Download The novel cytochrome c6 of chloroplasts

Journal of Experimental Botany, Vol. 57, No. 1, pp. 13–22, 2006
doi:10.1093/jxb/erj023 Advance Access publication 29 November, 2005
REVIEW ARTICLE
The novel cytochrome c6 of chloroplasts: a case of
evolutionary bricolage?
Christopher J. Howe1,*, Beatrix G. Schlarb-Ridley1, Juergen Wastl1,†, Saul Purton2 and Derek S. Bendall1
1
Department of Biochemistry, University of Cambridge, Downing Site, Tennis Court Road, Cambridge CB2 1QW, UK
2
Department of Biology, University College London, Gower Street, London WC1E 6BT, UK
Received 14 June 2005; Accepted 21 October 2005
Abstract
Cytochrome c6 has long been known as a redox carrier
of the thylakoid lumen of cyanobacteria and some
eukaryotic algae that can substitute for plastocyanin
in electron transfer. Until recently, it was widely accepted that land plants lack a cytochrome c6. However,
a homologue of the protein has now been identified in
several plant species together with an additional isoform in the green alga Chlamydomonas reinhardtii. This
form of the protein, designated cytochrome c6A, differs
from the ‘conventional’ cytochrome c6 in possessing
a conserved insertion of 12 amino acids that includes
two absolutely conserved cysteine residues. There are
conflicting reports of whether cytochrome c6A can substitute for plastocyanin in photosynthetic electron
transfer. The evidence for and against this is reviewed
and the likely evolutionary history of cytochrome c6A is
discussed. It is suggested that it has been converted
from a primary role in electron transfer to one in
regulation within the chloroplast, and is an example of
evolutionary ‘bricolage’.
Key words: Arabidopsis, bricolage, Chlamydomonas, cytochrome, endosymbiosis, immunophilin, plastocyanin, photosystem, thioredoxin, thylakoid.
Introduction
Cytochrome c6 is a low molecular weight soluble redox
carrier found in oxygenic photosynthetic bacteria and in
eukaryotic algae, where it is widely distributed among
green, red, and ‘brown’ (i.e. those that contain chlorophyll c) algal lineages (Sandmann et al., 1983; Kerfeld and
Krogmann, 1998). It is located in the thylakoid lumen,
and functions in photosynthesis in the transfer of reducing
equivalents from cytochrome f of the cytochrome b6 f
complex to Photosystem I (Merchant and Dreyfuss, 1998).
In oxygenic photosynthetic prokaryotes, where the photosynthetic membranes also contain a cytochrome oxidase,
cytochrome c6 also serves as an electron donor to this
complex (Lockau, 1981; Obinger et al., 1990; Moser et al.,
1991; Nicholls et al., 1992). The mature cytochrome c6
polypeptide typically comprises 83–90 residues, with a
single haem, and has a midpoint redox potential of 335–390
mV. Under copper-replete conditions, many cyanobacteria
and green algae such as Chlamydomonas replace cytochrome c6 with the copper-containing redox carrier plastocyanin, synthesis of which is repressed under copper
starvation (Wood, 1978; Ho and Krogmann, 1984). Although the structures of the two proteins are different, they
are similar in size and midpoint redox potential, reflecting
their similar functions. There is considerable variation across
species in the pI of both cytochrome c6 and plastocyanin.
However, in any one species, the pIs of both proteins are
similar, again reflecting their similar functional interactions
with other proteins (Ho and Krogmann, 1984; De la Rosa
et al., 2002). It was widely accepted that land plants lacked
cytochrome c6, having replaced it with plastocyanin
(Kerfeld and Krogmann, 1998). Nevertheless, recent studies have shown that plants do have a form of cytochrome c6.
There are differing views in the literature of its function.
In this review, the question of whether the ‘plant’ form of
the protein functions as a substitute for plastocyanin and,
if it does not, what its role might be, is discussed.
Discovery of cytochrome c6A in plants
The model whereby land plants had abandoned cytochrome
c6 in favour of plastocyanin became significantly more
* To whom correspondence should be addressed. E-mail: [email protected]
y
Present address: BASF-AG, Agrochemical Division, D-67056 Ludwigshafen, Germany.
ª The Author [2005]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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14 Howe et al.
complicated in 2002 when two groups reported nearly
simultaneously the existence of a modified form of
cytochrome c6 in a range of land plants. This modified
form of the cytochrome has subsequently been named
cytochrome c6A for reasons that are discussed below, and
this nomenclature will be used in the rest of this review. The
report of Wastl et al. (2002) was based on searching of
plant genomic and EST databases with a cyanobacterial
cytochrome c6 sequence. This identified homologues of
cytochrome c6 coding sequences in a number of land
plants, including Arabidopsis, rice, wheat, barley, potato,
and soybean. Although this report did not demonstrate the
existence of the cytochrome at the protein level, the fact that
the gene was transcribed (and therefore represented in EST
databases) and conserved indicated that it encoded a functional protein. Remarkably, all the predicted amino acid
sequences for the plant protein contained a conserved
insertion of 12 amino acids compared with the bacterial
and algal proteins. This insertion occurs in a region corresponding to a loop in the known three-dimensional structure
of the bacterial and algal proteins. For brevity, the insertion will be referred to as the LIP (Loop Insertion Peptide).
It contains two cysteine residues that are conserved in all
the sequences studied. These sequences came from a wide
range of plant species, and it was suggested that a disulphide
bridge (or bridges) involving the cysteine residues was an
important part of the function of the protein. Homology
modelling, based on the structure of cytochrome c6 from
the eukaryotic green alga Monoraphidium, allowed a structure for the cytochrome c6A to be proposed (Fig. 1). This
structure suggested that the surface properties of cytochrome c6A from Arabidopsis are quite different from
those of Arabidopsis plastocyanin, implying that cytochrome c6A does not function as a simple substitute for
plastocyanin in the light reactions of photosynthesis. In
addition, it was noted that several of the EST libraries from
which cytochrome c6A sequences had been identified were
derived from non-photosynthetic tissue.
About the same time as the report of Wastl et al. (2002),
a separate study independently reported the existence of the
same plant homologue of cytochrome c6. In this case, the
initial discovery of cytochrome c6A had been as a protein
interacting with the chloroplast immunophilin FKBP13 in a
yeast two-hybrid assay (Gupta et al., 2002a; Buchanan and
Luan, 2005). Gupta et al. (2002a) showed that the mRNA
for cytochrome c6A was present in leaf tissue of Arabidopsis, with protein (as determined by western analysis using
antibodies to an over-expressed glutathione S-transferase
fusion protein) present in leaf and (at lower abundance) root
tissue. Gupta et al. (2002a) also expressed the protein in the
cyanobacterium Synechocystis sp. PCC 6803 and provided
evidence that it could stimulate photosynthetic electron
transport in inside-out thylakoid membrane vesicles from
Arabidopsis. They identified an Arabidopsis line with a
T-DNA insertion mutation for cytochrome c6A, but this
strain had no visible phenotype even if homozygous plants
were grown under conditions of copper deprivation.
(Interestingly, no increase in levels of cytochrome c6A was
observed in wild-type plants under copper deprivation.)
When plastocyanin levels were depleted using RNAi in
an otherwise wild-type background, the resulting plants
were significantly smaller. Depletion of plastocyanin
levels by RNAi in the cytochrome c6A mutant plants was
lethal. Gupta et al. (2002a) interpreted all these data as
indicating that cytochrome c6A was a functional substitute
for plastocyanin in photosynthetic electron transfer in
Arabidopsis.
However, Weigel et al. (2003b) subsequently showed
that if both of the plastocyanin genes of Arabidopsis were
inactivated by stable frameshift mutations resulting from
transposon insertion, the resulting plants were unable to
grow photoautotrophically, even if cytochrome c6A was
Fig. 1. Homology model of Arabidopsis thaliana cytochrome c6A (left panel) based on that of cytochrome c6 from Monoraphidium braunii (right
panel). The haem is indicated in purple and the cysteines of the LIP are indicated in yellow. The models were prepared with Modeller4 and displayed
with Molscript (Kraulis, 1991).
Cytochrome c6 of chloroplasts
artificially overexpressed at the same time using the
Cauliflower Mosaic Virus 35S promoter. Fluorescence
data indicated a block in electron transfer downstream of
the cytochrome b6 f complex, consistent with the loss of
plastocyanin function. This study was carried out using
a different ecotype from that used by Gupta et al. (2002a),
but Weigel et al. (2003b) argued that the role of cytochrome
c6A was unlikely to be fundamentally different between the
two ecotypes, and that their own results showed the protein
could not substitute for plastocyanin in vivo. This disagreement was further addressed by Molina-Heredia et al. (2003),
who showed that cytochrome c6A could not substitute for
plastocyanin in vitro. Their study used Arabidopsis cytochrome c6A expressed in Escherichia coli, thus avoiding
any possibility of contamination with cytochrome c6 when
using a cyanobacterium as the expression host. The protein
expressed in E. coli showed spectroscopic characteristics
expected of a functionally-folded protein. However, it was
found to have a redox midpoint potential for the haem over
200 mV lower than that for plastocyanin from Arabidopsis
(365 mV) or cytochrome c6 from the eukaryotic green alga
Monoraphidium (358 mV). The low midpoint potential of
the Arabidopsis cytochrome c6A makes it extremely unlikely that it could oxidize cytochrome f (its substrate if it
substituted for plastocyanin) efficiently in vivo, because
cytochrome f has a midpoint potential similar to that of
plastocyanin or cytochrome c6. Although the redox potential of cytochrome c6A would, in principle, allow it to reduce
photosystem I, Molina-Heredia et al. (2003) also showed
that cytochrome c6A was at least 100 times less efficient at
doing so in vitro than plastocyanin. This was not unexpected, given the very different surface electrostatic
potentials predicted for cytochrome c6A and plastocyanin.
However, increasing the ionic strength of the reaction
conditions to screen the likely charge repulsion between
cytochrome c6A and Photosystem I failed to increase the
rate of reaction significantly, suggesting that charge repulsion was not the only factor prohibiting the reaction. It
is not clear why the midpoint potential of cytochrome c6A
is so different from cytochrome c6. The difference is not
due to the LIP, since its removal, or mutation of the
cysteine residues in it to serine, did not greatly affect
either the midpoint potential or the reactivity of the protein
towards Photosystem I in vitro (Wastl et al., 2004a).
More doubt was cast on the ability of cytochrome c6A to
substitute for plastocyanin in higher plants by the observation that the mRNA expression profile of a set of over
3000 nuclear genes in a homozygous Arabidopsis cytochrome c6A mutant was significantly different from the
profiles in mutants lacking either or both functional
plastocyanin genes (Weigel et al., 2003a). However, this
study also noted that transcript levels of some genes for
the photosynthetic machinery were similarly altered in
the cytochrome c6A mutant and the plastocyanin single
mutants, and interpreted this as consistent with the notion
15
that cytochrome c6A plays a role in some aspect of the
regulation of photosynthesis.
Cytochrome c6A in algae
It is also now clear that cytochrome c6A is not restricted
to higher plants, as searching of the complete genome
sequence of the green alga Chlamydomonas reinhardtii
showed that this organism contains a gene (designated
CYC4) for cytochrome c6A (Wastl et al., 2004b) in addition
to its much-studied cytochrome c6 (Gorman and Levine,
1966; Merchant and Bogorad, 1987). The cytochrome c6A
gene has also been identified in sequence data (http://
genome.jgi-psf.org/chlre2/chlre2.download.ftp.html) from
the multicellular green alga Volvox carteri (S Purton,
unpublished observation). However, not all eukaryotic
algae contain cytochrome c6A, as the red algal and diatom
genomes do not contain a recognizable gene for it (Wastl
et al., 2004b). The presence of both cytochrome c6A and
cytochrome c6 genes in Chlamydomonas also adds weight
to the proposal that cytochrome c6A is not a simple substitute for plastocyanin, as it would presumably be redundant in the presence of a conventional cytochrome c6.
Questions raised
This survey of the experimental information available about
cytochrome c6A raises several interlinked questions. For
clarity and convenience, it is important to decide what the
most appropriate name should be. A second question is how
the existence and taxonomic distribution of cytochrome c6
and cytochrome c6A evolved. The remaining questions relate to the function of the protein. Where is it located, what
is its structure, and what is its function?
Nomenclature
Initially, the protein was referred to simply as cytochrome
c6 or ‘the cytochrome c6-like protein’. Weigel et al. (2003a)
subsequently proposed the term cytochrome cx, as the
protein had a different function from conventional cytochrome c6. However, this nomenclature runs the risk of
confusion with the c-type ‘haem x’ discovered in the
cytochrome b6 f complex (Kurisu et al., 2003; Stroebel
et al., 2003) and the cytochrome cx of purple photosynthetic bacteria (Jones et al., 1990). The name cytochrome
c6A seems the most appropriate because it distinguishes
the protein from cytochrome c6, while reflecting its origin
from the cytochrome c6 family (Wastl et al., 2004b).
Evolution
A model for the evolution of cytochrome c6A is shown in
Fig. 2. The earliest electron donors both to Type I and to
Type II reaction centres (as seen today in, for example,
16 Howe et al.
Fig. 2. Proposed evolutionary pathway for cytochrome c6A. The diagram supposes that the ancestor to Photosystem I was transferred laterally into the
oxygenic photosynthetic bacterial lineage, although other origins for two photosystems in these organisms have been proposed (Allen, 2005).
Chlorobium and Rhodobacter, respectively) were probably
c-type cytochromes (Mathis, 1994; Itoh et al., 2002; Meyer
and Cusanovich, 2003). It therefore seems likely that the
original electron donor to Photosystem I in oxygenic
photosynthetic bacteria was also a c-type cytochrome,
which gave rise to present-day cytochrome c6.
As atmospheric oxygen levels rose after the development
of oxygenic photosynthesis, free iron would have become
less readily available at the earth’s surface, copper would
have become more available, and this would have provided
a selective advantage for the development of the coppercontaining redox protein plastocyanin (Österberg, 1974;
Wood, 1978; De la Rosa et al., 2002). The effect that the
amount of iron required for synthesis of cytochrome c6 has
on the cell’s physiology presumably depends on its
stoichiometry compared with the other components of the
photosynthetic electron transfer chain, and may not be large
(Raven et al., 1999). It is also likely that there would have
been geographic heterogeneity in iron and copper levels, as
well as variation with depth. However, iron-limitation is
known to have an important effect on the metabolism of
present-day cyanobacteria (Ferreira and Straus, 1994;
Laudenbach et al., 1988; Laudenbach and Straus, 1988;
Bibby et al., 2001; Boekema et al., 2001), so this explanation for the selective advantage of developing plastocyanin remains possible.
Based on 13C isotope signatures of carbonate in rocks,
indicative of Form I Rubisco (found in oxygenic photosynthetic organisms), oxygenic photosynthesis developed some
2.7–3.0 billion years ago (reviewed by Nisbet and Fowler,
2004). Data on iron deposition and sulphur isotope fractionation indicate a major increase in oxygen levels around
2.3 billion years ago (Bekker et al., 2004; reviewed by
Nisbet and Fowler, 2004). Phylogenetic analyses of presentday oxygenic photosynthetic bacteria (CJ Howe, unpublished results; Battistuzzi et al., 2004) indicate that the
radiation of this group occurred more recently than 2.3
billion years ago (although the group itself has existed for
longer). Thus if the development of plastocyanin was
associated with the increase in oxygen levels at 2.3 billion
years ago, both electron transfer systems would have been
present at the time of the radiation of present-day oxygenic
Cytochrome c6 of chloroplasts
photosynthetic bacteria. This proposal is consistent with
the observation that Gloeobacter, widely regarded on
ultrastructural grounds as well as sequence-based studies as
diverging early among the extant cyanobacteria (Nelissen
et al., 1995; Honda et al., 1999), has both cytochrome c6 and
plastocyanin (Nakamura et al., 2003). Presumably the
maintenance of either or both of the two redox carriers in
different extant species reflects the environments they have
occupied during their evolution, and the consequences of
lateral gene transfer.
Plastids are thought to have arisen about 1.5 billion years
ago (reviewed by Falkowski et al., 2004), by which time
both cytochrome c6 and plastocyanin would have been in
existence according to the arguments above on oxygen
levels. The chlorophyte lineage contains both cytochrome
c6 and plastocyanin, so is most likely to have derived
from an endosymbiont containing both of these proteins.
Duplication and divergence of cytochrome c6 gave rise to
cytochrome c6A. This probably took place in a chloroplastcontaining lineage, rather than prior to endosymbiosis, as
cyanobacteria do not appear to have cytochrome c6A.
(However, it is possible that the duplication occurred
earlier, in the prokaryotic ancestor to chloroplasts, and
a prokaryotic cytochrome c6A remains to be discovered.)
The cytochrome c6 form was then lost from the lineage that
gave rise to land plants after the divergence from the
Chlamydomonas lineage. (This may indicate that the
environments initially colonized by land plants contained
adequate levels of copper. Furthermore, genuine copper
starvation is relatively unusual in the wild among presentday land plants.) Cytochrome c6A and plastocyanin were
retained in the land plant lineage.
Plastocyanin appears to be absent from red and brown
algae (Sandmann et al., 1983), and the complete genome
sequences of the red alga Cyanidioschyzon merolae and
the diatom Thalassiosira pseudonana do not contain a gene
for cytochrome c6A or for plastocyanin. The significance of
these observations for the origin of cytochrome c6A depends
on whether a polyphyletic or a monophyletic origin of
plastids is assumed (Lockhart et al., 1999; Stiller et al.,
2003; McFadden and van Dooren, 2004). If polyphyletic
origins of plastids are assumed, these observations can be
most simply explained if the red algal lineage (and others
derived subsequently by serial endosymbiosis) was derived
from a symbiont possessing only cytochrome c6, and not
plastocyanin. This lineage never developed cytochrome
c6A. If a monophyletic origin is assumed, the simplest
explanation is that loss of plastocyanin in the red/brown
lineage and duplication of cytochrome c6 in the green
lineage happened after the separation of the two lineages.
Other possibilities cannot be excluded, such as a monophyletic origin with an early duplication of cytochrome c6
prior to divergence of the red and green lineages, together
with subsequent loss in (at least some of ) the non-green
lineages, but these are more complex.
17
Location
Knowing where cytochrome c6A occurs, both in the whole
plant and at the subcellular level, will help greatly in
understanding its function. Using northern blots, Gupta
et al. (2002a) showed that mRNA for cytochrome c6A was
present in leaves, but barely detectable, if at all, in roots of
plants grown in a greenhouse under long-day conditions.
Using western blots with antibodies raised against the
protein expressed in E. coli, they similarly showed that
the protein was present in leaves, and at lower levels in
roots. When the gene was placed under the control of a
constitutive promoter, transcripts were found both in roots
and leaves, but the protein appeared to be predominantly
located in leaves, with an electrophoretic mobility that
suggested it was post-translationally processed. Gupta et al.
(2002a) also showed that the protein could be recovered
in a thylakoid luminal fraction isolated from protoplasts of
over-expressing plants. It was not found in the stromal
fraction, although the efficiency of recovery of stromal proteins was not indicated. Although these data favour a location in the thylakoid lumen, they do not exclude other less
likely possibilities, for example, that the protein is distributed between the thylakoid lumen and stroma (or even
that it is mitochondrial, with a fraction mis-targeted to the
chloroplast under over-expression conditions). A location
in the lumen is consistent with the interaction between
cytochrome c6A and the luminal immunophilin FKBP13.
Apart from these observations, there is little direct
information on the expression or localization of the protein.
A number of the EST databases from which cytochrome
c6A sequences were identified by Wastl et al. (2002) were
derived from non-photosynthetic tissue, such as anthers,
suggesting that the transcript is present at significant
levels in such tissue. Searching of microarray databases
(Zimmermann et al., 2004) also indicates the presence of
transcripts for cytochrome c6A (as well as plastocyanin) in
anthers of Arabidopsis. In general, however, cytochrome
c6A transcripts are low in non-photosynthetic tissue (roots,
petal, stamen, senescing leaf ) and higher in photosynthetic
tissue. This relative pattern is similar to that seen for
plastocyanin, but with the cytochrome c6A transcripts less
abundant.
Predictions as to the subcellular location of cytochrome
c6A from the putative targeting sequence are inconclusive,
with different algorithms variously predicting the protein
to be in the chloroplast or the mitochondrion (Wastl
et al., 2004a). Proteomic analyses of the thylakoid lumen
have failed to detect cytochrome c6A (Peltier et al., 2002;
Schubert et al., 2003). It is unlikely that this failure was due
to the size or isoelectric point of the protein, since
plastocyanin was detected in these studies, yet has a lower
predicted molecular mass and a more extreme isoelectric
point than cytochrome c6A (10.5 kDa and 4.4 for plastocyanin compared with 11.8 kDa and 5.1 for cytochrome
18 Howe et al.
c6A). This suggests that cytochrome c6A is of low abundance under the conditions used for those studies (but
perhaps present at higher levels under stress conditions).
More general proteomic studies of plastids, including
differentiated chloroplasts and undifferentiated heterotrophic plastids from a tobacco BY-2 cell culture have likewise
failed to detect cytochrome c6A (Baginsky et al., 2004;
Friso et al., 2004; Kleffmann et al., 2004). It has also not
been reported from other subcellular compartments, such
as mitochondria (Millar et al., 2001).
Overall, therefore, it seems likely that, under ‘normal’
conditions cytochrome c6A is present at low levels in
chloroplasts from green tissue, and may well be located in
the thylakoid lumen. It is generally present at even lower
levels (presumably in the plastid) in non-photosynthetic
tissue. However, this model is based on a limited amount
of data, and more detailed analyses, such as a more
direct demonstration of the subcellular location and a study
of the expression under different developmental and stress
conditions, are needed.
Structure
No experimentally-determined three-dimensional structure
of cytochrome c6A is available yet, although a structure
has been modelled based on homology to cytochrome
c6 proteins whose structure is known (Fig. 1). Aspects of
this have already been described. The 12 amino-acid insertion of cytochrome c6A occurs as an extension to a loop
region of cytochrome c6, but is not very distant from the
haem group. The pI of Arabidopsis cytochrome c6A is 5.1,
compared with 4.4 for Arabidopsis plastocyanin, and
the electrostatic potential of cytochrome c6A indicates a
much more positively charged surface than for plastocyanin (Molina-Heredia et al., 2003). It will be important
to confirm this structure experimentally and to determine
any structural differences between the oxidized and reduced
forms of the protein.
Function
A role as a simple substitute for plastocyanin seems
unlikely, given the redox characteristics of the protein
(albeit expressed in a heterologous system), its inability to
reduce Photosystem I in vitro, the lethality of plastocyanin
mutants, the RNA microarray data described above, the
failure to observe induction of cytochrome c6A under
copper-deprivation, and the presence of a cytochrome c6A
gene in Chlamydomonas, which also has a conventional
cytochrome c6. The contrary evidence of Gupta et al.
(2002a, b) could be explained if gene inactivation in
RNAi lines were incomplete (as is usually the case), and
the cytochrome c6A used for reconstitution experiments
contained cyanobacterial protein as contamination or was
modified in some way. In any case, reactions can occur
in vitro that do not necessarily occur in vivo.
So what does cytochrome c6A actually do? The following
is known about the ‘physiology’ of the protein. It is
expressed in chloroplast-containing tissue, at low levels
under ‘normal’ growth conditions, and is probably located
in the thylakoid lumen. It may also be present, at lower
levels, in non-photosynthetic tissue. Loss of the protein
leads to changes in transcript levels for, among others,
proteins involved in photosynthesis. Loss in plants that
already have reduced levels of plastocyanin is lethal. These
observations suggest a regulatory role, as originally suggested by Wastl et al. (2002), in response to stress such
as a disturbance of the light reactions of photosynthesis.
Under ‘unstressed’ conditions, there is no need for the protein, so cytochrome c6A mutants have little phenotype. However, under serious stress conditions, including a major
depletion of plastocyanin, the absence of cytochrome c6A
is lethal, presumably because the organism is unable to
respond appropriately.
How might the molecule perceive and transmit a signal?
There are three obvious features; the haem, the LIP (which
is absent from the conventional cytochrome c6), and the
apparent ability of the protein to interact with the chloroplast immunophilin FKBP13 in a two-hybrid screen (Gupta
et al., 2002a; Buchanan and Luan, 2005). The binding of
haem requires specific residues which are conserved in all
cytochrome c6A proteins, suggesting that haem remains an
important part of its function. The LIP is also highly
conserved, with - -C-P-G-CTFG identical from Chlamydomonas to higher plants. Interestingly, this sequence is
similar in the spacing of the cysteine residues to an insertion
EICDINGKC (in spinach) in the gamma subunit of land
plant chloroplast ATP synthases that mediates the regulation of ATP synthase by thioredoxin. This insertion is
absent from the cyanobacterial gamma subunit (reviewed
by van Walraven and Bakels, 1996). Disulphide bridges are
also known to be a mechanism for the perception and
response to oxidative stress in other systems (Paget and
Buttner, 2003). The reported interaction with an immunophilin is particularly interesting (although it should be noted
that two-hybrid systems can often give false positive
results). Immunophilins typically have peptidyl-prolyl cistrans isomerase (PPI) activity, and there are a number of
them in the thylakoid lumen (Romano et al., 2005). The PPI
activity of the immunophilin AtFKBP13 from the Arabidopsis thylakoid lumen can be modulated in vitro by a
thioredoxin (Gopalan et al., 2004), and the precursor of
AtFKBP13 also interacts with the Rieske Fe-S protein of
the photosynthetic electron transfer chain (Gupta et al.,
2002b). At least one luminal immunophilin, TLP40, has
been shown to participate in intraorganellar signalling as
it is involved in modulating the phosphorylation state of
a number of Photosystem II polypeptides, in addition to
having a conventional PPI activity (Fulgosi et al., 1998).
There are a number of different ways that cytochrome
c6A might participate in regulation through the haem, the
Cytochrome c6 of chloroplasts
19
Fig. 3. Schematic representation of possible signalling pathways for cytochrome c6A. The grey cylinder represents cytochrome c6A, and the blue arrows
possible signalling or electron transfer pathways. IP indicates the immunophilin-binding function (whose structural basis is unknown), SS indicates the
cysteine residues in the LIP, and the star indicates the haem. The yellow crescent indicates a disulphide-containing substrate, and the blue circle indicates
plastocyanin (PC). Fig. 3A shows general pathways. Figure 3B shows two specific models proposed in the text.
LIP, or immunophilin-binding (Fig. 3A). One of the three
elements might simply perceive a signal and then transmit
it to another molecule without the involvement of the
other two. However, these possibilities would not satisfactorily explain the existence of all three elements. Another
possibility is that one element receives a signal and transmits it through one of the other two to a signalling pathway.
For example, oxidation or reduction of one component
might lead to oxidation or reduction of the other. Assuming
it is similar to the midpoint potential of the disulphides in
thioredoxin and thioredoxin-regulated enzymes, the midpoint redox potential of the putative disulphide bridge in
the LIP is likely to be of the order of ÿ300 mV (Knaff,
2000). If electron transfer took place between the disulphide
bridge and the haem, it would therefore be expected to
require reduction of the former by another redox carrier
(such as a thiol) followed by reduction of the haem by the
cysteines and regeneration of the disulphide bridge. The
haem could then be reoxidized by another redox carrier
(perhaps plastocyanin, as discussed below). Electron flow
from cysteines to haem would probably be consistent with
the distance between them predicted from the homology
model. However, haem reduction or oxidation is a oneelectron process, whereas the reduction or formation of the
disulphide bridge is a two-electron process, with a reactive
intermediate generated after single electron transfer. Such
an intermediate would need to be suitably safeguarded.
Regulatory pathways involving only two of the three
elements again leave the role of the third unexplained.
The most attractive possibility is that all three features
are involved, perhaps with the protein functioning in the
integration of different signals. For example, the haem
might serve to transmit a signal based on the redox state
of some component of the photosynthetic machinery, and
its ability to do this might be influenced by a conformational change in the protein that depends on the LIP or on
immunophilin-binding. Alternatively, the LIP may function
in signalling, with its role likewise modified in response to
the redox state of the haem or immunophilin-binding. It is
suggested that the protein is used to sense both the redox
state of the inter-photosystem electron transfer chain via the
haem, and the state of the thioredoxin pool (which might
reflect the overall flux through the chain) via the LIP, in
order to transmit a signal through an immunophilin (Fig.
3B). For example, a change in oxidation state of the haem or
the LIP might lead to a conformational change that modifies
the ability of cytochrome c6A to bind to an immunophilin,
either by directly altering the immunophilin binding site or
causing the release of cytochrome c6A from some other
complex and freeing it to bind with an immunophilin. Many
processes in chloroplasts are regulated in response to the
redox state of the plastoquinone pool, and thus the poise
20 Howe et al.
of the electron transfer chain in general. These processes
include the phosphorylation of light-harvesting and other
proteins of the thylakoid membrane (Allen et al., 1981) and
chloroplast gene expression (Pfannschmidt et al., 1999).
The midpoint potential of the haem of cytochrome c6A
would allow it to be reduced by the plastoquinone pool, or
something closely linked to it, and would be consistent with
the protein having a role in these regulatory processes.
Furthermore, many chloroplast functions are influenced
by thioredoxins, including thylakoid protein phosphorylation (Carlberg et al., 1999). Although they are generally
located in the stroma, at least one thioredoxin-like protein,
Hcf164, is associated with the thylakoid membrane and
lumen (and, interestingly, is required for the biogenesis of
the cytochrome b6 f complex and is similar to bacterial
proteins that are required for maturation of c-type cytochromes (Lennartz et al., 2001)). There is also at least one
putative thiol disulphide transporter in the thylakoid
membrane, CCDA, which may serve to link the state of
the luminal thioredoxin pool to that in the stroma (Page
et al., 2004). It has also been suggested that cytochrome
c6A may interact with stromal redox proteins on its way into
the thylakoid lumen (Weigel et al., 2003b). Given the
implication of immunophilins in regulatory processes as
well as folding (Fulgosi et al., 1998), cytochrome c6A
provides an attractive link to redox processes in the
chloroplast. It is, of course, possible that a signal might
move in a different direction through the molecule. For
example, it might serve to detect the activity of the protein
folding machinery, and use this to modulate photosynthetic
electron transfer.
A possible regulatory role of a different kind is prompted
by the observation that FKBP13 is activated by oxidation of
disulphide bridges within the molecule (Gopalan et al.,
2004). Cytochrome c6A might therefore oxidize FKBP13
(or other disulphide containing proteins), reducing the
disulphide bridge in the cytochrome. This, in turn, could
reduce the cytochrome’s haem group, and the electrons
could be passed from that onto another redox carrier, such
as plastocyanin, with a higher midpoint potential (i.e. a
stronger oxidant) as shown in Fig. 3B. In other words,
cytochrome c6A would form part of a short electron transfer
chain that served to oxidize and regulate other proteins,
with the electrons being ‘disposed’ of into the photosynthetic electron transfer chain. (A close and stable complex between cytochrome c6A and its substrate might help
avoid damage caused by the single-electron transfer intermediate.) Given the thiol disulphide transporter referred
to above, cytochrome c6A might also help to regulate the
thiol balance of the stroma.
More distant roles for the protein have also been
suggested, including a speculation that it functions in
apoptosis (Weigel et al., 2003a). It was not detected as
an up-regulated protein in heat- or senescence-induced cell
death in Arabidopsis cell culture (Swidzinski et al., 2004),
but this may reflect the small size of the protein. The
preferred model for the role of the protein is therefore in
modulating the function of the immunophilin FKBP13 and
perhaps other proteins. Cytochrome c6A either directs
a response to the redox poise of luminal thiols and the
electron transfer chain, or it provides the actual electron
transfer pathway for oxidative regulation of disulphide
proteins. If the role of cytochrome c6A has indeed been
switched from an ancestral one in electron transfer to
a regulatory one, it is an excellent example of evolutionary
‘tinkering’ or bricolage (Jacob, 1977). The protein was no
longer needed for dealing with copper starvation (either
because there was an existing functional cytochrome c6, as
in Chlamydomonas, or because copper starvation was rare
in plants) and was then used for a different function. No
cytochrome c6A has yet been identified in a cyanobacterium.
This may simply be because the protein failed to evolve
until after chloroplasts were formed. It is possible that
cytochrome c6A controls a process that does not occur in
cyanobacteria, or that the process is controlled differently,
or not at all.
Although the functional models are speculative, they
help us to identify important areas for further study of this
protein. More information is clearly needed on its subcellular location, and its location within whole plants, as
well as the range of conditions under which it is expressed,
both in land plants and in Chlamydomonas. Structural
data are needed to help us to understand the possible interactions between the haem and the LIP as well as possible
conformational changes on oxidation or reduction. Data
are needed on the nature of the interaction with immunophilin(s) and data are also needed on how chloroplast
control systems, especially those linked to the plastoquinone pool and to thioredoxins, are disturbed in plants
deficient in cytochrome c6A, given the exciting possibility
that this protein plays a key role in regulatory pathways in
the thylakoid.
Acknowledgements
Work on cytochrome c6A in the authors’ laboratories was supported
by the EU ‘Transient’ network, and by the Biotechnology and
Biological Sciences Research Council. We thank Euan Nisbet for
helpful discussions. CJH is grateful to Trish McLenachan, Peter and
Toby Lockhart, and the Allan Wilson Centre, Massey University,
NZ, for their hospitality during the writing of this review.
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