584th Meeting
Held at University College. Swansea,
26 and 27 September 7979
BIOCHEMICAL ASPECTS OF LIGHT
TRANSDUCTION AND EMISSION
Colloquium organized and edited by
T. J. Walton (Swansea)
Light-Absorption and Transduction in Higher-Plant Chloroplasts
RICHARD P. F. GREGORY
Department of Biochemistry, University of Manchester, Manchester M13 9PL, U.K.
This review attempts to summarize some recent approaches to a problem that has existed
since Emerson & Arnold (1932a,b) showed that energy captured by chlorophyll could
be conveyed to reaction centres that were very sparse in relation to the chlorophyll
(1/2400),and that the efficiency of the transfer could approach 90% (Emerson, 1958).
The losses include the effects of thermal deactivation and fluorescence as well as any
wasted absorption in ‘disconnected’ chlorophyll. The 90 % efficiency would be good
even in a crystal, where the inevitable irregularities serve as traps for the dissipation of
energy. There is no indication of crystallinity in the chlorophyll, and so the chloroplast
must rely on other structural features.
Absorption
The view of earlier biochemists such as Willstatter & Stoll (1918) (cited by Rabinovitch, 1945b) appears to have been that chlorophyll (bound to protein) was an active
catalyst for COr fixation (chlorophyll catalysis remained the view of Warburg et al.,
1969). After 1932 the chlorophyll of the ‘enzyme’ had to be imagined as connected to a
mass of 2400 chlorophyll molecules serving as an antenna. Suggestions that the chlorophyll was dissolved in the lipid part of the membrane failed to account for the red-shift
in both absorption and fluorescence with respect to solutions in organic solvents, and
the idea of a protein ‘carrier’ has a long history (see Rabinovitch, 1945a). The two-lightreaction theory arose around 1960, based in part on the phenomena of enhancement,
where each light-reaction possessed its own antenna, the antennae being neither completely separate nor united, On the other hand the demonstration by Kok (1961) of
chlorophyll P-700, the photochemically active chlorophyll at the heart of Photosystem
I, reinforced the concept of chlorophyll activated by a specific protein. Perhaps this
present review would be better regarded as more a discussion about protein than chlorophyll.
Several protein-chlorophyll complexes were indeed isolated, although some of the
first were the water-soluble preparations from Chenopodium album (see e.g., Yakushiji
eta/., 1963) and other sources, of less relevance than those prepared by means of anionic
and non-ionic detergents. A brief summary of photosynthetically relevant particles is
given in Table 1. Two complexes were soon well established: the reaction-centre complex of photosystem I1 and a chlorophyll a+b protein (Thornber et al., 1967). Lesswell-characterized preparations [‘Photosystem I1 complex’ (PS-I1 complex) ;see Table 11
have been obtained containing chlorophyll P-680, the photoactive chlorophyll that is
the analogue of chlorophyll P-700 in Photosystem I1 (Doring et a/., 1967; Floyd et af.,
VOl. I
Table 1. Comparison of the chlorophyll-protein complexes in higher-plant chloroplasts
es: 1, Brown et al. (1974); 2, Genge et al. (1974); 3, Malkin (1975); 4, Shiozawa et al. (1974); 5 , Thornber et al. (1967); 6, V
71); 7, Vernon et a f . (1972); 8 , Wessels & Borchert (1978); 9, Wessels & Voorn (1972).
x
Chlorophyll content
Preparation
MoLwt.
Protein
mol.wt.
CPII
Anionic detergent
27000-35000
21 000-25000
-
Anionic detergent
45 000
39000-42000
Designation
TSF2a
FIII
Triton X-100
Triton X-100
Digitonin
CPI
Anionic detergent
-
Lauryldimethylamine N-oxide
HP700
Triton X-100
Triton X-100
-
313
b 650;
a 662, 670, 677, (684)
a
71 000
Reaction centre
None
None
None
a
alb = 8
a/b = 1.6
96 OOO
-
Spectral types
a
110000
-.
a/b
a 662,669, 677, 686
Delayed-light
Photochemically
active
P-700/chlorophy11=
1/40
P-700/chlorophyll=
1/40
P-700/chlorophyll=
1/40; photochemically active
P-700 cytochromes
Re
584th MEETING, SWANSEA
1225
1971). These three complexes together account for most of the chlorophyll, but there is
still the possibility of the existence of ‘free pigment’ in solution, not yet to be dismissed,
since it has proved very difficult to predict the behaviour of a pigment bed unless some
sort of solution is assumed.
The photoactive chlorophylls P-700 and P-680 absorb maximally at the long-wavelength end of the operative wavelength ranges of Photosystems I and I1 respectively.
Since they have lowest energy of excitation, they serve as ‘sinks’ or traps for excitation
energy. It is assumed that the shift in the absorption arises from environmental effects.
There is in fact a considerable degree of spectral differentiation within the chlorophyll
bed. Techniques such as absorption spectroscopy and low-temperature fluorescence
(see, e.g., Cho & Govindjee, 1970u,b) show multiple components that must be due to
the chlorophyll molecules existing in a range of environments. Groups can be identified
from even-order differentials of absorption spectra (Butler & Hopkins, 1970qb);see also
Litvin & Sineshchekov, 1975) and computer-based fitting of Gaussian curves (of predetermined number but variable position, amplitude and half-width) to chloroplast
absorption spectra (French, 1972). There is room for improvement in the correlation
between the two methods. It is t o be noted that there is more than one type of chlorophyll
even in one type of complex.
The chlorophyll a+b complex was eliminated from any photochemical role by its
absence from certain mutants thaf still carried out both photoreactions (Thornber &
Highkin, 1974). The assignment of it to an antenna function is supported by its great
abundance in normal plants, being half, or even more, of the total chlorophyll (Thornber
1975). The term ‘light-harvesting complex (LH complex)’ is useful for distinguishing
this component, since the term ‘antenna’ can also be applied to the chlorophyll in the
PS-I1 complex apart from the chlorophyll P-680 itself. The ‘antenna of PS-I1 complex’
is the source of the variable fluorescence at 685 nm, and can be identified with chlorophyll
a-678.
Transmission
There is a well-characterized reaction-centre complex (PS-I complex) of Photosystem
I that carries chlorophyll P-700 and other chlorophyll molecules that form its antenna.
However, there are only 14 chlorophyll molecules/complex, whereas the chlorophyll
P-700/chlorophyll a ratio is 1/40-45 (Dietrich & Thornber, 1971). These authors
suggested that only one particle in three is really a PS-I complex and differs from the
other antenna-I complexes by the inclusion of a small chIorophy11-P-700-containing
peptide. The 1/40-50 ratio (confirmed by Malkin, 1975; Wessels & Borchert, 1978)
seems high when the ratio in chloroplasts is 1/400, and Photosystem I is believed to take
only a minor share of energy in the LH complex-PS-I1 complex aggregate. Of the 600
chlorophyll molecules in the photosynthetic unit, it is unlikely that 540 are preferentially
related to Photosystem 11. Arntzen (1978) stresses the point that crude particles of Photosystem I activity prepared by means of digitonin contain one chlorophyll P-700 molecule/
110-150 chlorophyll a molecules, in which case the true antenna-I complex is lost during
treatment with anionic detergent.
Electron-microscopic studies of thylakoid membranes freeze-fractured into E and P
leaflets have revealed particles that protrude from the fracture faces (Staehelin et ul.,
1976). The E leaflet carries large (15nm) particles (EF) that are interpreted as PS-IIcomplex particles surrounded by LH complex. There are smaller (8 nm) particles (PF)
in the P leaflet that could be PS-I complex together with antenna-I particles. Fig. 1
indicates the roles of the four postulated chlorophyll-protein particles, LH complex,
PS-I1 complex, PS-I complex and antenna-I complex, which may be expected to contain
most if not all of the chlorophyll. The structural arrangement of the chlorophyll that
will account for the efficient transmission of energy must be elucidated by protein
crystallography. The only pigment-protein complex for which this has been done,
however, is the antenna complex from the green bacterium Chlorobium limicolu, which
Fenna & Matthews (1975) showed to contain seven bacteriochlorophyll molecules very
evenly spaced but with no obvious orientation. If this applies generally it will answer
Vol. 7
1226
BIOCHEMICAL SOCIETY TRANSACTLONS
Lumen
>
J
Stroma
Fig. 1. Relationships between the protein-chlorophyll complexes
The rectangles represent the complex on the basis that L H complex represents 60%
of the total chlorophyll, PS-I complex 5 %, antenna-1 complex 10% and PS-11 complex
5 %. The dashed rectangle represents the larger Phososystem i unit (chlorophyll
P-700/total chlorophyll = 1/50). The stippling represents ion-sensitive binding, and
the arrows show the directions of energy transmission. The two leaflets of the thylakoid
membrane are indicated, but the Figure is not a structural model.
the question how chlorophyll molecules can be close enough to ensure efficient transfer
by the Forster (1948) (R-6) mechanism without allowing any molecules to approach
closer than 4nm, given by Porter (1978) as a limit below which excitation-quenching
occurs.
Energy transfer between complexes requires that they approach closely as Fig. 1
indicates. Such an association is obvious in the L H complex-PS-I1 complex aggregate,
but not in the PS-I complex-antenna-I particles in the PF-particle set. However, evidence
for transmission from the E F to the PF particles is provided by the phenomenon of
‘spill-over’, in which excitation in PS-I1 complex can under some conditions migrate to
PS-I complex when the PS-11-complex reaction centres are blocked. This is shown, for
example, by fluorescence, which increases when the traps are blocked, but declines
again if Photosystem I activity is allowed, showing that energy is removed from the
PS-11 complex. This is regulated by cations such as Mg2+ and K+, which have been
shown to affect the thickness of the granal thylakoid membrane and to decrease the
PS-I1 complex-PS-I complex contact (see the review by Papageorgiou, 1975).
The same ionic effects appear to regulate the contacts between the photosynthetic
units themselves, probably at the level of the LH complex. The addition of Mg2+ to
solutions of isolated L H complex causes precipitation of large-scale aggregates (Burke
et al., 1978). Whether this applies in the chloroplast can perhaps be established by c.d.
measurements. L H complex and PS-I complex have characteristic c.d. spectra (Gregory
et al., 1972; Scott & Gregory, 1975), and these can be distinguished in grana-less but
otherwise active preparations of chloroplasts. In addition to the spectra of the complexes, there is a prominent negative c.d. at 680nm that resembles that seen in the Mgz+aggregated L H complex, suggesting that specific aggregation is present in the chloroplast.
It is, however, not necessarily always due to L H complex, since the 680nni effect is still
seen in the mutant of barley that lacks that complex (Canaani & Sauer, 1978). The c.d.
technique is a rapid non-destructive method applicable to active fragments.
if grana are present, c.d. reveals a much larger signal, which can be interpreted
(Raps & Gregory, 1975) as due to a large-scale interaction; this may relate t o the kind
of helical architecture suggested by Paolillo (1970) from his electron-microscopic
observations.
1979
584th MEETING, SWANSEA
1227
Transduction
The transduction of excitation energy to chemical energy is achieved by the photoactive chlorophyll molecules of the reaction centres. The process can be generalized as in
the following sequence:
y pax
e- from
other
donors
1
y + pa x
_ _ _Light
_ _ _or_ _ _ _ - _ _ y_
j
energy transfer
(ps)
pa* x
- _ _ _ _ _ _ Y Pa’+ xj
(ns)
b:Iipt
other ors
x
c-----------.----------------------------y pa’+
(m)
where Pa stands for chlorophyll P-700 or P-680, Y is the immediate donor and X the
immediate acceptor. Component Y I , (commonly labelled Z) is not identified, but it may
be the same as the manganese-containing four-step accumulator S described by Kok
et al. (1970), in which:
4eso _4_x_le_-_steps
_ - - -+- - s
_ _ _ _ _ _ _ so + 0
~
2
Component X, I may well be the couple plastoquinone-plastosemiquinone anion, from
the spectroscopic agreement of component X-320 (Stiehl & Witt, 1968) with the semiquinone prepared by pulse radiolysis (Bensasson & Land, 1973). However, classically,
the primary acceptor is known as component Q (quencher), because of the dependence
of the PS-11-complex fluorescence on the degree of its reduction (Duysens & Sweers,
1963). The donor to chlorophyll P-700 is likely to be the copper-protein plastocyanin,
although there are some difficulties. Component X, is only known from e.s.r. studies
at very low temperatures as a signal possibly due to an iron complex (Evans e t al., 1975).
Subsequent acceptors are the ‘bound ferredoxins’ known from their e.s.r. spectra.
These may be related to the spectroscopic entity P-430 described by Ke (1973). The first
chemically defined acceptor is ferredoxin itself.
The energy of a quantum of light of 680nm is 1.82 eV (175kJ.einstein-I). In each
light-reaction the difference in the redox potentials of the identifiable products gives a n
indication of the chemical work done. Thus in Photosystem I1 there is a gap of some
0.7V between Oz and plastoquinone, and a similar value is found for the difference
plastocyanin-ferredoxin in Photosystem I (0.76V). These values can be improved to
approx. 1.OV if guesses are made of the potentials of the primary donors and acceptors.
The loss of nearly half the incoming energy represents the difference between a practical
productive system and a theoretical static equilibrium, and need not be dismissed as a n
imagined ‘inefficiency’!
Arntzen, C. J. (1978) Curr. Top. Bioenerg. 8, 111-160
Bensasson, R. & Land, E. J. (1973) Biochim. Biophys. Acta 325, 175-181
Brown, J . S . , Alberte, R. S. & Thornber, J. P. (1974)Proc. Int. Congr. Photosynth. 3rd 19511962
Burke, J. J., Ditto, C. L. & Arntzen, C. J. (1978) Arch. Biochrm. Biophys. 187, 252-263
Butler, W. L. & Hopkins, D. W. (1970a)Photucheni. Photobiol. 12,439-450
Butler, W. L. & Hopkins, D. W. (19706) Photochem. Photobiol. 12, 451-456
Canaani, 0 . D. & Sauer, K. (1978) Biochim. Biophys. Acta 501,545-551
Cho, F. & Govindjee (1970a) Biochim. Biophys. Acta 205, 371-378
Cho, F. & Govindjee (19706) Biochim. Biophys. Acta 216, 151-161
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Doring, G . , Stiehl, H. H. & Witt, H. T. (1967) Z . Naturfursch. Teil B 22, 639-644
Duysens, L. N. M. & Sweers, H . E. (1963) in Studies in Microalgae andPhoyosynthetic Bacteria
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Emerson, R. (1958) Annu. Rev. Plant Physiol. 9, 1-24
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BIOCHEMICAL SOCIETY TRANSACTIONS
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Litvin, F. F. & Sineshchekov, V. A. (1975) in Biophysics of Photosynthesis (Govindjee, ed.),
pp. 619-661, Academic Press, New York
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Photochemical Reactions Centre of Photosynthetic Bacteria
R I C H A R D J. COGDELL
Department of Botany, University of Clasgow, Clasgow GI2 8QQ, Scotland, U.K.
The light-absorbing pigments in bacterial photosynthesis are organized into ‘units’.
Most of these pigments (bacteriochlorophyll and carotenoids) serve as a light-harvesting antenna funnelling the absorbed light-energy to the reaction centres. The reaction
centres then ‘trap’ the absorbed light-energy in the primary photochemical reaction by
using it to separate charge in an endothermic oxidation-reduction reaction.
Methods are now available for isolating and purifying reaction centres from a variety
of photosynthetic bacteria (Gingras, 1979). The best characterized of these is the reaction
centre prepared from Rhodopseudomonas sphaeroides by using the zwitterionic detergent lauryldimethylamine N-oxide.
1979