DETERGENT EFFECT ON CYTOCHROME b559 ELECTRON PARAMAGNETIC
RESONANCE SIGNALS IN THE PHOTOSYSTEM II REACTION CENTRE.
Y uela, I.1*, Ga cía-Rubio, I.2, Roncel, M.3, Ma ínez, J.I.2, Rami o, M.V.1, O ega, J.M.3,
Alonso, P.J.2 and Pico el, R1.
1 Es ación Expe imen al de Aula Dei, Consejo Supe io de In es igaciones Cien í icas,
Apdo. 202, E-50080-Za agoza, Spain.
2 Ins i u o de Ciencia de Ma e iales de A agón, Consejo Supe io de In es igaciones
Cien í icas-Uni e sidad de Za agoza, Plaza San F ancisco s/n, E-50009-Za agoza,
Spain.
3 Ins i u o de Bioquímica Vege al y Fo osín esis, Uni e sidad de Se illa-Consejo
Supe io de In es igaciones Cien í icas, Ame ico Vespucio s/n, E-41092-Se illa, Spain.
Co esponding au ho : I. Y uela; [email p o ec ed],
Fax: +34 976 716145,
phone: +34 976 716058
Keywo ds: cy och ome b559, de e gen , elec on pa amagne ic esonance,
pho osys em II, eac ion cen e, edox po en ial.
1
ABSTRACT
De e gen e ec on Cy och ome b559 om spinach pho osys em II was s udied
by elec on pa amagne ic esonance (EPR) spec oscopy in D1-D2-Cy b559 complex
p epa a ions. Va ious n-dodecyl-β-D-mal oside concen a ions om 0 o 0.2% (w/ )
we e used o s abilise he D1-D2-Cy b559 complexes. Low spin heme EPR spec a
we e ob ained bu gz ea u e posi ions changed depending on de e gen condi ions.
Redox po en iome ic i a ions showed a unique edox po en ial cy och ome b559 o m
(E ‘m = +123-150 mV) in all he D1-D2-Cy b559 complex p epa a ions indica ing ha
de e gen does no a ec his p ope y o he p o ein in hose condi ions. Simila e ec
on Cy och ome b559 EPR spec um was obse ed in mo e in ac pho osys em II
p epa a ions independen ly o hei agg ega ion s a e. This inding indica es ha
changes due o de e gen could be a common phenomenon in pho osys em II
complexes. Resul s a e discussed in e ms o he en i onmen each de e gen p o ides
o he p o ein.
2
1. INTRODUCTION
Cy och ome (Cy ) b559 is an in eg al componen o pho osys em (PS) II eac ion
cen e (RC). I s p esence is c i ical o he biogenesis and s able assembly o he PSII
RC, bu i is no in ol ed in he p ima y elec on anspo in PSII. D1-D2-Cy b559
complexes wi h he minimum polypep ide composi ion ha a e able o pe o m e icien
ligh -induced p ima y cha ge sepa a ion s ill bound Cy b559.1 Ha sh ea men s a e
equi ed o sepa a e Cy b559 om his complex 2,3 and he igh ela ionship wi h he
D1/D2 he e odime ha binds he essen ial co ac o s o PSII p ima y pho ochemis y
sugges s an essen ial ole o Cy b559. Howe e , despi e many s udies pe o med
du ing he las decades4-6 he exac unc ion(s) o his hemop o ein is s ill unclea .
Cy och ome b559 consis s o wo small polypep ides, he α (9 kDa) and β (4.5 kDa)
subuni s wi h a ansmemb ane α-helical domain and he e odime ic s uc u e.6,7 Two
his idine esidues wi hin he hyd ophobic domain o each polypep ide ac as ligands o
heme i on.
One o he mos in iguing p ope ies o Cy b559 is he ema kable a iabili y o
he midpoin edox po en ial (E’m) o he heme g oup. In i s na u al memb ane
en i onmen i exhibi s a labile high-po en ial (HP, E’m ≈ +380 mV) and a s able low-
po en ial (LP, E’m ≈ +20 - +200 mV) o m. An in e media e po en ial (IP) was also
epo ed in chlo oplas s, hylakoids and in ac PSII memb anes.6,8-12
Heme i on o Cy b559 in PSII RC is known o display he ypical low-spin
elec on pa amagne ic esonance (EPR) signals gx ≈ 1.5, gy ≈ 2.3 and gz ≈ 3.0.
Howe e , sligh ly di e en gz alues o Cy b559 ha e been obse ed depending on he
p epa a ion and ex en o pu i ica ion o samples.6 These changes in EPR spec a we e
connec ed in he pas wi h changes be ween di e en edox o ms o Cy b559. Indeed, i
has been sugges ed ha HP and LP o ms o Cy b559 can be dis inguished by hei
EPR spec a. The HP o m o Cy b559 was associa ed o a gz a ound 3.01-3.08 based
on EPR measu emen s in chlo oplas s and PSII memb anes wi h high con en o his
o m. On he o he hand, LP Cy b559 o m was ela ed o a gz in he 2.93-3.04 ange.
3
The gz alue o 2.93 was measu ed in he isola ed Cy b559.6,13-17 Ne e heless, a
s aigh o wa d ela ionship be ween edox po en ial and EPR signal is no clea . O he
ac o s ha we e sugges ed o in luence he EPR gz posi ion we e he pu i ica ion
deg ee o he sample and he hyd ophobici y o heme en i onmen .6,14
In o de o elucida e ac o s con olling EPR signal, we s udy he e in de ail EPR
spec a o oxidised LP Cy b559 in he D1-D2-Cy b559 complex and in mo e in ac PSII
p epa a ions. Ou esul s clea ly demons a e ha de e gen s modi y he EPR spec um
o Cy b559 and sugges ha EPR signal a ia ions and midpoin edox po en ial could
no be di ec ly ela ed. The way de e gen s a ec EPR pa ame e s is discussed in
e ms o a mo e gene al concep o hyd ophobici y e e ed in.14
4
2. MATERIALS AND METHODS
P epa a ion o PSII memb anes.- Highly en iched-PSII memb anes we e isola ed
om ma ke spinach acco ding o Be hold e al.18 Samples we e suspended in 0.4 M
suc ose, 15 mM NaCl, 5 mM MgCl2 and 50 mM 2-(N-mo pholino) e hanesul onic acid
(Mes)-NaOH, pH 6.0, ozen in liquid ni ogen and s o ed a –80ºC un il use. PSII
memb anes exhibi ed oxygen e olu ion a es o 520 ± 30 μmol o O2 mg Chl-1 h-1 using
DCBQ as a i icial elec on accep o .
P epa a ion o D1-D2-Cy b559 complexes.- A s anda d D1-D2-Cy b559 complex
p epa a ion con aining six chlo ophyll (Chl) molecules pe RC was isola ed om highly
pu i ied oxygen-e ol ing PSII memb anes om ma ke spinach18 acco ding o he
p ocedu e o Nanba and Sa oh1 and modi ied by Mon oya e al.19 This me hod makes
use o a Toyopea l TSK-DEAE column. Samples loaded in he column we e washed
wi h 0.05% (w/ ) T i on X-100 un il abso bance a 417 nm was highe han ha a 435
nm. De e gen was subsequen ly exchanged by n-dodecyl-β-D-mal oside (β-DM) a
di e en concen a ions anging om 0 o 0.2% (w/ ) o 0.15% (w/ ) suc ose
monocap a e. De e gen eplacemen was done un il T i on X-100 abso bance a 280
nm was lowe han 0.01. Then, D1-D2-Cy b559 complexes we e elu ed wi h a linea sal
g adien in he same bu e and ac ions we e collec ed a 1 ml/min.
This me hod was also used wi h h ee di e en modi ica ions desc ibed in he
li e a u e.20-22 The a ia ions, conce ning basically he column and washing bu e
condi ions a e he ollowing: i) Toyopea l TSK-DEAE column, 1% (w/ ) T i on X-100; ii)
Toyopea l TSK-DEAE column, 1% (w/ ) T i on X-100 and 1.5% (w/ ) au ine ; iii) Q-
Sepha ose Fas -Flow (Pha macia) column and 0.15% (w/ ) T i on X-100. The β-DM
concen a ion in he elu ion bu e was 0.1% (w/ ) o hese la e samples and
de e gen eplacemen was done as explained abo e. All isola ed D1-D2-Cy b559
complex p epa a ions con ained six Chl pe RC.
5

D1-D2-Cy b559 complexes we e isola ed a di e en pH in he 5.5-7.7 ange by
changing he pH o he washing bu e . Fo pH 5.5-6.5 and pH 7.0-7.7 Mes-NaOH and
is(hyd oxyme hyl)aminome hane (T is)-HCl bu e s, espec i ely, we e used. Desal ed
D1-D2-Cy b559 complex p epa a ions we e p epa ed by a 2 h dialysis agains he sal
ee elu ion bu e ee o sal using a 30,000 kDa cu -o dialysis ube (Spec apo ).
All isola ion p ocedu es we e done in da kness in a cooled chambe a 4 ºC.
Samples we e hen ozen in liquid N2 and s o ed a –80 ºC. Pigmen composi ion o
isola ed D1-D2-Cy b559 complex p epa a ions was de e mined as desc ibed in
Eijckelho and Dekke .23 Cy och ome b559 con en was calcula ed om he di hioni e-
educed minus e icyanide-oxidised abso p ion di e ence spec a using an ex inc ion
coe icien o 21.0 mM-1cm-1 a 559 nm.1
P epa a ion o PSII co e complexes.- PSII co e complex samples we e p epa ed
ollowing he me hod desc ibed in24 wi h some modi ica ions. An ion exchange
Toyopea l TSK-DEAE column was used. The column was washed a 2 ml/min o 2 h
and subsequen ly he co e complexes we e elu ed wi h a linea sal g adien in 50 mM
Mes-NaOH, pH 6.5 wi h 0.03% (w/ ) T i on X-100. Addi ionally, PSII co e complexes
we e elu ed om he column a e de e gen exchange wi h 0.1% (w/ ) β-DM in he
same bu e . De e gen exchange was done un il T i on X-100 abso bance a 280 nm
was lowe han 0.01. Pigmen ed ac ions we e concen a ed in Cen ipep (Amicon)
ubes.
Isola ion o monome ic and dime ic PSII RC and co e complexes.- . Monome ic
and dime ic complexes we e isola ed by suc ose densi y g adien cen i uga ion. To do
ha , PSII RC we e suspended in 50 mM Mes-NaOH, pH 6.5 o 50 mM T is-HCl, pH 7.2
and 0.1% β-DM (w/ ).25 PSII co es we e suspended in 25 mM Mes-NaOH, pH 6.5, 10
mM NaCl, 5 mM CaCl2 and 10 mM NaHCO3 and incuba ed wi h β-DM o a inal
concen a ion o 1.25% (w/ ).26 The solubilised PSII RC and PSII co e samples we e
homogenised, loaded on o a eshly p epa ed 0.1-1.0 M suc ose g adien and
6
cen i uged a 90,000xg in a Beckman SW41 swing-ou o o o e nigh and 75,000xg in
a Beckman SW28 swing-ou o o o 22 h, espec i ely. The suc ose g adien bu e
composi ion was 10 mM NaCl, 50 mM Mes-NaOH, pH 6.5 o 50 mM T is-HCl, pH 7.2
and 0.1% (w/ ) β-DM o PSII RC samples and 10 mM NaCl, 5 mM CaCl2, 25 mM Mes-
NaOH, pH 6.5, and 0.03% (w/ ) β-DM o PSII co es. The chlo ophyll- ich ac ions
we e hen emo ed om he suc ose g adien s, ozen in liquid ni ogen and s o ed a -
80 ºC.
Po en iome ic edox i a ions.- Po en iome ic edox i a ions we e ca ied ou
unde a gon a 12 ºC using D1-D2-Cy b559 complex samples (5 μM Chl) in 50 mM
Mes-NaOH, pH 6.5, by ollowing he abso bance changes a 559 minus 570 nm
induced by sequen ial addi ion o aliquo s o 0.1 M sodium di hioni e. The
measu emen s we e pe o med in an Aminco DW-2000 UV-Vis spec opho ome e
using he dual wa eleng h mode. Samples we e p e iously oxidised wi h 25 μM
po assium e icyanide. The edox po en ial in he eac ion cell we e
simul aneously measu ed wi h a po en iome e (Me h om He isau, Swi ze land)
p o ided wi h a combined P -Ag/AgCl mic oelec ode (C ison Ins umen s, Spain)
p e iously calib a ed agains a sa u a ed solu ion o quinhyd one (E’m, pH 7, +280
mV a 20 ºC). In addi ion o e icyanide (E’m, pH 7, +430 mV) he ollowing edox
media o s we e used: 10 μM 1,4-benzoquinone (E’m, pH 7, +280 mV), 20 μM
2,3,5,6- e ame hyl-p-phenylendiamine (E’m, pH 7, +240 mV), 20 μM 1,2-
naph hoquinone (E’m, pH 7, +145 mV), 2.5 μM N-me hyl-phenazonium
me hosul a e (E’m, pH 7, +80 mV), 10 μM N-me hyl-phenazonium e hosul a e (E’m,
pH 7, +55 mV) and 20 μM e ame hyl-p-benzoquinone (E’m, pH 7, +5 mV).
EPR measu emen s.- Samples we e concen a ed (0.5 – 1.2 mM Chl) in Cen ipep-30
and Cen icon-30 (Amicon) ubes o EPR measu emen s. Con inuous wa e EPR
spec a we e eco ded wi h a B uke ESP380E spec ome e wo king a he X-band
7
( equency abou 9.6 GHz). Typical measu emen s we e achie ed a 8 K wi h 1.46 μW
mic owa e powe (which ensu es no sa u a ion e ec s on he signal) and 1 mT o
modula ion ampli ude.
8
3. RESULTS
E ec o n-dodecyl-
β
-D-mal oside on Cy b559 EPR signal.
The in luence o β-DM on Cy b559 EPR spec um was obse ed in a s anda d
D1-D2-Cy b559 complex p epa a ion con aining six Chl pe RC. D1-D2-Cy b559
complexes we e isola ed in he p esence o a ious β-DM concen a ions anging om
0 o 0.2% (w/ ) ( o de ails see Ma e ials and Me hods). No pigmen and polypep ide
composi ion a ied among samples a e de e gen ea men s. All samples displayed a
ypical low spin heme EPR signal, wi h p incipal alues o he g enso being a ound gz
≈ 3.0, gy ≈ 2.3 and gx ≈ 1.5.6 Al hough all spec a we e simila , some di e ences we e
de ec ed in hei gz ea u es, wi h no changes a gx and gy posi ions. Following Taylo ’s
model27 i is expec ed ha gy alue emains nea ly una ec ed when a small shi in gz
alue occu s. Besides, conside ing ha he gx ea u e is e y b oad, i is no possible o
de ec small changes in i as hose obse ed o gz. In e media e ield ea u e in he
EPR spec a s ay a gy = 2.26 o all samples and, when he high ield ea u e is
de ec ed, a gx alue o 1.53 is ob ained. Figu e 1 shows he de e gen e ec on he low
ield ea u e in EPR spec a o Cy b559. The D1-D2-Cy b559 complex sample
suspended in he p esence o 0.1% (w/ ) β-DM displays a gz ea u e wi h a maximum
a 2.98 and a ull wid h a hal maximum (FWHM) o 13 mT (Fig. 1A,a). On he o he
hand, D1-D2-Cy b559 complex samples in he p esence o lowe β-DM concen a ion
(0.03% (w/ ) β-DM) display a gz ea u e wi h a maximum a 2.93 ha seems o be
asymme ic showing a smoo he dec ease owa ds lowe ields and FWHM o 15 mT
(Fig. 1A,b). An EPR signal wi h simila gz maximum alue has been epo ed in he
li e a u e o D1-D2-Cy b559 complexes isola ed ollowing he same p ocedu e.28
The dependence o gz posi ion on β-DM concen a ion is shown in Fig. 1B. The
concen a ion a which gz peak shi s (0.03-0.06% (w/ )) is abo e he c i ical micelle
concen a ion (c.m.c.) o β-DM.29 A c.m.c. de e gen monome molecules sel -
9
ABBREVIATIONS
Chl, chlo ophyll; c.m.c, c i ical micelle concen a ion; Cy , cy och ome; D1 and D2, co e
polypep ides o he pho osys em II eac ion cen e; DCBQ, 2,6-dichlo obenzoquinone;
E’m, midpoin po en ial; EPR, elec on pa amagne ic esonance; FWHM, ull wid h a
hal maximum; HP, high po en ial; IP, in e media e po en ial; LP, low po en ial; Mes, 2-
(N-mo pholino)e hanesul onic acid; PS, pho osys em; RC, eac ion cen e; T is,
is(hyd oxyme hyl)aminome hane.
REFERENCES
[1] O. Nanba and K. Sa oh, Isola ion o a pho osys em II eac ion cen e consis ing o
D1 and D2 polypep ides and cy och ome b559, P oc. Na l. Acad. Sci. USA, 1987,
84, 109-112.
[2] X.S. Tang, K. Fushimi and K. Sa oh, D1-D2 complex o he pho osys em II eac ion
cen e om spinach. Isola ion and pa ial cha ac e iza ion, FEBS Le ., 1990, 273,
257-260.
[3] W.R. Widge , W.A. C ame , M. He modson, D. Meye and M. Gulli o , Pu i ica ion
and pa ial amino acid sequence o he chlo oplas cy och ome b-559, J. Biol.
Chem., 1984, 259, 3870-3876.
[4] W.A. C ame and J. Whi ma sh, Pho osyn he ic cy och omes, Annu. Re . Plan
Physiol., 1977, 28, 133-172.
[5] J. Whi ma sh and H.B. Pak asi, Fo m and unc ion o cy och ome b559, in Oxygenic
Pho osysn hesis: The Ligh Reac ions, ed. D.R. O and C.F. Yocum, Kluwe
Academic Publishe s, Do d ech , The Ne he lands.1996, pp. 249-264.
[6] D.H. S ewa and G.W. B ud ig, Cy och ome b559 o pho osys em II, Biochim.
Biophys. Ac a, 1998, 1367, 63-87.
[7] A. Zouni, H.-T. Wi , J. Ke n, P. F omme, N. K ausse, W. Saenge and P. O h,
C ys al s uc u e o pho osys em II om Synechococcus elonga us a 3.8 Å
esolu ion, Na u e 2001, 409, 739-743.
16

[8] P.R. Rich and D.S. Bendall, The edox po en ials o he b- ype cy och omes o
highe plan chlo oplas s, Biochim. Biophys. Ac a, 1980, 591,153-161.
[9] J.M. O ega, M. He ás and M. Losada, Redox and acid-base cha ac e iza ion o
cy och ome b559 in pho osys em II pa icles, Eu . J. Biochem., 1988, 171, 449-
455.
[10] O. Kaminskaya, J. Ku eck, K.D. I gang, G. Renge and V.A. Shu alo , Redox
and spec al p ope ies o cy och ome b559 in di e en p epa a ions o pho osys em
II, Biochemis y, 1999, 38,16223-16235.
[11] N. Mizusawa, T. Yamashi a and M. Miyao, Res o a ion o he high-po en ial o m o
cy och ome b559 o pho osys em II occu s ia a wo-s ep mechanism unde
illumina ion in he p esence o manganese ions. Biochim. Biophys. Ac a, 1999,
1410, 273-286.
[12] M. Roncel, J.M.O ega and M. Losada, Fac o s de e mining he special edox
p ope ies o pho osyn he ic cy och ome b559, Eu . J. Biochem., 2001, 268, 4961-
4968.
[13] J. Be gs om and T. Vannga d, EPR signals and o ien a ion o cy och omes in he
spinach chlo oplas hylakoid memb ane, Biochim. Biophys. Ac a, 1982, 682, 452-
456.
[14] L.K. Thompson, A.-F. Mille , C.A. Buse , J.C. de Paula and G.W. B ud ig,
Cha ac e iza ion o he mul iple o ms o cy och ome b559 in pho osys em II,
Biochemis y, 1989, 28, 8048-8056.
[15] C. Be homieu, A. Boussac, W. Män ele, J. B e on and E. Nabed yk, Molecula
changes ollowing oxido educ ion o cy och ome b559 cha ac e ized by Fou ie
ans o m in a ed di e ence spec oscopy and elec on pa amagne ic esonance:
pho ooxida ion in pho osys em II and elec ochemis y o isola ed cy och ome b559
and i on p o opo phy in IX-bisimidazole model compounds, Biochemis y, 1992,
31, 11460-11471.
17
[16] G.T. Babcock, W.R. Widge , W.A. C ame , W.A. Oe ling and J.G. Me z, Axial
ligands o chlo oplas cy och ome b559: Iden i ica ion and equi emen o a heme-
c oss-linked polypep ide s uc u e, Biochemis y, 1985, 24, 3638-3645.
[17] L.I. K ish alik, G.S Che epano , and W.A. C ame , The edox p ope ies o
cy och omes b imposed by he memb ane elec os a ic en i onmen , Biophys. J.,
1983, 65, 184-195.
[18] D.A. Be hold, G.T. Babcock and C.F. Yocum, A highly esol ed, oxygen-e ol ing
pho osys em II p epa a ion om spinach hylakoid memb anes, FEBS Le ., 1981,
134, 231-234.
[19] G. Mon oya, R. Cases, I. Y uela and R. Pico el, Spec oscopic cha ac e iza ion o
wo o ms o he D1-D2-cy och ome b559 complex om suga bee , Pho ochem.
Pho obiol., 1993, 58, 724-729.
[20] I. Y uela, P.J.M. an Kan, M.G. Mülle and A.R. Holzwa h, Cha ac e iza ion o a
D1-D2-cy b559 complex con aining 4 chlo ophyll a/2 pheophy in a isola ed wi h
he use o MgSO4, FEBS Le ., 1994, 339, 25-30.
[21] I. Y uela, R. Tomás, M. Al onso and R. Pico el, E ec o he pH on he abso p ion
spec um o he isola ed D1-D2-cy och ome b559 complex o pho osys em II, J.
Pho ochem. Pho obiol. B:Biol., 1999, 50, 129-136.
[22] I. Y uela, E. To ado, M. Roncel and R. Pico el, Ligh -induced abso p ion spec a
o he D1-D2-cy och ome b559 complex o pho osys em II: e ec o me hyl
iologen concen a ion, Pho osyn h. Res., 2001, 67, 199-206.
[23] C. Eijckelho and J.P. Dekke , A ou ine o de e mine he chlo ophyll a, pheophy in
a and β-ca o ene con en s o isola ed Pho osys em II eac ion cen e complexes,
Pho osyn h. Res., 1997, 52, 69-73.
[24] P.J. an Leeuwen, M.C. Nie een, E.J. an de Mee , J.P. Dekke and H.J. an
Go kom, Rapid and simple isola ion o pu e pho osys em II co e and eac ion
cen e pa icles om spinach, Pho osyn h. Res., 1991, 28, 149-153.
[25] D. Zhele a, B. Hankame and J. Ba be , He e ogenei y and pigmen composi ion
o isola ed pho osys em II eac ion cen e s, Biochemis y, 1996, 35, 15074-15079.
18
[26] B. Hankame , J. Nield, D. Zhele a, E. Boekema, S. Jansson and J. Ba be ,
Isola ion and biochemical cha ac e isa ion o monome ic and dime ic pho osys em
II complexes om spinach and hei ele ance o he o ganisa ion o pho osys em
II in i o, Eu . J. Biochem., 1997, 243, 422-429.
[27] C.P.S. Taylo , The EPR o low spin heme complexes, Biochim. Biophys. Ac a,
1977, 491, 137-149.
[28] V.A. Shu alo , R. Fiege, U. Sch eibe , F. Lendzian and W. Lubi z, EPR s udy o
cy och ome in he D1-D2-Cy b559 complex, Biochim. Biophys. Ac a, 1995, 1228,
175-180.
[29] B. Gall and H. Schee , S abiliza ion o pho osys em II eac ion cen e s: in luence o
bile sal de e gen s and low pH. FEBS Le ., 1998, 431, 161-166.
[30] D.F. Ghano akis and C.F. Yocum, Pu i ica ion and p ope ies o an oxygen
e ol ing eac ion cen e complex om pho osys em II memb anes, FEBS Le .,
1986, 197, 244-248.
[31] R.M. Ga a i o and S. Fe guson-Mille , De e gen s as a ool in memb ane
biochemis y, J. Biol. Chem. 2001, 276, 32403-32406.
[32] M. le Mai e, P. Champeil and J.V. Mölle , In e ac ion o memb ane p o eins and
lipids wi h solubilizing de e gen s, Biochim. Biophys. Ac a, 2000, 1508, 86-111.
[33] M. Seibe , Biochemical, biophysical and s uc u al cha ac e iza ion o he isola ed
Pho osys em II eac ion cen e complex, in The Pho osyn he ic Reac ion Cen e ,
ed. J. Deisenho e and J.R. No is, Academic P ess, San Diego, CA, 1993, ol. 1,
pp. 319-356.
[34] G. Mon oya, R. Cases, R. Rod íguez, M. Au ed and R. Pico el, De e gen -induced
e e sible dena u a ion o he pho osys em II eac ion cen e : Implica ions o
pigmen -p o ein in e ac ions, Biochemis y, 1994, 33, 11798-11804.
[35] F.A. Walke , B.H. Huynh, W.R. Scheid, and S.R. Os a h, Models
o he cy och omes b. E ec o axial ligand plane o ien a ion on he EPR
and Mössbaue spec a o low-spin e ihemes, J. Am. Chem. Soc., 1986, 108,
5288-5297.
19
[36] F.A. Walke , D. Reis and V.L. Balke, Models o he cy och omes b. EPR
s udies o low-spin i on(III) e aphenylpo phy ins, J. Am. Chem. Soc., 1984, 106,
6888-6898.
[37] R. Quinn, M. Nappa, and J.S. Valen ine, New i e- and six-coo dina e imidazole
and imidazola e complexes o e ic e aphenylpo phy in, J. Am. Chem. Soc.,
1982, 104, 2588-2595.
[38] J.C. Sale no, S. Yoshida and T.E. King, E ec s o p o ein-p o ein and p o ein-lipid
in e ac ions on heme si e con o ma ion in he mi ochond ial b cy och omes, J.
Biol. Chem., 1986, 261, 5480-5486.
[39] B. Lanne, B.G. Malms öm and T. Vännga d, The in luence o pH on he EPR and
edox p ope ies o cy och ome c oxidase in de e gen solu ion and in phospholipid
esicles, Biochim. Biophys. Ac a, 1979, 545, 205-214.
[40] M.A. Noo de mee , G.A. Veldink and J.F.G. Vliegen ha , Spec oscopic s udies on
he ac i e si e o hyd ope oxide lyase; he in luence o de e gen s on i s
con o ma ion, FEBS Le ., 2001, 489, 229-232.
20
FIGURE LEGENDS
Figu e 1. (A) Low ield egion o EPR spec a o oxidised Cy b559 heme in D1-D2-Cy
b559 complex samples elu ed om he column in he p esence o a) 0.1%
(w/ ) o β-DM; b) 0.03% (w/ ) o β-DM; c) 0.05% (w/ ) o T i on X-100; d)
0.15% (w/ ) suc ose monocap a e. (B) Dependence o he Cy b559 EPR gz
alue on β-DM concen a ion. EPR condi ions: empe a u e, 8K; mic owa e
powe , 1.46 μW. O he expe imen al condi ions a e desc ibed in Ma e ials
and Me hods.
Figu e 2. Low ield egion o EPR spec a o oxidised Cy b559 heme in D1-D2-Cy b559
complexes: a) sample elu ed om he column in he p esence o 0.03% (w/ )
T i on X-100; b) he same RC sample in (a) subsequen ly ea ed wi h 0.1%
(w/ ) β-DM h ough a DEAE-Toyopea l TSK-650S column o eplace T i on X-
100; c) sample elu ed om he column in he p esence o 0.1% (w/ ) β-DM
a e de e gen exchange; d) he same RC sample in (c) a e emo ing β-DM
de e gen . EPR condi ions: empe a u e, 8K; mic owa e powe , 1.46 μW.
O he expe imen al condi ions a e desc ibed in Ma e ials and Me hods.
Figu e 3. Po en iome ic edox i a ions o Cy b559 heme in D1-D2-Cy b559 complex
p epa a ions a pH 6.5 in he p esence o 0.03% (w/ ) β-DM concen a ion
(μ); 0.1% (w/ ) β-DM (F); 0.15% (w/ ) suc ose monocap a e (Ì).
Expe imen al condi ions a e desc ibed in Ma e ials and Me hods.
Figu e 4. EPR spec a a he gz egion o he oxidised Cy b559 heme in PSII co e
complex sample in he p esence o a) 0.03% (w/ ) T i on X-100; b) 0.1%
(w/ ) β-DM EPR condi ions: empe a u e, 8K; mic owa e powe , 1.46 μW.
O he expe imen al condi ions a e desc ibed in Ma e ials and Me hods.
21

22
200 220 240 260 280
b
c
d
a
Magne ic Field (mT)
A
0.0 0.1 0.2
2.90
2.93
2.96
2.99
g
z
alue
% (w/ )
β
-DM
Fig. 1
23
200 220 240 260 280
d
c
b
a
Magne ic Field (mT)
Fig. 2
24
-100 0 100 200 300
0
25
50
75
100
% Reduced Cy b559
Ambien edox po en ial (mV)
Fig. 3
200 220 240 260 280
b
a
Magne ic Field (mT)
Fig. 4
25