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Photosynthesis Research 73: 63–71, 2002.
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Research on photosynthetic reaction centers from 1932 to 1987
Roderick K. Clayton
Liberty Hyde Bailey Professor Emeritus of Biophysics, Section of Plant Biology, Cornell University, Ithaca, New
York; Mailing address: 176 Mountain Vista Circle, Santa Rosa, CA 95409, USA (e-mail: [email protected])
Received 4 July 2001; accepted in revised form 14 September 2001
Key words: W. Arnold, R.K. Clayton, J. Deisenhofer, L.N.M. Duysens, isolation of reaction centers, H. Michel,
photosynthetic bacteria, Rhodobacter sphaeroides, Rhodopseudomonas viridis, C.B. van Niel
Abstract
The history of research on photosynthetic reaction centers is outlined, starting with the implication of their existence through the discovery of the photosynthetic unit, as reported by R. Emerson and W. Arnold in 1932, and
culminating in the crystallization and X-ray analysis of the anoxygenic bacterial reaction centers, reported by J.
Deisenhofer, H. Michel, and coworkers, over the period 1982–1987. Reaction centers of purple photosynthetic
bacteria have received the most attention because they have been well purified and characterized. Structures of
cyanobacterial reaction centers of Photosystems I and II are now available from the laboratories of H. Witt and W.
Saenger.
Background
In 1932, R. Emerson and W. Arnold published two
landmark papers on the yield of photosynthetic oxygen evolution in the green alga Chlorella in response
to brief flashes of light (Emerson and Arnold 1932a,
b). The yield per flash was maximal if the flashes,
about 10 µs in duration, were separated by more than
about 40 ms. The yield amounted to no more than
one O2 evolved for every 2400 molecules of chlorophyll (Chl), even though the quantum efficiency under
optimal conditions must have been one O2 per eight
quanta absorbed (Emerson 1958). Thus most of the
Chl is not involved directly in the photochemistry
of photosynthesis. Instead, it acts as an antenna for
absorbing light and delivering energy to a relatively
small number of reaction sites where the photochemistry of photosynthesis begins. Emerson and Arnold
called this system of ca. 2400 Chls a ‘photosynthetic
unit’. In contemporary terms of one quantum driving a single photochemical act (electron transfer), the
size of the photosynthetic unit in Chlorella is 2400/8
or about 300 Chls since 4 electrons are transferred
twice from water to CO2 . A quantum absorbed any-
where in an antenna of 300 Chls, or collectively by
the entire ensemble, could drive a single act of electric
charge separation, presumably at a single photochemical site. Thus, the concept of a reaction center served
by a light-harvesting antenna emerged in 1932, but lay
fallow for lack of specific knowledge for decades.
The literature of the 1930s and 1940s was
burdened by much speculation about ‘water splitting’
(formation of H and OH radicals, or the equivalent,
from H2 O) and by the great quantum efficiency controversy: one O2 evolved per four quanta according to
O. Warburg and his adherents and one O2 per eight
quanta according to R. Emerson and others. Eventually, the value of one O2 per eight quanta became
accepted by ‘the majority’ and the polemics dried up
(see Govindjee 1999).
Meanwhile, C. B. van Niel (see van Niel 1941)
developed a formulation for the overall chemistry
of photosynthesis, which unified the description for
green plants and photosynthetic bacteria and gave a
new view of the role of water: In bacteria, CO2 +
2H2 A + light → (CH2 O) + 2A, where H2 A can be H2 S
or any of a variety of oxidizable organic substrates
and (CH2 O) represents carbohydrate. In green plants,
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H2 A is H2 O. Water serves as the oxidizable substrate.
This formulation emphasized the recognition of photosynthesis as an oxidation–reduction process, and put
‘water splitting’ in a new perspective (see Clayton
1980).
During this period, every well-equipped laboratory had a Warburg–Barcroft apparatus for measuring
gas exchanges related to life processes. In the 1950s,
a well-equipped laboratory had a spectrophotometer,
which could monitor changes in the optical absorption spectra of biologically significant molecules. This
could give a far greater variety of specific information
than could the measurements of gas exchange. Optical techniques flourished and the Warburg–Barcroft
apparatus began to gather dust.
Students of photosynthesis could record small
changes in the optical absorption spectra of chlorophylls, cytochromes, and other substances, induced
by illumination of plant or microbial materials. Such
measurements, combined with information from biochemical analyses, and the discovery of the Emerson
enhancement effect (reviewed in Govindjee 2000 and
see later) culminated in the renowned ‘Z-scheme’ for
oxygenic photosynthesis, first proposed in 1960 by R.
Hill and F. Bendall (1960) and given compelling experimental support by L. N. M. Duysens, J. Amesz
and B. M. Kamp (1961).
In this scheme there are two kinds of photosystems in green plants, each performing a specific act:
a single electron transfer driven by one quantum. System I makes a strong reductant, able to mediate the
assimilation of CO2 , and a weak oxidant, an oxidized
cytochrome (Cyt). System II makes a strong oxidant, able to mediate the evolution of O2 from water,
and a weak reductant, a reduced quinone. The two
systems are connected by electrons flowing from reduced quinone to oxidized cytochrome. Eight quanta
can thus form the four oxidizing equivalents that can
release one O2 from water, and the four reducing
equivalents needed for assimilation of one CO2 .
We were thus ready to visualize and begin to characterize the specific photochemical reaction centers
(RCs)1 implied by Emerson and Arnold’s discovery
of the photosynthetic unit. Each type, I and II, of
RC in green plants mediates the transfer of a single
electron at the expense of one quantum. From optical absorbance changes suggesting reversible lightinduced oxidation of Chl a, it could be interpreted
[first for System I (B. Kok 1956); later for System
II (Döring et al. 1969) in H. Witt’s laboratory] that
each involves Chl a in the specialized context of a RC,
acting as the primary electron donor.
Green plants, algae, and cyanobacteria (‘bluegreen algae’) all show the same pattern of two distinct
photochemical systems with two types of RC. The
green and purple photosynthetic bacteria fall into several categories, but each type has but one kind of RC
that engages in one kind of primary photochemistry.
The RCs of the non-sulfur purple bacteria Rhodobacter sphaeroides and Rhodopseudomonas viridis have
proven to be especially easy to extract and purify
and have been the most rewarding sources of detailed
information on chemical patterns, composition, structure, and physical mechanisms. Accordingly, I shall
outline briefly much of what we have come to know
about the RCs of green plant Systems I and II, and
then examine in more detail what we have learned
about the bacterial photosynthetic RCs. Early accounts
are reviewed in Clayton (1978, 1980), and Breton and
Vermeglio (1988).
The reaction centers of green plant Photosystems I
and II
In the 1950s, information leading to the recognition
of two types of photosystems in green plants came
largely from action spectra: wavelength dependence
of an effect caused by illumination. Such studies
yielded some surprising results. First, there was the
‘red drop’ effect, discovered by Emerson and Lewis
(1943): Light beyond 680 nm, well within the absorption band of Chl a in vivo, was peculiarly ineffective
for photosynthesis. This deficiency could be corrected
by mixing in some shorter wave light (the Emerson
enhancement effect; see Emerson et al. 1957). The
enhancing light was most effective with light absorbed
in accessory pigments (such as Chl b) as well as
Chl a absorbing around 670 nm (Emerson and Rabinowitch 1960; Govindjee and Rabinowitch 1960).
Second, light absorbed by accessory pigments (such
as phycobilins) was more effective in promoting Chl
a fluorescence than was light absorbed by Chl a itself
(Duysens 1952; a review by Blinks 1954). These phenomena could be explained by proposing the presence
of two forms of Chl a in vivo: ‘long wave,’ effective
beyond 680 nm, and ‘shorter wave,’ ineffective beyond 680 nm (see Govindjee and Rabinowitch 1960 for
a ‘shorter wave’ Chl a 670; also see French 1961).
The shorter wave form is the more strongly fluorescent
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and is served preferentially by phycobilins where such
pigments are present.
The longer wave or ‘far red’ forms, Chl a 680
and beyond, were found to mediate oxidation of endogenous Cyt or of an externally supplied electron
donor, and did not promote O2 evolution, explaining
the ‘red drop.’ The shorter wave Chl a 670 favored
reduction of oxidized Cyt or of an externally supplied
electron acceptor, coupled with O2 evolution (‘Hill
reaction’). These observations became crystallized in
the Z-scheme, and we could begin to inquire into the
natures of the two photosystems.
Meanwhile a wealth of information began to come
from the development of new instruments that could
detect changes of optical absorbance with unprecedented sensitivity. L.N.M. Duysens first applied his newly
constructed instrument to the study of photosynthetic bacteria (Duysens 1952, 1953); subsequently
he and others, including B. Kok (1956), H.T. Witt
and coworkers (1961), B. Chance (Chance and Smith
1955, Chance and Strehler 1957) and others detected minute light-induced changes in the absorption
of light by green plant tissues, as well as by photosynthetic bacteria. These changes reflected oxidation
of Chl a and Cyt, reduction of quinones, and other
events including shifts in the wavelengths of absorption maxima attributed to electric fields arising from
charge separation (oxidation–reduction). The optical
measurements often became more informative when
they were correlated with changes in the fluorescence
emitted by Chl in the material, since fluorescence
and photochemical utilization are alternative fates of
excitation energy in Chl.
Information has also come from increasingly refined measurements of electron paramagnetic resonance (EPR). In broken cell preparations, another dimension could be added to all of the foregoing observations by poising the material at specific ambient
oxidation–reduction potentials. This could reveal the
midpoint potential of an electron transfer event and
also the number of electrons involved.
In time, against a background of much more decisive information obtained with photosynthetic bacteria,
many of these observations came to be associated with
photochemical activities in or in proximity to the RCs.
These developments came mainly in the 1960s and
1970s. The results can be summarized as follows.
The RC of System II is analogous to those of
purple bacteria insofar as singlet excited Chl a 680,
P680, transfers an electron to pheophytin a (Pheo a)
and on to plastoquinones, QA and QB . But the elec-
tron donor to oxidized Chl a 680 is not a Cyt but ‘Z’
(now called YZ ), identified with a tyrosine residue in
a polypeptide ‘D1’ that is a part of the RC complex.
The Z, in turn, interacts with a manganese complex
that cycles through four progressive states and results
in the liberation of O2 from water (for references and
details, see Ke 2001).
The RC of System I resembles that of the purple
bacteria insofar as P700, a Chl a dimer (or ‘special
pair’; see later about P870), gives up an electron when
excited, and the oxidized P700 is re-reduced by a
Cyt (Cyt f) through the copper-containing plastocyanin. Excited P700 delivers electrons not to Pheo and
quinones, but to iron–sulfur complexes (ferredoxins).
So the reducing side of the System I RC is not like
that of the purple bacteria; rather it resembles that of
the green sulfur bacteria, making a reductant (reduced
Fe–S complexes) more electronegative than reduced
quinone (see, e.g., a review by Fromme 1999).
Perhaps the RCs of purple bacteria diverged in
two directions, becoming able to make a stronger
reductant, as in green plant System I and in green
sulfur bacteria, and a stronger oxidant, as in green
plant System II. The latter development would have
had enormous survival value, making water accessible as oxidizable substrate. This development altered
the planetary environment drastically and changed the
nature of all subsequent life on Earth. For a recent
study of reaction center evolution, see Xiong et al.
(2000).
The reaction centers of anoxygenic photosynthetic
bacteria
My own odyssey in photosynthesis research began
in collaboration with William Arnold (see my ‘Personal perspective,’ Clayton 1988; at the suggestion
of the editors, pictures of the author are shown in
Figure 1). Chance and M. Nishimura (1960) had
just shown that in the purple bacterium Chromatium
vinosum, light-dependent oxidation of a Cyt proceeds
at 77 K. Wondering if drying acted like low temperature, in limiting reactions that depend on diffusion,
we prepared dried films of membrane fragments from
Rhodobacter sphaeroides and looked for photochemical activity as evidenced by light-induced changes of
optical absorbance. We found, as had L.N.M. Duysens
earlier in cells of Rhodospirillum rubrum (Duysens
1952, 1953; Duysens et al. 1956), that light caused
changes, suggesting the photochemical oxidation of
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Figure 1. Four images of the author. Top left: from the Student Yearbook, California Institute of Technology, 1941, age 19. Top right: at the C.F
Kettering Research Laborotory, 1964. The instrument is part of a helium cryostat. Bottom left: at Cornell University, ca. 1972. Bottom right:
self portrait, 2000.
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Figure 2. Absorption spectrum (a) and spectra of light-induced absorbance changes (b, c) of reaction centers isolated from Rb. sphaeroides.
Trace b, reaction centers alone. Trace c, reaction centers plus an electron donor to prevent accumulation of oxidized bacteriochlorophyll during
illumination. Changes due to oxidation of the donor have been discounted. The increase at 1250 nm (trace b) shows formation of oxidized
bacteriochlorophyll. The features at 275 nm and 320 nm reflect reduction of ubiquinone. Reproduced from Clayton (1980).
bacteriochlorophyll (BChl) manifested mainly as a
small partial bleaching of the long wave absorption
band near 870 nm. The presumptive species responsible for the small change had already been termed
P870 by Duysens (1952). Similar changes were caused
by chemical oxidation and the effects of light and
oxidants were complementary. We found the lightinduced change to be reversible, even at 1.3 K (W.
Arnold and R.K. Clayton 1960). Other changes suggested that ubiquinone (UQ) is involved as an electron
acceptor (see Figure 2). At the time, we had no way
of knowing whether the effects we were seeing reflected a small change of absorbance in the antenna or a
large change in a specific minor component: BChl in
a reaction center. This question was answered through
a fortuitous and remarkably fortunate choice of material. We had developed a carotenoidless mutant of
Rb. sphaeroides, designated R26 (Clayton and Smith
1960), in order to study the irreversible photooxidative destruction of BChl, already known to occur in
carotenoidless Rsp. rubrum. (Incidentally, I had selected a constitutive high-catalase mutant in isolating
R26 because I wondered if that enzyme would protect
against the destruction of BChl. It did not.) In strain
R26, the major absorption bands at 800 and 850 nm
were missing, leaving a large band at 870 nm and
much smaller ones at 760 and 800 nm. The reversible
bleaching amounted to about four per cent of the total
absorbance at 870 nm, reflecting an unusually small
antenna for a photosynthetic unit.
When I accidentally left some mature cultures of
strain R26 in illuminated bottles for two weeks, I
saw that their slate blue color had changed to pink.
Companion cultures kept anaerobically in the dark
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did not show this change, nor did illuminated cultures of the parent strain that contained carotenoid
pigments. In the pink cultures nearly all of the BChl
had been converted, in an irreversible light-dependent
process, to bacteriopheophytin (Bpheo, BChl with the
magnesium atom removed). In this pheophytinized
material, the 870 nm band of BChl was much attenuated, and what remained showed almost complete
reversible light-induced bleaching. The magnitude of
this effect was the same as in the companion cultures
kept in the dark and in which the change amounted
to four per cent of the total absorbance. This showed
that the reaction occurred in a specific minor component in which the BChl had resisted conversion to
BPheo and not in the antenna as a whole. I identified this minor component hypothetically as part of the
photosynthetic RC (Clayton 1963).
Most of the BPheo could be washed away from
broken cell preparations of pheophytinized strain R26,
leaving an absorption spectrum showing maxima at
760 nm (residual BPheo), 800 nm (BChl a), and 870
nm (photoactive BChl a). I obtained a similar spectrum by illuminating cell membrane fragments in the
presence of the detergent Triton X-100. In this treatment the antenna BChl was degraded without harm
to the photoactive part. When D. Reed came to my
laboratory in 1967, he and I attacked the problem
of separating the RC component from the rest of the
photosynthetic membrane. We succeeded in this, using broken cell preparations treated with Triton X-100
in two ways: (1) centrifugation through a sucrose
density gradient and (2) fractional precipitation with
ammonium sulfate. We thus had our first relatively
crude RC preparation (Reed and Clayton 1968). G. Feher then obtained much cleaner preparations by using
lauryl dimethyl amine oxide instead of Triton X-100
as the detergent (see Feher 1971; and the Personal
perspective of Feher 1998).
In Feher’s laboratory and in mine the following
picture emerged (see Feher 1971; and G. Feher and
M. Okamura 1978), with Feher’s group providing the
greater part of the definitive characterization. The RC
in Rb. sphaeroides is a protein consisting of three subunits H, M, and L (for heavy, medium, and light)
weighing 28, 24, and 21 kilodaltons, respectively.
Photochemical activity resides in M and L; H can be
removed without loss of activity. Bound to this protein
are two molecules of Bpheo a and four of BChl a. The
latter are responsible for the absorption bands at 800
and 870 nm. The bound BPheo a contributes the band
at 760 nm. Other components are two molecules of
UQ and an iron atom. One of these quinones, UQ1 , is
tightly bound; the other, UQ2 , can be removed along
with the Fe atom by chemical treatments.
J.R Norris and J.J. Katz (1978) proposed the term
‘special pair’ rather than ‘dimer’ for the two molecules
of P870 to emphasize that the exact nature of their interaction was not known. Water appears to be involved
in the structure and function of RCs: exhaustive dessication causes loss of photochemical activity, restored
on rehydration (Clayton 1966).
The other two BChl molecules, responsible for
the 800 nm band, appeared to be mere voyeurs of
the photochemistry, but evidence has emerged for
their participation in the earliest phases of the photochemical electron transfer. One of the two BPheos
is definitely implicated as an early electron acceptor
in the process. Strictly speaking, the bands at 800
and 870 nm should be regarded as joint properties of
all four BChl, describable by a single wave function.
However, it is useful, and has not been misleading, to
regard the two pairs as separate entities, each with its
own properties and function.
For a time the view was defended that the primary
event is oxidation of Cyt coupled to reduction of
BChl, with oxidation of P870 regarded as an aberrant
reaction; perhaps a safety valve for dissipation of excess energy. Apart from the lack of any evidence for
the photochemical formation of reduced BChl, this
view was laid to rest in two ways. First, W. Parson (1968, 1978), exposing subcellular preparations
of Chr. vinosum to brief laser flashes, observed that
the oxidation of P870 was complete within 0.2 µs
and the oxidation of Cyt then paralleled the return of
P870 to its reduced form, with a half-time of about
2 µs. Second, a mutant of Rb. sphaeroides (strain
PM-8, isolated by W.R. Sistrom, B.M. Ohlsson and
J. Crounce in 1963) possesses a normal content of
antenna BChl but cannot grow photosynthetically. It
must be grown aerobically. This strain lacks P870;
there is no evidence that it has RCs (Clayton et al.
1965; Clayton and Sistrom 1966).
More has been learned from the fluorescence of
BChl, especially in isolated RCs. In RCs, fluorescence
accompanies the return of singlet-excited P870 to its
ground state. This process competes with other fates
of the excited state, mainly the photochemistry of photosynthesis: oxidation of P870 coupled with reduction
of UQ by way of earlier electron acceptors, BPheo and
perhaps BChl 800.
In photochemically active RCs, the photochemical
efficiency is close to unity and the efficiency (quantum
69
yield) of fluorescence is 3 × 10−4 (Clayton 1967;
Zankel et al. 1968; Wraight and Clayton 1973). If
fluorescence were the only pathway for dissipation of
singlet-excited P870, this excited state would have its
‘intrinsic’ lifetime τ 0 . With other pathways operating,
in this case photochemical utilization, the lifetime is
shorter, in proportion to the efficiency of the competing pathway. Then the ratio of actual lifetime τ to
intrinsic lifetime τ 0 will equal the quantum yield of
fluorescence; in this case τ /τ 0 = 3 × 10−4 . Because
the probability of an upward transition (P + hν → P∗ ;
absorption) is proportional to that of the corresponding
downward transition (P∗ → P +hν, fluorescence), the
intrinsic lifetime can be estimated from the integrated
area under the absorption band, in this case the 870 nm
band. This gives τ 0 =2 × 10−8 s and τ = 2 × 10−8 ×
3 × 10−4 or 6 × 10−12 s. We could thus estimate that
the first step in the photochemistry of photosynthesis,
with conversion of singlet-excited P870 to oxidized
P870, occurs in about 6 ps (Zankel et al. 1968; Clayton
1978). The validity of this estimate was confirmed
when the picosecond time domain became accessible
to spectroscopists.
The new technology also showed BPheo to be an
electron carrier between P870 and UQ. It could be
seen that BPheo becomes reduced within less than 10
ps after a flash, and passes its electron to the tightly
bound UQ1 with a half-time of 200 ps. Finally, some
small, rapid absorbance changes around 800 nm might
implicate the two molecules of B800 as the very earliest electron acceptors, perhaps in a biradical P+. B−
(reviewed in Clayton 1980).
At this state of knowledge, the RCs of three species of purple bacteria, the non-sulfur bacteria Rb.
sphaeroides and Rhodopseudomonas viridis and the
purple sulfur bacterium Chr. vinosum, show very similar properties, even though Rps. viridis does not
possess BChl a but BChl b, with long-wave absorption
maxima at 1020 nm (antenna) and 980 nm (special
pair, P980) (see Gingras 1978; and Blankenship et al.
1995).
Early research on RCs culminated in the period
1982–1987 with the crystallization and X-ray structural analysis of RCs from Rps. viridis (H. Michel
1982; J. Deisenhofer et al. 1984; H. Michel et al.
1986; J. Deisenhofer and H. Michel 1988, 1989). Similar success was then obtained with RCs from Rb.
sphaeroides by G. Feher and coworkers (Allen and
Feher 1984; Allen et al. 1988; and see Personal perspective by Feher 1998), by J.R. Norris and coworkers
(C.H. Chang et al. 1985; P. Gast and J.R. Norris 1984),
and by Arnoux et al. (1995).
Although the first membrane proteins to be crystallized were rhodopsin (Michel and Oesterhelt 1980)
and porin (Garavito and Rosenbush 1980), the work
with RCs from Rps. viridis earned Deisenhofer and
Michel (and R Huber) a Nobel Prize in 1988 because it
was of great significance for the future understanding
of structure and function in biological membranes.
Recently the research groups of H. T. Witt and W.
Saenger have crystallized and provided X-ray analysis
of RC complexes in Photosystem I (Krauss et al. 1993;
Jordan et al. 2001) and in Photosystem II (Zouni et al.
2001). X-ray analysis of the crystals of RCs from Rps.
viridis and Rb. sphaeroides revealed their structures
in exquisite detail, showing the positions of the chromophores and of bound UQ and Cyt. These definitive
results are consistent with many earlier conclusions
about RCs, drawn on a trail often littered with luck
and faith.
Acknowledgments
I am indebted to Charles McDougall and Jorjan
Powers for their expertise with computers. I thank
Govindjee and Colin Wraight for helpful comments
and for providing many references.
Note
1 I introduced the term ‘reaction center’ in 1963, but some students of photosynthesis were surely aware of the concept since
Emerson and Arnold’s discovery of the photosynthetic unit.
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