TALKING POINT
TIBS 23 – MARCH 1998
4 Hijikata, M. et al. (1991) Proc. Natl. Acad. Sci.
U. S. A. 88, 5547–5551
5 Hijikata, M. et al. (1993) J. Virol. 67, 4665–4675
6 Grakoui, A. et al. (1993) Proc. Natl. Acad. Sci.
U. S. A. 90, 10583–10587
7 Grakoui, A. et al. (1993) J. Virol. 67,
2832–2843
8 Houghton, M. (1996) in Fields Virology (Vol. 1)
(Fields, B. N., Knipe, D. M. and Howley, P. M.,
eds), pp. 1036–1041, Lippincott-Raven
9 Voss, T. et al. (1995) Protein Sci. 4, 2526–2531
10 Sommergruber, W. et al. (1994) Virology 204,
815–818
11 Pieroni, L. et al. (1997) J. Virol. 71, 6373–6380
12 Vallee, B. L. and Auld, D. S. (1990)
Biochemistry 29, 5647–5659
13 Springman, E. R. et al. (1990) Proc. Natl. Acad.
Sci. U. S. A. 87, 364–368
14 Reed, K. E. et al. (1995) J. Virol. 69, 4127–4136
15 Lipscomb, W. N. and Strater, N. (1996) Chem.
Rev. 96, 2375–2433
16 Rawlings, N. D. and Barrett, A. J. (1994)
Methods Enzymol. 244, 461–486
17 Rawlings, N. D. and Barrett, A. J. (1997) in
Proteolysis in Cell Functions (Hopsuhavu, V. K.,
Jarvinen, M. and Kirschke, H., eds), pp. 13–21,
IOS Press
–1.0
The origin and evolution of
oxygenic photosynthesis
P870*/P870+
P680*/P680+
–0.5
Robert E. Blankenship and Hyman Hartman
THE ADVENT of oxygen-evolving photosynthesis is one of the central events in
the development of life on Earth. Before
the evolution of this metabolic capability,
the atmosphere of the early Earth was
largely anaerobic1,2. The development of
advanced eukaryotic life forms did not
take place until the free oxygen in the
atmosphere rose to a sufficient level.
While significant questions remain over
when oxygenic photosynthesis began3–5,
no other known process, either biogenic
or non-biogenic, is capable of producing
the large quantities of molecular oxygen
that demonstrably changed the course of
life on Earth. Understanding the evolutionary origin of this metabolic process is
therefore of considerable importance.
All known oxygen-evolving photosynthetic organisms contain two photosystems linked in series6. Water oxidation is
carried out by photosystem II and ferredoxin reduction is mediated by photosystem I. Several groups of anoxygenic
(non-oxygen-evolving) photosynthetic
R. E. Blankenship is in the Department of
Chemistry and Biochemistry, Center for the
Study of Early Events in Photosynthesis,
Arizona State University, Tempe,
AZ 85287-1604, USA. H. Hartman is at the
Institute for Advanced Studies in Biology,
880 Spruce St., Berkeley, CA 94707, USA.
94
bacteria contain reaction center complexes that show clear evolutionary
relatedness to one or other of the two
photosystems found in oxygenic organisms and are thought to be more closely
related to the earliest photosynthetic
organisms7–10. However, no native organisms containing only a single photosystem are known that can oxidize water.
Also, all known oxygen-evolving organisms contain the photosynthetic pigment
chlorophyll a, while anoxygenic photosynthetic bacteria contain one of several
bacteriochlorophylls (a, b or g) as reaction center pigments, all of which absorb longer wavelength, and therefore
lower energy, light than does chlorophyll a. The major consequence of this is
that the excited state energy and therefore the redox potential span that can
be generated by bacteriochlorophyllcontaining organisms is significantly less
than in chlorophyll-containing organisms
(see Fig. 1).
Oxygenic photosynthesis is found in
cyanobacteria and related prokaryotes,
and in a variety of eukaryotic organisms
whose chloroplasts were formed by endosymbiosis of a simpler photosynthetic
organism11. The mechanism of oxygen
production in all known oxygenic photosynthetic organisms appears to be very
similar, and involves charge separation
0.0
Em′ (volts)
The evolutionary developments that led to the ability of photosynthetic
organisms to oxidize water to molecular oxygen are discussed. Two major
changes from a more primitive non-oxygen-evolving reaction center are
required: a charge-accumulating system and a reaction center pigment
with a greater oxidizing potential. Intermediate stages are proposed in which
hydrogen peroxide was oxidized by the reaction center, and an intermediate pigment, similar to chlorophyll d, was present.
Fe2+/Fe(OH)3
H2O2/O2
0.5
P870/P870+
H2O/O2
1.0
P680/P680+
1.5
Figure 1
Midpoint reduction potentials at pH 7 and
25⬚C (Em⬘) of various redox couples relevant to photosynthetic electron transfer.
All potentials shown are relative to the normal hydrogen electrode and are written
as reduced/oxidized forms of the redox
couple. The reaction depicted is: oxidized
+ e – → reduced. Hydrogen ions are not
shown. The vertical arrows on the right
indicate the photon excitation in the purple
bacterial reaction center (P870 → P870*)
and the photosystem II reaction center
(P680 → P680*). The length of these
arrows indicates the excitation energy and
also the redox span between the ground
and excited state redox potentials27. The
redox reactions in the text (Eqns 1 and 2)
are indicated as oxidations, although, by
convention, the midpoint potentials are
given for the reduction reaction. The reaction center potentials are taken from
Ref. 27, the Fe2+/Fe(OH)3 potential from
Ref. 26 and the H2O and H2O2 potentials
from Ref. 34.
Copyright © 1998, Elsevier Science Ltd. All rights reserved. 0968 – 0004/98/$19.00
PII: S0968-0004(98)01186-4
TALKING POINT
TIBS 23 – MARCH 1998
by a chlorophyll a-containing photosynthetic reaction center and charge accumulation by a Mn–protein complex prior
to the actual conversion of water into
molecular oxygen12–15. Water is a very
stable compound, and its oxidation to
molecular oxygen requires a powerful
oxidizing agent (Eqn 1):
2H2O → O2 ⫹ 4H⫹ ⫹ 4e ⫺
(1)
The midpoint reduction potential for
this process is E m⬘ = ⫹0.82 V (Fig. 1); thus
an oxidant with redox potential greater
than 0.82 V is needed to decompose water
into molecular oxygen.
Two major evolutionary developments
are required before water oxidation can
take place: the development of an oxidant
with a sufficiently high redox potential
and the ability to collect and store oxidizing equivalents, formed by the photochemical events in the reaction center,
that transfer at most one electron per
photon absorbed. Neither of these properties is found in any of the known anoxygenic photosynthetic reaction centers,
nor are any intermediate forms known.
The midpoint redox potential of P680, the
reaction center photoactive chlorophyll
of photosystem II, is greater than 1 V
(Refs 12, 13), fully half a volt above the
bacteriochlorophyll-containing reaction
centers found in anoxygenic bacteria16.
The simultaneous evolutionary development of both the high redox potential
species needed to oxidize water and the
charge accumulation system needed to
carry out the four-electron chemistry of
water splitting (Eqn 1) would seem impossible, as each characteristic is almost
certainly the result of several independent molecular changes in the reaction
center proteins. What is needed is a series
of transitional forms, in which one of
these capabilities arises first in a simplified, but still functional, form and the
other characteristic then arises later.
Olson17 proposed a series of nitrogen
compounds of increasing redox potentials
as possible transitional electron donors to
a reaction center that gradually increased
its redox potential as the more easily oxidized species were depleted. However,
only one of these compounds is able to be
oxidized by any known photosynthetic
reaction center, and this proposal also
does not provide a plausible transition
to the present Mn-containing system in
which the electron donor is water.
Here, we propose an evolutionary scenario that provides for functional intermediate forms that both benefited the
organisms that contained them and link
to the current system. The first proposal
is that hydrogen peroxide may have
been a transitional electron donor on the
early Earth and that the current oxygenevolving complex may be structurally
related to Mn-containing catalase enzymes. The second proposal is that a
pigment related to chlorophyll d may have
been an intermediate pigment between
bacteriochlorophyll-containing reaction
centers and chlorophyll a-containing reaction centers.
Hydrogen peroxide as a transitional
electron donor
Hydrogen peroxide is capable of being
both an oxidant and a reductant. The oxidation of hydrogen peroxide to oxygen
can be carried out by a modestly oxidizing species (Em⬘ = 0.27 V), which is fully
within the oxidative capabilities of reaction centers from existing anoxygenic
photosynthetic bacteria (Fig. 1).
H2O2 → 2H⫹ ⫹ 2e ⫺ ⫹ O2
(2)
A bacteriochlorophyll-containing reaction center is thermodynamically capable of oxidizing hydrogen peroxide to
molecular oxygen. This oxidation is similar to part of the mechanism of catalase
enzymes. The reaction cycle of catalase
involves both oxidation and reduction
of hydrogen peroxide18. Mn catalase enzymes are known that have a binuclear
metal center that is structurally similar
to half of the proposed geometry of the
tetranuclear Mn center that makes up
the photosynthetic oxygen-evolving complex18,19 (Fig. 2). This structural similarity
has been previously noted18–20. Under
certain conditions, the photosynthetic
Mn complex can act as a catalase21,22. The
possibility of hydrogen peroxide serving
as a transitional electron donor has also
been suggested previously20,22,23.
An association of an anoxygenic photosynthetic reaction center and a Mn catalase enzyme might produce a primitive
system that evolved oxygen, using hydrogen peroxide as an electron donor. Subsequent developments could convert
the binuclear Mn site to a tetranuclear
site capable of accumulating up to four
oxidizing equivalents. The gene sequence
for the Mn catalase from Lactobacillus
plantarum has recently been published24.
There are no obvious sequence similarities to the loop regions of the D1 protein,
which is thought to provide the majority
of the ligands to the Mn cluster in photosystem II. However, the precise ligands
to the Mn in both the oxygen-evolving
center and the Mn catalase have not yet
Figure 2
Proposed structures for the oxygen-evolving
center (top) and Mn catalase active site
(bottom), redrawn from Refs 14 and 18,
respectively. The blue atoms are Mn, the
red atoms are O and the yellow atoms
are C. Some additional bonds to the Mn
centers have been omitted for clarity.
been conclusively identified, so a definite conclusion as to whether a distant
homology exists is not yet possible.
Additional sequences from Mn catalases
will be useful in this analysis.
Was hydrogen peroxide present on
the early Earth? The early atmosphere is
thought to have been mildly reducing or
neutral in overall redox balance1,2. Water
photolysis by UV light can produce hydrogen peroxide, which then might be concentrated by rainfall in certain protected
environments25. Thus, the presence of
hydrogen peroxide on the early Earth is
possible, although only a small amount
might have accumulated due to its intrinsic reactivity. Therefore, it seems unlikely that hydrogen peroxide was ever
the principal electron donor to the primitive photosystem because of the limited
amounts available. A more likely candidate for an early electron donor is Fe2+,
which was present in substantial quantities in the Archean oceans1. Purple
photosynthetic bacteria that can oxidize
ferrous iron to the ferric form have recently been described, although the enzyme complexes that catalyze this oxidation have not yet been identified26.
95
TALKING POINT
H 3C
H 3C
TIBS 23 – MARCH 1998
O
A
H 2C
H
N
N
CH3
H
B
CH2CH3
H 3C
CH
A
N
Mg
H 3C
N
N
C
CH3
H 3C
N
H
E
ROOC
O
CH3OOC
O
CH3
N
N
B
CH2CH3
H 3C
A
H
N
CH3
N
Mg
H 3C
N
N
D
C
CH3
H
N
CH2CH3
C
CH3
N
D
E
CH3OOC
B
Mg
H 3C
E
HH
ROOC
O
(b) Chlorophyll a
H
O
A
CH3
E
(a) Bacteriochlorophyll a
H 3C
C
HH
CH3OOC
H 3C
CH2CH3
N
D
HH
ROOC
B
Mg
N
D
H
CH3
HH
O
(c) 3-Acetyl-chlorophyll a
ROOC
CH3OOC
O
(d) Chlorophyll d
Figure 3
Chemical structures of (a) bacteriochlorophyll a and (b) chlorophyll a. Differences in the structures are shown in red. Chemical structures of (c) 3-acetyl-chlorophyll a and (d) chlorophyll d.
R is the phytyl tail.
We propose that the Mn catalase developed to detoxify the hydrogen peroxide
that was present in a local environment
on the early Earth. This catalase was
then recruited to extract electrons from
the hydrogen peroxide, producing the
first oxygen-evolving complex. Finally,
the dimanganese center evolved to become the four-manganese center of the
oxygen-evolving center so that it could
extract electrons from water.
The development of a highly oxidizing
reaction center
While the scenario described above for
the origin and evolution of the Mn center
provides a plausible way in which the
early reaction center might begin to produce oxygen from hydrogen peroxide, it
does not address the issue of how the
highly oxidizing species that is needed
to split water developed. To understand
this issue, it is important to appreciate
that the essence of the primary electron
transfer process in photosynthesis is an
oxidation of the excited state of the reaction center primary donor pigment to
generate a reduced acceptor molecule
and an oxidized donor27. The effective
redox potential of the excited state is
a function of the ground state redox
96
potential of the donor and the excitation
energy. The ground state redox potential of the primary donor in purple bacterial reaction centers is approximately
+0.5 V. This potential is determined by
both the intrinsic redox behavior of the
bacteriochlorophylls and the details of
their environment in the reaction center
protein. Recent work has shown that the
redox potential of the primary donor
bacteriochlorophyll a in reaction centers
from the purple bacterium Rhodobacter
sphaeroides can be raised dramatically
by engineered mutations that increase the
number of hydrogen bonds to the bacteriochlorophyll28. However, a necessary
result of the increase in redox potential
of the donor is that the potential of the
excited state becomes less negative in
parallel, because the redox span (i.e. the
difference in redox potentials between the
ground and excited states) is determined
by the energy of the excitation photon,
which is at 870 nm in Rb. sphaeroides
(Fig. 1). If the excited state potential is
increased too much, then the quantum
yield of electron transfer falls, because
the energy of the reduced acceptor now
lies above the energy of the excited state
of the donor. In fact, this has been shown
to occur in engineered mutants in which
the potential of the donor has been
greatly increased29. While changing the
environment of the primary donor can
substantially modulate the potential of
the primary donor, that in itself is not
enough to create a working reaction center with both a highly oxidizing potential
of the donor and a sufficiently reducing
potential of the excited state. The only way
to do both these things is to increase the
photon energy by shifting the absorption
maximum from the near infrared into
the visible region of the electromagnetic
spectrum. This assumes that the redox
properties of the electron acceptors are
unchanged. The overall reaction center
structure and especially the acceptor systems are very similar in photosystem II
and the purple bacterial reaction centers.
The chemical structures of chlorophyll a and bacteriochlorophyll a are
shown in Fig. 3. There are two differences, which are shown in red. The first
is the oxidation state of ring B, which is
oxidized in chlorophyll a and reduced,
converting the double bond into a single
bond, in bacteriochlorophyll a. The second difference is the presence of the
acetyl substituent at the 3 position in
ring A in bacteriochlorophyll a instead
of the vinyl found in chlorophyll a. The
spectral differences between these two
pigments are primarily due to the first of
these two structural differences.
How might bacteriochlorophyll a be
converted into chlorophyll a? In the modern biosynthetic pathways of these pigments, chlorophyll is an intermediate on
the way to bacteriochlorophyll30. The biosynthetic pathway of both these pigments
includes a reduction of ring D, with the
formation of a single bond instead of a
double bond. In bacteriochlorophyll biosynthesis, an additional reduction of
ring B takes place. This reaction is catalyzed by a set of three enzymes (coded
by the bchlX, bchlY and bchlZ genes) that
are clearly homologous to the enzymes
(coded by the chlL, chlN and chlB genes)
that reduce ring D in the more primitive
oxygen-evolving organisms. Burke and
coworkers31 proposed that the ancestral
reductase was non-specific and reduced
both rings B and D, producing bacteriochlorophyll without intermediate chlorophyll formation (however, see Ref. 32). A
small change in substrate specificity of
one or more of these primitive enzymes
such that ring B is no longer reduced
would produce a situation in which the
chlorophyll-like pigment 3-acetyl-chlorophyll a is produced (Fig. 3c). This pigment is nearly identical to chlorophyll d,
which has recently been reported as the
TALKING POINT
TIBS 23 – MARCH 1998
Conclusions
The proposed scenario is that the ability to oxidize hydrogen peroxide by an
ancestral bacteriochlorophyll-containing
reaction center similar to the purple
bacterial complex was the first step in
the progression that led to the oxygenevolving complex of photosystem II. The
second step was the conversion from
bacteriochlorophyll to chlorophyll, which
raised the redox potential of the reaction
center pigment sufficiently to oxidize
the very weak electron donor water. Additional evolutionary steps that led to two
linked photosystems were also needed
to produce the present system found in
all oxygenic photosynthetic organisms.
These steps either could have followed
the steps described above, or possibly
could have preceded them.
Acknowledgements
The authors thank Drs John Olson,
James Allen and Wayne Frasch for helpful discussions. Supported by a grant
from the Exobiology program of NASA.
This is publication 342 from the Arizona
State University Center for the Study of
Early Events in Photosynthesis.
References
1 Schopf, J. W. and Klein, C., eds (1992) The
Proterozoic Biosphere, Cambridge University Press
2 Kasting, J. F. (1993) Science 259, 920–926
3 Doolittle, R. F. et al. (1996) Science 271, 470–477
4 Castresana, J. and Saraste, M. (1995)
Trends Biochem. Sci. 20, 443–448
5 Martin, W. F. (1996) BioEssays 18, 523–527
–1.0
Stage 1
Stage 2
Stage 3
P*
P*
P*
Stage 4
P*
–0.5
Em (volts)
principal pigment in a newly discovered
oxygenic prokaryotic photosynthetic organism33. Chlorophyll d has an in vivo
absorption maximum at only a slightly
longer wavelength than chlorophyll a
(716 nm versus 680 nm). This modest
change in the biosynthetic enzyme specificity would produce a reaction center
donor pigment that is structurally similar
to bacteriochlorophyll but with a redox
potential that is capable of oxidizing water.
The acetyl group would fit the preexisting hydrogen-bonding interactions for the
bacteriochlorophyll a. Finally, the ability
to make the acetyl group at the 3 position
was lost and the reaction center pigment
became chlorophyll a. Information on the
pathway and enzymes involved in the
biosynthesis of chlorophyll d would be
important in evaluating this proposal.
Figure 4 shows the stages of development of the reaction center protein
from purple bacterial complex to photosystem II. The intermediate stages involve the association of the Mn catalase
and the intermediate pigment similar to
chlorophyll d.
0.0
H 2 O2
D
D
0.5
P
H2O2
D
P
H2O
P
1.0
P
BChl
DRed
DOx
Chl d
BChl
DRed
Mn Cat
DOx
H2O2
O2
DRed
Chl a
Mn
OEC
DOx
H2O2
H2O
O2
O2
Figure 4
Evolutionary stages of oxygen evolution capacity (OEC). Four stages are depicted, although
additional intermediate stages undoubtedly also existed. For each stage, the upper diagram
shows an energetic picture, and the lower diagram a schematic of the reaction center protein
in the photosynthetic membrane. Stage 1: the energetics are the same as those found in contemporary purple bacterial reaction centers. The pigment is bacteriochlorophyll (BChl) a, and
an external donor (D), possibly Fe2+, reduces the oxidized reaction center special pair pigment (P). Stage 2: the energetic and pigment composition are the same as stage 1, but the
Mn catalase is present and can donate electrons to P, although probably with low efficiency.
Stage 3: the pigment has changed to chlorophyll (Chl) d or a similar pigment, and the Mn
catalase (Mn Cat) has become more closely associated with the reaction center. The higher
excitation energy of the chlorophyll d pigment increases the redox potential of P. Stage 4: the
pigment has converted to chlorophyll a and the Mn center has become fully incorporated into
the reaction center protein. The known ground and excited state redox potentials for purple bacteria reaction centers were used for stages 1 and 2 and those of photosystem II for stage 4,
calculated according to Ref. 27. The redox potentials of stage 3 were estimated using the
in vivo excitation energy of chlorophyll d (Ref. 33), assuming that the redox potential of the
excited state is unchanged from that of stage 2.
6 Ort, D. R. and Yocum, C. F., eds (1996) Oxygenic
Photosynthesis: The Light Reactions, Kluwer
7 Olson, J. M. and Pierson, B. K. (1987) Int. Rev.
Cytol. 108, 209–248
8 Blankenship, R. E. (1992) Photosynth. Res. 33,
91–111
9 Nitschke, W. and Rutherford, A. W. (1991)
Trends Biochem. Sci. 16, 241–245
10 Mulkidjanian, A. Y. and Junge, W. (1997)
Photosynth. Res. 51, 27–42
11 Whatley, J. M. (1993) Int. Rev. Cytol. 144,
259–299
12 Debus, R. J. (1992) Biochim. Biophys. Acta
1102, 269–352
13 Britt, R.D. (1996) in Oxygenic Photosynthesis:
The Light Reactions (Ort, D. R. and Yocum, C. F.,
eds), pp. 137–164, Kluwer
14 Yachandra, V. K., Sauer, K. and Klein, M. P.
(1996) Chem. Rev. 96, 2927–2950
15 Rögner, M., Boekema, E. J. and Barber, J.
(1996) Trends Biochem. Sci. 44–49
16 Blankenship, R. E., Madigan, M. T. and Bauer,
C. E., eds (1995) Anoxygenic Photosynthetic
Bacteria, Kluwer
17 Olson, J. M. (1970) Science 168, 438–446
18 Penner-Hahn, J. E. (1992) in Manganese Redox
Enzymes (Pecoraro, V., ed.), pp. 29–45, VCH
19 Dismukes, G. C. (1996) Chem. Rev. 96,
2909–2926
20 Rutherford, A. W. and Nitschke, W. (1996) in
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Origin and Evolution of Biological Energy
Conversion (Baltscheffsky, H., ed.),
pp. 143–175, VCH
Frasch, W. D. and Mei, R. (1987) Biochim.
Biophys. Acta 891, 8–14
Bader, K. P. (1994) Biochim. Biophys. Acta
1188, 213–219
Samuilov, V. D. (1997) Biochemistry (Moscow)
62, 451–454
Igarashi, T., Kono, Y. and Tanaka, K. (1996)
J. Biol. Chem. 271, 29521–29524
McKay, C. P. and Hartman, H. (1991) Origins
Life Evol. Biosphere 21, 157–163
Widdle, F. et al. (1993) Nature 362, 834–836
Blankenship, R. E. and Prince, R. C. (1985)
Trends Biochem. Sci. 10, 382–383
Lin, X. et al. (1994) Proc. Natl. Acad. Sci. U. S. A.
91, 10265–10269
Woodbury, N. W. et al. (1995) Chem. Phys. 197,
405–421
Porra, R. J. (1997) Photochem. Photobiol. 65,
492–516
Burke, D. H., Hearst, J. E. and Sidow, A. (1993)
Proc. Natl. Acad. Sci. U. S. A. 90, 7134–7138
Lockhart, P. J. et al. (1996) Proc. Natl. Acad.
Sci. U. S. A. 93, 1930–1934
Miyashita, H. et al. (1996) Nature 338, 402
George, P. (1965) in Oxidases and Related
Redox Systems (Vol. 1) (King, T. E., Mason, H. S.
and Morrison, M., eds), pp. 3–36, Wiley
97