Kinetics of Electron Transfer between Q A and Q B in Wild Type and
Herbicide-Resistant Mutants of Chlamydomonas reinhardtii
A n ton y R . C ro fts*, Irene B aroli, D avid K ra m e r, and Shinichi T a o k a
Biophysics Division and *D epartm ent o f M icrobiology, University o f Illinois at
U rbana-Cham paign, U rb ana, IL 61801, U .S .A .
Z. Naturforsch. 4 8 c , 2 5 9 - 2 6 6 (1993); received December 10, 1992
Electron Transfer, Kinetics, Tw o-Electron G ate, Herbicide Resistance, Photosystem II
We have investigated the electron transfer kinetics for reduction o f plastoquinone by photo­
system II in six m utant strains o f Chlamydomonas reinhardtii by following the decay o f the
high fluorescence state after flash activation, and com pared the separate reactions of the twoelectron gate with those o f a wild type strain. By analysis o f the electron transfer kinetics, and
separate measurement o f the equilibrium constant for stabilization o f the bound semiquinone
after one flash, we have been able to deconvolute the contributions o f rate constants and equi­
librium constants for plastoquinone binding and electron transfer to the overall process. Two
mutations, S 264 A and A 251 V, led to a marked slowing o f kinetics for reduction of plastoqui­
none to the bound semiquinone. In S 2 6 4 A , the second electron transfer was also slower, but
was normal in A 2 5 1 V. In m utant G 2 5 6 D , the electron transfer kinetics were normal after the
first flash, but slowed after the second. In mutants L 2 5 7 F , V 2 1 9 I, and F 2 5 5 Y , the electron
transfer kinetics after both flashes were similar to those in wild type. We discuss the results in
terms of a model which provides a description o f the mechanism o f the two-electron gate in
terms of measured kinetic and equilibrium constants, and we give values for these parameters
in all strains tested.
Introduction
Eight different am ino acid substitutions th at
con fer resistance to a variety o f PS II herbicides
have been identified to d ate. All o f them are clus­
tered between residues 211 and 271 o f the D 1 sub­
unit o f PS II. The m ost frequent m u tatio n s, which
m arkedly increase to leran ce tow ard s the herbi­
cides atrazine and d iuron, are localized a t serine26 4. In toleran t m utants o f higher plants S e r-2 6 4 is
replaced by glycine, w hereas in the green alga
Chlamydomonas reinhardtii and cy an o b a cte ria the
com m on substitution is by alanine. T his repeated
loss o f S er-264 is highly significant, indicating th at
it m ight play an im p o rtan t role in herbicide bind­
ing. T rebst [1] and B ow yer et al. [2] suggested th at
S er-264 contributes to the binding o f inhibitors o f
the urea-atrazin e fam ily to the Q B site by hydrogen
bonding o f its side ch ain h ydroxyl grou p to the
N H group in the herbicide m olecule, in an alogy
with the observed H -bon d in g o f the herbicide terbutryne to Ser-223 in the L subunit o f bacterial
reaction centers. Tietjen et al. [3] p rop osed th at
S er-264 would stabilize the structure o f the Q B site
Reprint requests to A. R. Crofts, Biophysics Division,
156 D avenport Hall, 607 S. Mathews, U rb an a, IL
6 1 0 8 1 ,U .S .A .
Verlag der Zeitschrift für N aturforschung,
D -W -7400 Tübingen
0 9 3 9 -5 0 7 5 /9 3 /0 3 0 0 - 0 2 5 9 $ 0 1 .3 0 /0
by H -bonding to H is-252. C ro fts et al. had p re­
viously suggested th at this histidine residue could
also be involved in binding o f the p ro to n th at sta ­
bilizes the Q b" semiquinone in the pocket [4],
Bow es et al. [5], on the basis o f kinetic m eas­
ured in m ultiflash experim ents, suggested th at the
am ino acid substitution S 2 6 4 G in the higher plant
Amaranthus hybridus affected the reaction s o f the
2-electro n gate by decreasing the rate o f electron
tran sfer from Q A~ to Q B by an o rd er o f m agnitude.
T he w ork by O rt et al. [6] supported the idea th at
the m u tation led to a lower ap parent equilibrium
co n stan t (A^app) fo r the reaction Q a Q b ^ Q a Q b >
which resulted in a higher quasi-steady state level
o f Q a ". T a o k a and C rofts [7] m easured a decrease
in A app
lnn from direct m easurem ents o f the b ack -reaction, and showed th at this was due mainly to a
m uch higher dissociation co n stan t for quinone
from the Q B site in the m u tant. T he forw ard rate
co n stan t for electron transfer was 2 - 3 times larger
in the m u tan t strain, but the effect o f this p a ra m e ­
ter in increasing the value o f ATapp was offset by the
larger effect o f the quinone dissociation co n stan t
in decreasing the value. The intrinsic equilibrium
co n stan t for the sharing o f an electron between
Q a Q b an d Q aQ b was ° f sim ilar m agnitude in
m u tan t and wild type.
In the S 2 6 4 A m u tan t o f Chlamydomonas rein­
hardtii, an alm ost unchanged equilibrium co n cen -
Unauthenticated
Download Date | 7/31/17 2:05 PM
260
tration o f Q A“ was observed by fluorescence, o x y ­
gen em ission and therm olum inescence m ethods
[8]. E rick so n et al. [9] studied the effect on the
PS II a ccep to r side o f the substitutions S 2 6 4 A ,
V 2 1 9 I , F 2 5 5 Y , G 2 5 6 D and L 2 7 5 F . Only m u­
tants S 2 6 4 A and G 2 5 6 D showed a m arked de­
crease in the rate o f Q A~ reoxidation , m easured by
following the decay o f fluorescence yield directly
on ag ar plates. These results were supported by
those o f G ovindjee et al. [10]. H ow ever, care
should be taken in analyzing these d a ta , since the
dark red ox state o f the PS II quinone accep tors
was not determ ined p rior to the experim ents. In
dark adapted algal cells, ab ou t 5 0 % o f the PS II
reaction centers have a single reduced Q B~, and
this would lead to the observation, after one actin ­
ic flash, o f the con volu tion o f the kinetic d ata from
tw o reaction s: Q ^ Q b ^ Q a Q b
and Q a “Qb ^
QaQb2- , which have different half times [11]. Thus,
if the initial red ox state o f the system is unknown,
no reliable conclusions can be draw n from the ex ­
perim entally m easured kinetics ab out the discrete
kinetic p aram eters o f the individual reactions.
In the present w ork, wild type C. reinhardtii and
herbicide-resistant m u tan t strains with substitu­
tions in D 1 o f S 2 6 4 A , V 2 1 9 I , F 2 5 5 Y , G 2 5 6 D ,
L 2 7 5 F and A 251 V, have been studied under co n ­
ditions in which the photosystem II quinone a c ­
cep tors were oxidized before the experim ents by
treatm en t with benzoquinone. F ro m the d eco n v o ­
lution o f kinetic traces o f Chi a fluorescence decay
and the m easurem ent o f the ap parent equilibrium
co n stan t for electron transfer between Q A and Q B,
values for the intrinsic equilibrium co n stan t, the
dissociation co n stan t o f quinone from the Q B
binding site, the forw ard and reverse rate co n ­
stants for the electron transfer between the two
p lastoquinone accep to rs, and the on- and off-rate
con stan ts for binding o f plastoquinone were ca lcu ­
lated for wild type and resistant strains. The m u­
tants th at m ost significantly differed from wild
type were S 2 6 4 A and A 251 V, whereas the oth er
strains presented m ore m inor differences.
M ethods
Benzoquinone treatment o f intact cells
C om m ercially available /^-benzoquinone was re­
crystallized by sublim ation. Cell suspensions co n ­
A. R. Crofts et al. • Electron Transfer between Q A- Q B
taining 5 - 1 0 |im chlorophyll in the grow th m edi­
um were incubated at ro o m tem peratu re in the
presence o f 100 |_im benzoquinone for 5 min and
then centrifuged for 5 min at 3 0 0 0 * g. The pellet
o f treated cells w as resuspended in benzoquinonefree m edium con tain in g 0 .1 m sorbitol, 10 m M
K C1, 10 m M M g C l2, 100 jim N H 4C1 as uncoupler
and 2 0 mM H epes, pH 7.0. T he treated cells were
kept in com p lete darkness during the treatm ent
and until the experim ents were perform ed, since
light caused an irreversible quenching o f fluores­
cence.
Kinetics o f electron transfer
E xp erim en tal m ethods were essentially as de­
scribed by R o b in so n and C ro fts [4, 11, 12], except
th at the fluorescence p h o to m eter used was an im ­
p roved version. T he new ap p aratu s was equipped
with three m easuring flash lam ps, allowing m eas­
urem ent o f three time points in the sub-m s range in
each experim ent. In addition, by using a com m on
light guide for actin ic and m easuring flashes, and a
co m m o n reference channel for co rre ctio n o f v aria­
tions in flash intensity, the signal to noise ratio
was im proved, and co rrectio n fo r the overlapping
con trib u tion o f the actinic flash to fluorescence al­
lowed m easurem ent closer to the actinic flash.
Cells a t ap p ro x. 5 jaM Chi were suspended in 0.1 m
sorb itol, 10 m M KC1, 10 m M M gC l2, 100 |iM
N H 4C1 as uncou p ler and 20 m M H epes, pH 7.0,
with o th er additions as indicated. O ther details are
given in F ig u re legends and the text.
Experimental rationale
T he equilibrium and rate co n stan ts for the fo r­
w ard and reverse electron tran sfer and for binding
and dissociation o f plastoquinone at the Q B site
were calcu lated using the follow ing assum ptions
[4 ,7 ,1 1 ,1 2 ] :
i) T he ratio o f am plitudes o f fast and slow c o m ­
p onents o f the rapid biphasic d ecay kinetics o f Q A_
follow ing an actinic flash was assum ed to reflect
the ratio o f the initial fraction o f Q A Q B to Q A[vacant] centers.
ii) The d issociation co n stan t o f plastoquinone
from the Q B site in the dark w as assum ed to re­
m ain unchanged after an actinic flash.
iii) Binding o f plastoquinone to the Q B site was
assum ed to follow a pseudo-first o rd er reaction ki­
netics when the p ool was initially oxidized (ten ­
Unauthenticated
Download Date | 7/31/17 2:05 PM
A. R. Crofts et al. ■ Electron Transfer between QA_ Q B
261
fold excess o f oxidized plastoquinone over re a c ­
tion center).
The two following equations are solutions o f the
rate equations derived from the m odel
m echanism . By doing the experim ent a t different
pH values, we can also determ ine the pK values
which describe the role o f proton s in the m ech a­
nism, but this m ore extensive d ata set has so far
been collected only in Amaranthus hybridus and its
S 2 6 4 G m u tan t [7].
+ r2 = kon (1 + A J B q)
+ kBA{ \ + K app( l + A J B 0)}
r \ ' r 2 = K n '^ B A
(1 +
A J B J (1 +
(1 )
* a p p ).
(2)
Variables r, and r2 are the slow and fast rate
co n stan ts obtained directly from the experim ental
d ata by fitting the kinetic traces o f fluorescence de­
cay after one actinic flash to tw o exponential c o m ­
ponents, and a residual; A0 and B0 are the am pli­
tudes o f the slow and fast com p on en t, respectively,
o f the fitted kinetic traces, also obtained from the
tw o-exponential fit. K0 , the plastoquinone disso­
ciation con stan t, is calculated as the ratio A J B 0
(assum ption i) above).
Kapp is the ap parent equilibrium co n stan t for
sharing an electron between Q A and Q B, defined (if
p roton ated states are included) as:
K
[QaQb"] + [QaQb (H +)]
app
[Q a "1 + [Qa '( H +)] + [QA- Q B] + [Q a 'Q b(H +)] '
F o r simplicity, we can ignore the effects of proton­
ation, and treat Kapp at any fixed pH as determined by
the equilibrium constants for electron transfer, and
the dissociation o f Q from the site:
[Q aQ b~]
app
[Q a '] + [Q a 'Q b]
= K e/(\ + K 0).
(3)
A gain, for simplicity we have ignored the c o n tri­
bution o f the quinone p ool, so th at
*o
= Q a /Q a 'Q b
— A J B0 —k0ff/kQn.
Ke
= Q a'Q b /Q a 'Q b
= ^ab/^ba
A lso,
so th at
K ^ = k AB/kBA-(\+Ao/Bo)-\
(3 ')
The apparent equilibrium co n sta n t, K.dpp, ca n be
determ ined experim entally from the ratio o f the
half-tim e o f the back reaction S2P Q AQ B“ - ^
S ,P Q a Q b to that o f the back reactio n S2P Q A~ -^
S tP Q A. The m easured p aram eters therefore allow
us to determ ine the rate co n stan ts and equilibrium
con stan ts which define the tw o-electron gate
Results and Discussion
In o rd er to allow an analysis o f the separate
reaction s o f the tw o-electron gate, it w as necessary
to oxidize the quinone pool, and p rim ary and sec­
o n d ary bound quinone accep tors, as described
above. R epresentative kinetic curves fo r electron
tran sfer through the tw o separate reaction s leading
to p ro d u ctio n o f Q H 2 are shown in Fig. 1 (first
flash) and 2 (second flash). P aram eters for the kinet­
ics a fter the first flash, m easured from the co rrected
fluorescence decays, are sum m arized in Table I . The
rate o f the back reaction from S2P Q AQ B~ -^
S ,P Q aQ b was m easured from the rephasing o f the
binary oscillation, as suggested by R obinson and
C ro fts [11, 12]. The results for wild type and sever­
al m u tan t strains are shown in Fig. 3. The kinetic
p aram eters and equilibrium con stan ts derived
from these m easurem ents are sum m arized in T a ­
ble II.
T he kinetic p aram eters obtained here for wild
type C. reinhardtii are in agreem ent with those re­
ported for pea chlorop lasts [11, 12] and for Ama­
ranthus hybridus [7], If the co n cen tratio n o f p lasto ­
quinone in the m em brane is assumed to be 5 m M
[4], then the true dissociation co n stan t for p lasto ­
quinone from the Q B site, K0' = K0 -[PQ], ca lcu la t­
ed from this w ork for wild type C. reinhardtii is
2 m M , which is in the range calculated by C ro fts
et al. [4],
A m o n g the am ino acid substitutions covered by
this study, tw o clear groups can be established:
1)
M u tation s th at con fer herbicide resistance to
different extents but th at do not affect m arkedly
the electron transfer between the plastoquinone
acce p to rs o f PS II. These m utations are V 2 1 9 I ,
F 2 5 5 Y , L 2 7 5 F and G 2 5 6 D (T able II, and see
F ig . 1 and 2). It is im portan t to note th a t, in the
case o f G 2 5 6 D , a slow er electron transfer rate has
been reported previously by two different groups
[9, 10]. In both cases, the decay o f fluorescence was
m easured with repeated illumination o f the samples
and sh o rt dark periods between actinic flashes,
Unauthenticated
Download Date | 7/31/17 2:05 PM
A. R. C rofts et al. • Electron Transfer between QA- Q B
262
A
C
Wild type
Mutant Mz2 (A251V)
time (ms)
B
D
Mutant DCMU4 (S264A)
Mutant Ar204 (G256D)
time <ms>
Fig. 1. Kinetics o f [QA~] decay following the first actinic flash in dark adapted, benzoquinone treated C. reinhardtii
cells. A: Wild type; B: S 2 6 4 A (strain D C M U 4); C: A 251 V (strain M z2); D: G 2 5 6 D (strain A r2 0 4 ). Other strains
showed kinetics similar to wild type. Insets: Same data shown in a 2 ms time scale to show the fast component. In all
the traces shown, circles represent actual data and solid lines are the best non-linear squares fit o f the data using the
equation in Table I. Parameters obtained from the fits are given in Table I. See “Experimental rationale” for details.
which leads to a partial and uncontrolled red u c­
tion o f the plastoquinone pool. In ou r own exp eri­
m ents, the kinetics after the second flash (o r after
scram bling the tw o-electron gate) are ab o u t a fa c­
to r o f two slow er than in wild type (F ig . 2). The
substitution o f G ly -256 by asp artate introduces a
negative ch arge in the Q B region, and it could be
expected th at this would seriously affected the ion ­
ic environm ent and possibly the p roton binding
p roperties o f the Q B binding site. H ow ever, in our
Q b site m odel, G ly -2 5 6 is positioned facing the
strom al aqueous phase rath er than m aking co n tact
with the p lastoquinone. It should also be noted
th at V 2 1 9 I shows a significantly slow er electron
transfer after the second flash than wild type, and
a m ore m arked binary oscillation (n o t shown).
2)
M u tatio n s th at con fer herbicide resistance
and considerably slow down the electron transfer
Unauthenticated
Download Date | 7/31/17 2:05 PM
A. R. Crofts et al. ■Electron Transfer between QA~ Q B
A
Wild type
263
B
M utant DCM U4 (S264A)
1.0
t
-------------------------------------------------------------
i
o
iO
k
o
o
I völ
0.0-1----- ------ i----- ------ r----- ------ t----- ------ r0.0
(J
M utant M Z2 (A251V)
2.0
D
4.0
6.0
8.0
M utant A r204 (G256D)
1 .0 ---------------------------------------------------i
o
[ vö]
o
o.o-i
0.0
----- »----- ------ <----- ------ 1----- ------ >“
2.0
4.0
6.0
8.0
time (ms)
w
M u tan t D rl8 (V219I)
[ Vö)
tim e (ms)
O
2nd flash
A
12th Hash
Fig. 2. Kinetics o f [QA ] decay after the second (circles)
and the 12th (triangles) actinic flash. A: Wild type;
B: S 2 6 4 A (strain D C M U 4 ); C: A 2 5 1 V (strain M z2);
D: G 2 5 6 D (strain A r2 0 4 ); E: V 2 1 9 I (strain D r 18). Oth­
er strains showed kinetics similar to wild type.
Unauthenticated
Download Date | 7/31/17 2:05 PM
A. R. Crofts et al. • Electron Transfer between Q A~ Q E
264
A
Wild type
B
Mutant DCMU4 (S264A)
Mutant Mz2 (A251V)
D
Mutant Ar204 (G256D)
1.4
0.6
-
-
0.2
0.2
4
6
8
flash number
1 sec
10 sec
5 sec
20 sec
30 sec
10
12
4
6
8
flash number
60 sec
100 sec
200 sec
400 sec
no prcflash
40 sec
Fig. 3. Rephasing o f the binary oscillations o f the two-electron gate as a function of the dark time after a pre-illuminating flash. Binary oscillations o f QA~ level, measured from normalized variable fluorescence, observed at 200 us
after each of a series o f 12 actinic flashes fired at 1 Hz. Traces have been offset for clarity. The times between the
preflash and the first actinic flash are shown above. A: Wild type; B: S 2 6 4 A (strain D C M U 4); C: A 251 V (strain
M z2); D: G 2 5 6 D (strain A r204). Other strains showed oscillations similar to wild type.
Unauthenticated
Download Date | 7/31/17 2:05 PM
A. R. C rofts et al. ■Electron Transfer between QA~Q B
265
Table I. Kinetic data obtained from the deconvolution o f fluorescence decay
traces after correction for the non-linear relationship of [QA~] and the level o f flu­
orescence [14]. Param eters shown in the table are the result o f the non-linear last
squares fit o f the corrected fluorescence data with an equation o f the form:
[Q a'] = A0 exp ( - r xi) + B0 exp ( - r2t) + 1 - (A0 + Ba),
where t is time, A0 and B0 are the amplitudes, and r, and r2 the rate constants, of
the slow and fast components, respectively. The slowest kinetic phase is consid­
ered constant in the milliseconds time scale (ty2 = 2 —3 sec, depending on strain)
and is indicated here as a residual component (T - ( A0 + B0)). Values tx and t2 are
half-times calculated directly from r, and r2.
Strain
A0
Bo
(slow)
(fast)
Wild type
0.19
0.47
S264A
0.37
0.15
Residual
r\
h
[ms ']
[ms]
0.34
0.82
0.85
0.48
0.24
2.89
0 - A 0~ B 0)
h
[ms ']
[MS]
5.05
137
16.3
42
G 256D
0.28
0.42
0.30
0.67
1.03
5.51
V 219I
0.18
0.56
0.26
0.58
1.19
6.74
102
A 251 V
(2 com p)
0.52
0.19
0.29
0.42
1.65
1.51
459
A 251 V
0.67
—
0.33
0.65
1.06
-
0.29
0.39
0.32
0.90
0.77
4.93
(1 com p)
F255Y
126
140
Table II. Param eters o f the two-electron gate in wild type and herbicide-resistant mutants o f C. reinhardtii. The half
time o f the back reaction from Q A“ to S2 in the presence of D C M U (^ /2(Q A ))■> was measured directly from the correct­
ed fluorescence decay. The half time of the back reaction from Q B“ to S2 (/, 2(Q B“)), was measured from the rephasing
time o f the binary oscillations o f the two-electron gate (Fig. 3). The dissociation constant for plastoquinone from the
Qb site, K0 = A J B 0, is given without units, as a relative value for com parison between wild type and mutants. Multi­
plication o f K0 by [PQpooJ (approx. 5 m M ) would give the true dissociation constant in m M units.
Strain
*1/2(Qa )
[sec]
?1/2(Qb
[sec]
Wild type
2.7
25
0.40
8.3
11.6
S264A
2.4
20
2.50
7.3
25.6
G 256D
2.7
25
0.67
8.3
13.8
)
* 0
* .P P
(*)
^A B
^B A
^ o ff
^on
[ms ']
[ms ']
[ms ']
[ms-1]
0.37
0.34
0.86
0.56
0.60
0.24
4.66
0.34
0.47
0.70
4.29
15.0
V 219I
2.0
40
0.32
19.0
25.1
6.25
0.25
0.19
0.60
A 251 V
(2 com p)
3.0
15
2.47
4.0
13.9
n.d.
n.d.
n.d.
n.d.
F255Y
2.5
25
0.40
9.0
15.7
3.73
0.24
0.84
1.07
L275F
2.7
20
2.50
6.4
9.36
3.46
0.37
0.39
0.84
between the plastoquinone accep to rs, as in the
case o f S 2 6 4 A and A 2 5 1 V . In S 2 6 4 A , only a
slight ch ange in the ap p aren t equilibrium con stan t
for electron transfer was observed (T able II), al­
though a larger fraction o f the fluorescence did not
decay in the m s range (presum ably because o f a
higher fractio n o f n o n -Q B-centers). The slow elec­
tron tran sfer in this m u tan t can be ascribed to
a high dissociation co n stan t for plastoquinone
rath er than a ch ange in K.dpp. T he high value o f K 0
is associated with an increased fractio n o f centers
in the state Q A • [vacant] in the dark. This suggests
th at S er-264, besides being involved in the binding
o f inhibitors, also plays an im p o rtan t role in plas­
toquinone binding. A lso, both the on- and off-rate
con stan ts for binding and dissociation o f plasto-
Unauthenticated
Download Date | 7/31/17 2:05 PM
266
A. R. Crofts et al. • Electron Transfer between Q A- Q B
quinone are increased in this m u tan t (T ab le II). In
centers in the Q a Qb state, how ever, the electron
transfer reaction proceeds 3.5 tim es m ore rapidly
than in wild type (T ab le II). Assum ing structural
h om ology with the b acterial reactio n cen ter, the
loss o f the S er-2 6 4 hydroxyl grou p , shown to p ro ­
vide a H -bond to the bound quinone in Rhodopseudomonas viridis [13], m ight be expected to re­
sult in a w eaker binding, o r binding to an altern a­
tive site in these m u tan t strains. The m ore rapid
rate for the bound quinone m ight reflect a sh orter
bonding distance to the N o f H is-2 1 5 , which p ro ­
vides a H -bon d to the o th er quinone = 0 group at
the proxim al end o f the binding pocket, since reac­
tion rates are strongly dependent on distance for
electron transfer reaction s. In effect, the m u tation s
m ay “ push” the plastoquinone m olecules from the
reaction cen ter clo ser together, allowing fo r a fast­
er electron transfer. A gainst this hypothesis is the
fact th at the electron transfer after the second flash
is considerably slow er than wild type (F ig . 2),
though this m ight be explained by a rearran g e­
m ent o f the p ock et on reduction to form the semiquinone. W ithin the above h ypothetical fram e­
w ork, the difference in rate o f the slow phase be­
tween S 2 6 4 A and S 2 6 4 G (co m p are r e f [7] and
this w ork) m ight reflect a w eaker binding o f the
quinone to the altern ative H -b o n d d o n o r at the
distal end o f the binding pocket in the form er
strain, due to steric hindrance introduced by a
greater bulk o f the alanine than the glycine side
chain.
M u tan t A 2 5 IV had a sm aller Ka p p than wild
type and probably a decreased am o u n t o f n o n -Q B
centers, as indicated by the sm aller residual flu­
orescence at long times after the first flash. The ki­
netics o f electron transfer after one flash were c o n ­
siderably slower than th at o f the wild type o r the
oth er m utants (F ig . 1), but the kinetics after the
second flash were norm al (F ig . 2). In fact, the de­
cay o f [Qa ‘ ] after the first flash could be d eco n v o ­
lv e d with alm ost equal confidence into either tw o
exponential com ponents o f h alf tim es 4 5 9 |as and
1.65 ms respectively, o r only one exponential c o m ­
ponent o f ty2 = 1.06 ms (Table I). If the latter fit is
taken, it would indicate th at alm ost all the centers
in A 251 V are in the Q A- [vacant] state in the d ark ,
and th at K0 is correspondingly greater (and KE
also greater to keep K.dpp at the m easured value)
than shown in T able II. The substitution o f A la251 by the m ore bulky valine residue w ould then
lead to a m uch weaker binding o f plastoquinone.
A ssum ing the largest value o f KE found in the
oth er strains (KE= 26), K0 would be — 5.5 an d,
given the few points in each kinetic trace, we
would be unable to deconvolute sep arate co n trib u ­
tions from bound and vacan t centers.
[1] A. Trebst, Z. N aturforsch. 42c, 7 4 2 - 7 5 0 (1987).
[2] J. R. Bowyer, M . Hilton, J. Whitelegge, P. Jewes,
P. Camilleri, A. R. Crofts, and H. H. Robinson, Z.
Naturforsch. 45 c, 3 7 9 - 3 8 7 (1990).
[3] K . G. Tietjen, J. F. Kluth, R. Andree, M. Haug,
M. Linding, K . H. Muller, H. J. Wroblowsky, and
A. Trebst, Pesticide Sei. 31, 6 5 - 7 2 (1991).
[4] A. R. Crofts, H. H. Robinson, K. Andrews, S. Van
Doren, and E. Berry, in: Cytochrom e Systems
(S. Papa et al., eds.), pp. 6 1 7 - 6 2 4 , Plenum Publ.
Corp., New Y ork 1987.
[5] J. Bowes, A. R . Crofts, and C. J. Arntzen, Arch.
Biochem. Biophys. 200, 3 0 3 - 3 0 8 (1980).
[6] D. R. Ort, W. H. Ahrens, B. M artin, and E. W. Stoller, Plant Physiol. 72, 9 2 5 - 9 3 0 (1983).
[7] S. Taoka and A. R. Crofts, in: Current Research in
Photosynthesis (M . Baltscheffsky, ed.), Vol. I, pp.
5 4 7 -5 5 0 , Kluwer, The Netherlands 1990.
[8] A .-L . Etienne, J.-M . Ducruet, G. Ajlani, and C. Vernotte, Biochim. Biophys. A cta 1015, 4 3 5 - 4 4 0 (1990).
[9] J. M. Erickson, K. Pfister, M. Rahire, R. Togasaki,
L. Mets, and J.-D . Rochaix, The Plant Cell 1, 361 —
371 (1989).
[10] Govindjee, P. Eggenberg, K. Pfister, and R. J. Strasser, Biochim. Biophys. A cta, in press 1992.
[11] H. H. Robinson and A. R. Crofts, F E B S Lett. 153,
2 2 1 -2 2 6 (1 9 8 3 ).
[12] H. H. Robinson and A. R. Crofts, in: Advances in
Photosynthesis Research (C. Sybesma, ed.), Vol. I,
pp. 4 7 7 - 4 8 4 , Martinus Nijhoff/Dr. W. Junk, The
Hague 1984.
[13] H. Michel, O. Epp, and J. Deisenhofer, E M B O J. 5,
2 4 4 5 -2 4 5 1 (1986).
[14] P. Joliot, P. Bennoun, and A. Joliot, Biochim.
Biophys. Acta 305, 3 1 7 -3 2 8 (1973).
Unauthenticated
Download Date | 7/31/17 2:05 PM