Measurement of photosynthetic electron transport in Porphyridium by EPR electron paramagnetic resonanc

B Kok

EC Weaver

JR Norris

English

E. C. Weaver

Crossref

Intermediate Electron Transport in Porphyridium: EPR Studies

Weaver, E. C.

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.0
Paper given at the II International Congrese on Photosynthesis,,
Stresa ,  Italy. June 24-29 ., 1971
INTERMIATE ELECTRON TRANSPORT IN FORPMIDIUM • EPR STMIES
Ellen C. Weaver	 ^'t
Associate Professor	 NOV 197,
Biological Sciences Department
sIASA
Ian Jose State College
San Jose ,  California 95114 20N
EPR (electron paramagnetic resonance) is a form of spectroscopy
in which the measuring beam has a wavelength of 3 cm. ,  and thus has
no effect on Uving matter. Photosynthetic organjsms, or their chloro-
plasts or chromatophores ., become paramagnetic when illuminated with
visible light. What this means Is diagrammed in Figure 1; paramagnetism
ie due to unpaired electrons. It is (,'f9te of the first detectable events
-"T- enough electrons e unnaafter the absorption of a photon, f 	 	 ar	 red
10
U 0
	
0
N u	 (or there are enough free radicals of a given species), one sees an
U
absorption peak at a given ratio of magnetic field and microwave
frequency which is expressed as a so-called &-value arid is analogous
to wavelength in light spectroscopy.
gz
zc\	 0	 Where h Planck's constant,.) is the frequency in hz, is
X0z
LU	 Bohr magneton, and H,, is the d. c. magnetic field in gauss. Most0
C" free radicals occur at a g value of 2.00. M
<
z
All
	
Z09 M103 A1110VA	
plants have a light-induced EPR signal 1 (1) with similar
characteristics in a stem state. The e presence of this signal is
*Ref (J)L prm-ldes the baelzround information on light-induced EPR
signals Jz pbotosynthetic org&aisms. Ref. (2) , describes experimental
techniques.
4 M
I M
n' r
2-
dependent on a functioning photosystem I reaction center, since it is
absent in the Seenedesnms mutant lacking a photosystem I reaction
center (3). The cross section for signal I formation is approxi-
mately 3.15 chlorophyll molecules in a photoey6tem 1 sub,ahloroplast
particle derived from spinach, and in this preparation the quantum re-
quirement for spin production is one (4). These facts, plug a body
of evidence from the laboratories of Vernon and others (5, 6) make iti
highly probable that the signal, can be equated with oxidized P 00' It
should be noted that bulk chloropIxqll is a n observed; only the reaction
?	 center undergoing oxidation-reduction is detected.
Our interest in these signals lies in what they can tell us
about photosynthetic electron transport. By observing the oxidation
state of P700, under the influence of system 1 and/or system 2 light,
we can infer what is happening between the two photosystems. The
GsLL '^^ i are to nMC.! AV'f'atvf^ nnmr^lot»na.^• .,+^. 4. _ &I.-Se a8.*s..__-2 t%—_—extent vvuap-3-cow tat&v C.iJ LOW N11V^^i TdOtJ.4/UU L.L'(JJA
fluorescence yield measurements, being on the system 1 side of the
chain rather than the system 2 side.
The results reported herein were obtained with intact cells
of Por.Rh3Tidium c r^uentwn. Light of wavelength I (
	
680 to 703 nm)
or wavelength II ("I _ 550 M) could be alternated or superimposed on
the cuvette.
A signal. is :!readily produced (Fig. 2) with less than saturating
red light; green light about twice as intense produces less signal.
The signal resulting when the two beams are combined is considerably
smaller, than that with light I alone, indicating a reduction in the
steady-state number of oxidized reaction centers by the electrons
.. 	 i.
 
i
i;f
4 ,
E
..t
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being transferred kvom photosystem 11 by green light. If DCMU (3-3 1
 * 41
dichlorc phenyl 1, 1 di metlWI urea) is added, then the combined beams
increase the signal. When the signal is light saturated,
and with the addition of DCMU, the concentration of spins is approxi-
mately 1 per 200 chlorophyll molecules.
In figure 3, the amplitude of the signals as a function of
intensity of light I is shown in the upper curve, and with the addition
of a, low intensity of green light, in the lower. At moderate intensities,,
the reduction effected by light 11 is relatively constant.
The amplitude of the signal produced by a less than saturating
red light is reduced by increasing amounts of green :ijo-t t, as
illustrated in Figure 4. A weak green light (3 . 102
 ergs o cm-1-sec-l) c"'arj
reduce the signal, some 30%, although by itself It has no detectable
EPR effect.
let us now LOOK at the i net^.ca or siignal decay i.n` Line dark after
a steady state has been reached Mg . 5). The time course is apparent
first ordor with a ti of 0.3 sec. If light II is substituted for
light T, the decay is speeded to an extent dependent on the intensity
of light II; the time required for the signal to drop to half its.
original amplitude is 0.1 to 0.05 sec.
i	 Rise kinetics are more complex, since the net rate of signal,
.,i
formation represents a. balance between oxidation and reduction. Electrons
return to the reaction center along a cyclic pathway as well as being
•	 Y
funneled in from photosystem II (7). The size of the reducing pool
between the photosystems profoundly affects the rate of accumulation of
g?oc ox. . The rate of rise is strongly ;influencedi
 by the length of the
s
t
Y
x
/'
j
preceding dark time (Fig. b), by the intensity and wavelength of the
exciting light (cf. ref. 8). It can also be slowed by preiliu dmtion
with syst em II light.
The kinetics of signal amplitude following the superposition
of light I,Z upon light I (as was illustrated for steady-state nignals
in Fig. 1) can be informative (Fig. 7). A signal is produced by
illumination with ?.fight I; the addition of light II produced first a
rapid drop, and then a slower one, resulting after some 15 sec. in
a signal pane--third the amplitude of the original one. The fast drop
is an immediate reduction, the slow one perhaps the filling of an
intewinediate pool and/or some conformational change which facilitates
electron transport. The two phases may be indicative of the two
pools, A1. and A2, postulated by Kok, Jeliot and McGloin (9). If the
intensity of light, II is increased (Fig. 7b), there is a transient
drop
	
car 7 e	 T J-	 __ _- Ie 	i+I A ..^f+ 	em .. a	 ^i. -._.► 	-3 k ._.!1 _ _ t - A. L L_! o .,,. .._ ... r'z.^ I ^y NR.i. 4Mlavu va +. •	 itu.a u Op ^ oia.Lu InCa. 1.C:ciliCS Lnai
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some of the green photons were going auto system I oxidation. In
i
}	 addition, the signal resulting after exposure to light 12 reaches a
r
.E
higher steady state than that after dark, which indicates to us that
perhaps light-JI has induced the state II described by Duysens and
Talens (10) in their fluorescence studies (aee also Fig. 7c). Probably
Light II is doing three things: reducing P700, also contributing to
its oxidation, and third, bringing about a change of state. A +,hi ty-»
second dark period restores the original. conditions. If light II is
switched on and' ,off whJ+le light I remains on, a repeating set of
transients is obtained as
-
 illustrated in Fig. 7c. Note that the am-
plitude of tho signal increae 'es with repeated exposures to light II,.
r
I
eft
/E
-5-
The rate of passage of electrons between the two photosystems has
a substantial temperature coefficient. If the same sort of experiment
is performed at VC, green light is far more effective in producing
signal I than it is when the temperature is 25°C (Figure 8), meaning
that it is less effective in generating a reductant. If light II is
superimposed upon light I at the Lower temperature, the initial drop
is as fast and as large, but it is followed by a rise in signal rather
than a continued drop. Continued transfer of electrons is no;; taking
place rapidly enough to keep P 700 reduced.
We are confident that valid estimations of pool sizes and tempera-
ture coefficients of the rate limiting steps will soon be at hand.
fL
FIGURE CAPTIONS
Fig. 3. A schematic representation of the transition between the
diamagnetic state of material and the paramagnetic state.
The arrows represent electrons.
Fig. 2 Signals produced by 680 nm light (1.5 x 104 ergs • em 2• sec-1),
550 nm light (3 a 104 ergs'cffi 2-sec'l), and the two beams
combined.
Fig. 3 Amplitude of signal I as a function of the intensity of light
I, (680 nm), with and without the addition of light II
(550 nm). The arl r,-f indicates where the two light beams
are of equal intensity.
Fig. 4 Amplitude of signal 1, generated by light I (680 nm, 1.5 x 104
ergs'em 2. sec.1) with increasing intensity of light II (550 nm)
added.
Fig. 5 Amplitude of signal I as a function of time. An arrow t indicates
a given light is turned on, and ^j indicates it is turned off.
Decay of the signal is faster when light II is on than in the dark.
Fig. 6 Rate of rise of signal I as a function of the length of preceding
dark period.
Fig. 7 Amplitude of signal I as a function of time. In (7a) lights
I and II are of equal intensity. ^^ (7b) the intensity of light II
has been doubled. In (7c) light I is kept on and light II is
switched on and off at ten second intervals.
Fig. 8 Amplitude of signal I as a function of time. The only difference
in the upper and lower traces is temperature. See text.
tl
LITERATURE CITED
(1) Weaver, Ellen 5. EPR studies of free radicals in photosyrthetic
systems. in Ann. Rev. of Plant Physiology 19. (1965)
(L. Machl.is, W. Briggs, R. Park, eds.) Annual Reviews, Inc.
Palo Alto.
(2) Weaver, Ellen 0. and Harry E. Weaver. Techidques for studying
light-induced paramagnetism in photosynthetic materials by
means of EPR spectroscopy. in Methods in Enzymology,,
(A. San Pietro, ed.) in press.
(3) Weaver, Ellen C. and Norman I. Bishop. Photosynthetic mutants
separate electron paramagnetic resonance signals of Scenedes=s.
Science 140, 1-3.  (1963 )
(4) Weaver, Ellen C. and Harry E. Weaver. Paramagnetic unit in spinach
subchloroplast particles: Estimatioi. of size. Science 165,
906-907. (1969)
(5) Vernon, Leo P., Bacon Kra, Sakae Katoh, A. San Pietro, and
Elwood R. Shaw. Properties of subchloroplast particles prepared
by the action of digitonin, Triton X-100, and sonication.
in Energy Conversion by the Photosynthetic Apparatus. Brookhaven
Symposia in Biology: No. 19 (1966), pp. 102-114. (John Olson, ed.)
(6) Norris, J. R., R. A. Uphaus, H. L. Crespi and J. J. Katz. Electron
spin resonance of chlorophyll and the origin of signal I in
photosynthesis. Proc. Natl. Acad. Sci. U.S. 6$, 625-628 (1971).
(7) Weaver, Ellen C. Kinetic behavior of electron paramagnetic resonance
signal I. II. Comparison of wild type and mutant (ac-206)
Chlamydomonas reinhardt . Plant 'Pbys. 44.9 1535-1511. (1969).
t
01
4
d
LITERATURE, CITED (continued)
(8) Wearer, Ellen C. Kinetic behavior of EPR signal I: Induction
effects in intact algae. Photochem. Photobiol. 7. 93-100.
(x,968).
(9) Kok,, Bessel, Pierre Jol.iot and Marion P. McGloin. Electron
transfer between the photoacts. in Prog. in Photosynthesis,
pp. 1012»1056.
(10) Duysens, L. N. M. and A. Talena. Reactivation of reaction center II
by a product of photoreaction 1. in progress in Photosynthesis
Research Vol. 11, 1073-1081. (H, Metzner,, ed.) (1961).
(Publication sponsored by International Union of Biological
Scienoes)
3
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ACKNOWLEDGMMS
x
This work was supported in part by- the National Science
D.(
Foundation 0102-(31-1222) and the National Aeronautics and Space
Administration (#021-3430).
The author is grateful for the use of laboratory facilities
at NASA's Ames Research Center, Moffett Field, California, which
made the research possible. The author thanks Mr. Robert Baker for
technice). assistance.
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Intermediate electron transport in Porphyridium:  EPR studies

Intermediate electron transport in Porphyridium: EPR studies

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