e o he elec os a ic in e ac ions.
The cha ge in e ac ions a e expec ed o be a o able wi h neg-
a i e cha ges on C and p edominan ly posi i e ones on Pc.
Six p oline esidues a e loca ed in he in e ace, P o-37,
P o-38, P o-68, and P o-91 om Pc and P o-118 and P o-120
om C ep esen ing 17% o he esidues a he in e ace and
con ibu ing ⬃30% o he bu ied su ace a ea. P oline is a
esidue wi h e y low p opensi y in p o ein-p o ein in e aces
o igh complexes (3.8% (57–60)). Howe e , p oline is mo e
abundan in ansien p o ein complex in e aces (61). The
inabili y o P o o o m hyd ogen bonds may be employed in
his way o limi he a ini y in such complex. Fou glu amine
esidues a e ound in he in e ace, ep esen ing 13.7% o he
bu ied a ea (4.3% in ecogni ion si es o igh complexes).
These pola , uncha ged esidues su ounding he hyd ophobic
pa ch may enhance dissocia ion by acili a ing esol a ion o
he in e ace, as sugges ed p e iously by C owley and Ubbink
(33).
The bu ied su ace a ea o he in e ace has a s anda d size
o non-obliga e p o ein complexes (57–60). Bo h si es (1 and 2)
o Pc make con ac wi h he C su ace, bu ying a o al o
1100–1200 Å
2
, simila o he 1200–1400 Å
2
in he Pho midium
complex (31) and smalle han he 1720 Å
2
ound in he plan
complex (62).
Recen kine ic da a using si e-di ec ed mu an s o Nos oc Pc
ha e e ealed a la ge o e lap be ween he ecogni ion pa ches
o Pc o PSI (63) and C ,
2
in pa icula o Pc subs i u ions
a ec ing elec os a ic in e ac ions. Howe e , he e a e some
di e ences, acco ding o he e ec s obse ed o se e al sub-
s i u ions. Fo ins ance, A g-93 mu an s show a much la ge
e ec on he elec on ans e o PSI. A g-93 belongs o he
p oximal pa ch o si e 2, bu i does no make con ac wi h C in
he complex. The closes cha ged esidue o C (Glu-165) is 9 Å
away. Thus, he elec os a ic in e ac ion be ween hem may be
mode a e, and he kine ic e ec o eplacing A g-93 could be
a ibu able o mo e gene al elec os a ic in e ac ions, speci i-
cally du ing he encoun e phase o he complex o ma ion
p ocess, as desc ibed o he A g-93 o P. laminosum Pc (28, 29).
The mu a ion L14A has been shown o ha e a la ge e ec on
he eac i i y o Pc owa d PSI (63). On he o he hand, his
mu a ion has only mino e ec s (a 2- old dec ease in eac ion
a es) on he educ ion o Pc by C .
2
This is in ag eemen wi h
he loca ion o Leu-14 a he im o he ecogni ion si e. Indeed,
only a mino pa (2 Å
2
) o i s sol en -accessible su ace is
bu ied in he complex in e ace.
Elec onic Coupling—The s uc u e epo ed he ein is con-
sis en wi h as elec on ans e . Howe e , he a e age dis-
ance om i on o cadmium is he la ges (16.2 ⫾0.1 Å) ound
so a , wi h 11.0 and 13.9 Å in wo plan complexes (30, 65) and
15.0 ⫾2.0 Å o he complex o Pho midium (31).
To ge in o ma ion abou he esidues ha may be in ol ed
in coupling o elec on ans e be ween bo h p o eins, he bes
heo e ical pa hs in he ensemble o he ene gy-minimized
s uc u es o plan , Pho midium, and Nos oc we e analyzed
using G eenpa h Ve sion 0.971 (52). Pu a i ely impo an es-
idues a e shown in Fig. 6. In all spinach s uc u es Ty -1 and
His-87 (His-92 in Nos oc) a e impo an o he elec onic cou-
pling, wi h a “pa h leng h” alue (including bonds and h ough-
space couplings) o 14.2 ⫾0.4 Å. Simila esul s a e ound
using he s uc u e o he complex o popla Pc wi h u nip C
complex (65), al hough in his case he pa h leng h is la ge
(19 ⫾1 Å). In Pho midium he pa h leng h (19.4 ⫾0.5 Å) is
simila o ha in popla , bu all he bes pa hways ound
in ol e he a oma ic ing o Phe-3 in C .InNos oc, wo se s o
pa hways a e ound in he inal ensemble o s uc u es; one
(20.3 ⫾0.8 Å) in ol ing coupling ia Ty -1 in C and P o-91 and
His-92 in Pc and ano he (22.2 ⫾0.5 Å) ia Phe-3 in C and
His-92 in Pc. The ela i e couplings o bo h pa hways (3.6 ⫾
FIG.6.Impo an esidues o elec onic coupling o he en-
semble o he 10 bes con o ma ions o Pc-C complexes om
Nos oc (a), plan (b) (30)), and Pho midium (c) (31)). Diag ams
show he coppe a om o Pc, His-87 (His-92) coppe ligand, P o-86
(P o-91), and he heme g oup wi h he dis al ligand Ty -1 and Phe-3 (in
cyanobac e ial C ). Residue numbe s co espond o he o ganism ep-
esen ed in each panel. When wo di e en pa hways a e ound, he
esidues in ol ed in he one wi h be e coupling a e ep esen ed on
he le .
FIG.7. Compa ison o he s uc-
u es o PCd-C complexes, showing
he physiological cyanobac e ial sys-
ems o Nos oc sp. PCC 7119 (a) and
P. laminosum (c) and he con o ma-
ion be ween popla PCd and u nip
C (b). C is shown in ibbons, and Pc is
ep esen ed by he ensemble o he 10
bes con o ma ions shown as C
␣
aces.
The heme is in s icks, and he coppe s a e
shown as sphe es. Plan and Nos oc o ien-
a ions show a side-on binding mode,
whe eas Pho midium exhibi s a head-on
con o ma ion.
Plas ocyanin-Cy och ome In e ac ions 18913
0.6 ⫻10
⫺9
and 1.6 ⫾0.3 ⫻10
⫺9
) a e somewha smalle han
hose ound o he plan and Pho midium s uc u es (5.3 ⫾
0.8 ⫻10
⫺7
and 7 ⫾2⫻10
⫺8
, espec i ely).
A combined Mon eca lo and molecula dynamics calcula ion
on a he e ologous plan Pc-C complex (17) sugges ed wo elec-
on pa hways. The i s in ol es Ty -83 (in plan Pc). In all he
solu ion s uc u es epo ed including his one, he complex
con o ma ion is e y di e en om ha suppo ing his pa h-
way (con igu a ion E in Re . 17). The Nos oc Pc esidue equi -
alen o Ty -83 (Ty -88) is 12.5 Å away om he closes C
a om, and mu a ion o Ty -88 o ei he Ala o Phe does no
a ec he educ ion o Pc by C in Nos oc.
2
The second pa hway
was desc ibed o he heo e ical con o ma ion D, which is he
mos simila o he Nos oc s uc u e. I in ol es P o-86 in plan
Pc (P o-91 in Nos oc) ha should ecei e elec ons om a C
heme p opiona e. The ela i e coupling o he heo e ical com-
plex (1.8 ⫻10
⫺9
) is simila o hose ound o he Nos oc
p o eins. Howe e , in he Nos oc s uc u e he dis ance be-
ween P o-91 and he heme is ⬃7 Å, oo a o e icien di ec
elec on ans e , and ins ead, he sugges ed pa hway in ol es
Ty -1.
The Role o Elec os a ic In e ac ions—Pe haps he mos e-
ma kable aspec o he Nos oc s uc u e is he side-on binding
o Pc. This con o ma ion esembles he o ien a ion ound in he
plan complex a he han he one ound o he o he cya-
nobac e ial complex om Pho midium (Fig. 7). To es ablish he
signi icance o he di e en o ien a ions o Pc in he Nos oc and
Pho midium complexes, he sum o iola ions was calcula ed
o he Nos oc p o eins, wi h Pc in he o ien a ion o ha ound
o Pho midium. The sum is signi ican ly la ge han hose o
he bes s uc u es, wi h la ge minimum-dis ance and in e -
ace e ms. The pseudo-con ac es ain s show a smalle io-
la ion o His-92, bu he numbe o iola ions in he es o he
p o ein inc eases. To es whe he his o ien a ion co e-
sponded o a local minimum in he con o ma ional space, i was
used as he inpu s uc u e o a es ained igid-body docking
calcula ion. The calcula ion showed ha Pc changed i s o ien-
a ion owa d ha o he 10 bes s uc u es (sum o iola ions,
30.8 a bi a y uni s; .m.s.d. wi h he a e age o he 10 bes
s uc u es, 1.5 ⫾0.5 Å). I is concluded ha he expe imen al
da a a e su icien o make a clea dis inc ion be ween he
di e en o ien a ions.
The s uc u e o he Pho midium complex does no exhibi a
s ong ionic s eng h dependence (31), and also he elec on
ans e kine ics show only a weak sal dependence (28, 29).
Bo h he plan and he Nos oc complexes employ elec os a ics
bu wi h e e se cha ges. In plan , posi i e cha ges on C
in e ac wi h he nega i e cha ges on Pc, whe eas in Nos oc he
opposi e is obse ed. Such cha ge in e ac ions a e in acco d
wi h he kine ic s udies. These ha e demons a ed a s ong
ionic s eng h dependence o he educ ion eac ion o Pc by C
in i o o he plan sys em (21, 23–26, 28, 29), al hough he
physiological signi icance o elec os a ic in e ac ions is no
clea (15). Also o he Nos oc p o eins he eac ion is ionic
s eng h-dependen .
2
Thus, i appea s ha a side-on binding o
Pc is obse ed in complexes ha in ol e elec os a ic in e ac-
ions. I is no ewo hy ha in he p esen calcula ions, elec o-
s a ic es ain s we e no used in any way (con a y o he
plan case), and he p esen s uc u e was based en i ely on
NMR spec oscopy-de i ed es ain s.
I is also impo an o no e ha in all s uc u es de e mined
o Pc and C , he o ien a ion o Pc is such ha a sho elec on
ans e chain is c ea ed be ween he Q
o
si e o cy och ome b
6
and plas ocyanin (in ol ing he QH
2
, Rieske FeS clus e , C
heme, and Pc coppe ). Fig. 8 shows a model o he cy och ome
b
6
-Pc complex based on he s uc u e o cy och ome b
6
(6) and
he p esen s uc u e o Pc-C , ob ained a e alignmen o he
C molecules. I is clea ha C aces he lumen wi h he egion
a ound heme ligand Ty -1, eadily o ming a complex wi h Pc.
Acknowledgmen s—We a e g a e ul o C. Alba a´n o p o iding he
plasmid o C (pEAF-WT) and D s. M. He a´s and J. A. Na a o o
c i ical commen s. D . M. Hube is acknowledged o help wi h he EPR
spec oscopy.
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