CHAPTER
2
Molecular Architecture of Photosynthetic
Apparatus
Jayashree K. Sainis1 Rachna Agarwal1 and Michael Melzer2
1
Molecular Biology Division, BARC, Mumbai-400 085, India
2
Structural Cell Biology Group, Department of Physiology and Cell Biology, Leibniz Institute
of Plant Genetics and Crop Plant Research (IPK), Gatersleben 06466, Germany
ABSTRACT
The photosynthetic apparatus consists of the components of light and dark reactions, present in the thylakoids
and the stroma or cytosol. Photosynthetic reactions occur in a protein crowded and water limited environment in
vivo. Such an environment will necessitate that enzymes and proteins participating in light and dark reactions of
photosynthesis should be organized in the vicinity of each other to achieve maximum efficiency. We have been
working on the organization of Calvin cycle enzymes and were able to show that these enzymes are detected in
the neighbourhood of the thylakoid membranes in chloroplasts of higher plants and cyanobacteria. Functional
characterization, electron microscopy and proteome analysis of isolated thylakoids from cyanobacteria confirmed
the association of Calvin cycle enzymes to the membranes. The results suggest that Calvin cycle enzymes may
be distributed as thin layer on the surface of thylakoids, and it is conjectured that molecular architecture of
photosynthetic apparatus may be modular in nature. A module of photosynthesis would represent a discrete
structural and functional entity involving a small fraction of the components of photosynthetic apparatus
accomplishing a relatively autonomous function. Such a structure can be designated as a photosynthesome. The
information on molecular architecture of photosynthetic apparatus is discussed.
Keywords: Calvin cycle enzymes, Photosynthetic module, Soluble supercomplexes, Thylakoid membrane
association.
Abbreviations
RPI: Ribose phosphoisomerase, PGK: Phosphoglycerate kinase, RPK: Ribulose-5-phosphate kinase, GAPDH:
Glyceraldehyde-3-phosphate dehydrogenase RuBisCO: Ribulose-1,6-bisphosphate carboxylase/oxygenase.
30 Photosynthesis: Overviews on Recent Progress and Future Perspectives
chloroplast, and radial asymmetry in distribution of PSII, PSI and ATP synthase in photosynthetic
membranes of cyanobacteria is well established (Andersson and Andersson, 1980; Sherman et al.,
1994). Cytochrome b6f complex is uniformly distributed throughout the thylakoids. Separation of the
two pigment-protein systems is thought to be important in preventing unregulated excitation energy flow
between them. Without this, PS I, which is much faster than PS II, would disturb the balance of energy
distribution between the two photosystems. Fluorescence recovery after photobleaching (FRAP) and
confocal laser scanning microscopy (Mullineaux, 2004) have shown that phycobilisomes, the accessory
pigment protein complexes of cyanobacteria, are also mobile.
ENVIRONMENT WITHIN CHLOROPLASTS
The environment inside the chloroplasts is unusual. The soluble protein concentration in the stroma is
around 400 mg/ml, out of which around 250 mg/ml alone is due to RuBisCO (Robinson and Walker,
1981). A dense packing of large protein clusters was observed in the stroma portion of the chloroplast
using cryo-scanning electron microscopy, suggesting that there should be a tight packing of stromal
proteins, minimizing the random mobility (Süss and Sainis, 1997). However, in contrast to membrane
proteins, research on associations of soluble proteins did not find much appreciation. The stroma
harbours enzymes of several metabolic pathways such as oxidative pentose-phosphate cycle, nitrate
and ammonia assimilation, amino acid and fatty acid biosynthesis, ribosomes and the entire protein
synthetic machinery along with Calvin cycle enzymes. In addition, multiple copies of DNA, mRNA,
starch grains etc. also reside in the chloroplast. Furthermore, Dilley and Vernon (1965) had shown that
chloroplasts show reversible changes in volume in response to light conditions, resulting in changes
in ion composition and water content of stroma. The ultrastructure of thylakoid membranes was also
shown to change in response to light affecting the rates of electron transport and photophosphorylation
(Miller and Nobel, 1972). Thus exposure to light was found to result in an increase in macromolecular
crowding in chloroplast and a reduction in free water. These light dependent changes in the ultrastructure
of chloroplast would in turn regulate photosynthetic activities probably through changing the dynamic
associations of metabolic proteins. The efficiency of metabolism will be difficult to maintain in this
scenario unless there is an organization among sequential enzymes, which will facilitate the sequestering
of desired metabolites (Wolosiuk et al., 1993).
Therefore, the concept that the components of light and dark reaction are mutually exclusive in
function due to their exclusive spatial location needs to be revisited since the real situation in vivo
is far more complex than expected from in vitro experiments. Though according to the third law of
thermodynamics, entropy of the physical world always increases, the situation is reversed in living
systems, where organization or decrease in entropy are the key features of their survival.
SOLUBLE SUPERCOMPLEXES IN CHLOROPLASTS
Chloroplasts are capable of utilizing light energy for the synthesis of several organic compounds using
oxides of carbon, sulphur, phosphorus etc. Many of the enzymes involved in these processes have been
isolated from stroma of chloroplasts and are considered as functioning independently of each other. Among
these metabolic pathways in chloroplasts, enzymes of Calvin cycle have been extensively investigated,
albeit mainly in isolation. The existence of a CO2 fixing complex was predicted in the sixties (Van Noort
and Wildman, 1964). Muller (1972) showed that some of the CO2 fixing enzymes might be associated
in the form of a labile complex. Several years later, the associations among Calvin cycle enzymes were
Jayashree K. Sainis, Rachna Agarwal and Michael Melzer 31
discovered by a variety of procedures. Sainis and Harris (1986) and Sainis et al. (1989) observed the
association of phosphoriboisomerase (RPI) and phosphoribulokinase (RPK) with RuBisCO on a sucrose
gradient. Gontero et al. (1988) purified a functional five enzyme complex of the consecutive enzymes
of Calvin cycle, viz., RPI, RPK, RuBisCO, PGK and GAPDH by using DEAE Tris-acryl, Sephadex
G-200 and hydroxyapetite. Persson and Johansson (1989) had reported the partition behaviour of six
Calvin cycle enzymes viz. RuBisCO, PGK, GAPDH, TPI, aldolase and FBPase using countercurrent
distribution in the aqueous two phase system, which suggested a trend to exist as a protein-protein
complex among these enzymes. Nicholson et al. (1991) reported a stable complex between GAPDH
and RPK from chloroplasts. Süss et al. (1993) were able to isolate a multienzyme complex containing
RPI, RPK, RuBisCO, GAPDH, sedoheptulose-1,7-bisphosphatase and also Ferredoxin NADP reductase
(FNR) using molecular sieve and anion exchange chromatography. Mouche et al. (2002) used a multitechnique approach to study multienzyme complex of GAPDH and RPK. This bi-enzyme complex uses
ATP and NADPH produced by the primary reactions in photosynthesis.
Recently nearest-neighbour analysis was used to study co-localization of several Calvin cycle
enzymes in the stroma of pea chloroplasts. These studies indicated that carbonic anhydrase, RPK and
PGK occur in the neighbourhood of RuBisCO (Anderson and Carol, 2004); GAPDH, triose-P isomerase
and aldolase are also located close to one another; aldolase is located close to SBPase (Anderson et
al., 2005); and transketolase, xylulose-5-P 3-epimerase and RPI exist near each other (Anderson et al.,
2006).
Thus, there is adequate data from these in vitro studies to demonstrate that many of the sequential
Calvin cycle enzymes can be isolated as supramolecular complexes. Not much information, however, is
available about the supercomplexes of enzymes functioning in chloroplasts in other metabolic pathways
(Hrazdina and Jensen, 1992).
Fig. 2.1 Molecular architecture of multienzyme organization; A: Tight coupling of active sites of sequential enzymes of a
metabolic pathway. The tight coupling of active sites of sequential enzymes will not allow the intermediates to diffuse to
sequential enzymes of a pathway. B: Thin layer of sequential enzymes of a metabolic pathway on the surface of membranes. The
thin layer of sequential enzymes on the surface of membranes would facilitate channelling of intermediates among sequential
enzymes and avoid unwanted diffusion of substrates.
32 Photosynthesis: Overviews on Recent Progress and Future Perspectives
MEMBRANE ASSOCIATION OF
SOLUBLE ENZYMES
Such multienzyme supercomplexes would have
a diffusion limitation within the intracellular
soluble phases owing to their large sizes. The
traditional concept of multienzyme organization
revolves around the notion of interlocking the
active sites of sequential enzyme (Fig. 2.1 A).
This situation may not be very useful in vivo
as the intermediates have to diffuse to the
consecutive sequential enzymes and yet not
disseminate all over in the soluble phases in
the cell. This makes it plausible to hypothesize
that sequential enzymes would be structured
to optimize the diffusion of intermediates. If
organized as a thin layer on membranes, the
membrane will provide partially non-aqueous
environment, thereby reducing the superfluous
diffusion of intermediates and also helping
in channelling among sequential enzymes
(Fig. 2.1 B). In case of Calvin cycle, besides
CO2; ATP and NADPH, which are produced by
the components of light reactions residing in
thylakoid membranes are also required. Hence,
if the Calvin cycle enzyme supercomplexes
are positioned next to the machinery of light
reactions, ATP and NADPH would be easily
and efficiently channelized for photosynthetic
carbon assimilation pathway. Additionally, Fig. 2.2 Immunogold localization of RuBisCO in Synechococcus
thylakoid membranes may act as anchoring cells and maize chloroplasts. Predominant immunogold
localization of RuBisCO is seen adjacent to the thylakoid membranes
surfaces or a guide for organization of (A) in Synechococcus cells (5 nm protein-A gold) and (B) in maize
sequential enzymes of Calvin cycle. Figure 2.2 Bundle Sheath chloroplasts (10 nm protein-A gold). (C) Mesophyll
shows immunolocalization of RuBisCO in chloroplasts of maize did not show any labelling. Bar = 100 nm.
Synechococcus (Fig. 2.2 A) cells and in the
dimorphic chloroplasts of maize (Fig. 2.2 B and C). About 70-80% of gold particles were observed to be
located near thylakoid membranes. This was true for several other soluble enzymes of Calvin cycle and
other metabolic pathways. However, very few investigations were done on the association of Calvin cycle
enzymes with isolated thylakoid membranes. In 1967, Howell and Moudrianakis reported the structure
and properties of membrane-bound particle that are active in the dark reactions of photophosphorylation.
The dual localization of many of these enzymes was shown by Mori et al. (1984) and Hermoso et al.
(1992). The intricate and specific nature of these unusual interactions was subsequently evaluated by
Raghavendra et al. (1981) and Wolosiuk et al. (1993). Süss et al. (1993) and Sainis et al. (2003) predicted
the involvement of thylakoid membranes in the organization of Calvin cycle enzymes.
34 Photosynthesis: Overviews on Recent Progress and Future Perspectives
the coupling between photosynthetic electron transport and CO2 fixation for an enhanced efficiency
and more sensitive regulation of their function. Such an organization will also minimize unwanted
water diffusion into the enzyme layer, thus creating a non-polar microenvironment to assist in enzyme
catalysis.
These observations propose a paradigm shift in biology, leading to exploration of supramolecular
organizations of functionally related molecules, regarded as modules. A module is an organization
of macromolecules performing a synchronous function in a given metabolic pathway. The complex
metabolic reactions occurring inside the chloroplasts can be best described by the perception of the
molecular architecture in modular fashion. Figure 2.3 A and B show the modular organization of
different metabolic pathways in chloroplasts. Large metabolic networks can be imagined as a web of
several interconnected modules, which may or may not influence each other. Elucidating the physical
associations and the dynamics of the interactions in these predicted networks of modules would be a
challenge to the forthcoming technologies.
Networking of biochemical reactions by specific spatio-temporal associations of participating
components will answer several fundamental questions in biology. It will explain why several variants
of the same proteins, generated by multi-gene families, isozymes, isoforms, alternate splicing of genes
or variety of post-translational modifications are required. Single nucleotide polymorphisms may also
result in minor structural variations in proteins. The necessity for interaction with other proteins may
drive the need for such modifications without affecting the functionality. Several sites on the surface
of enzyme would be required for generation and maintenance of 3-D superstructures. Variations in
protein conformation will be tolerated as long as they do not affect the function of a given module,
putting a limitation on toleration of mutations. Networking of proteins in modules may also explain
the gene silencing that occurs in transgenics due to overexpression of a gene, resulting in suppression
of a given phenotype. Overexpression of one of the proteins in the network may result in differential
titration of interacting partners, and hence in inhibition of the phenotype. Modular biology will help to
understand the phenomenon of homeostasis where a perfect balance is maintained between anabolism
and catabolism, synthesis and destruction of proteins, and other macro- and micromolecules. Modules
would result in caging and isolation of intermediates to avoid unwanted reactions in living cells. Such
a microenvironment will explain how several competing and contrasting metabolic reactions occur
simultaneously in a crowded atmosphere in vivo. The differential degree of recruitment of modules
will explain the phenomenon of up and down regulation of metabolic processes as also metabolic
compensation. Thus metabolic regulation may have to be looked from a different perspective in the
background of modularity.
The finer details of the molecular architecture of modules, their interaction and organization may
decide the final form of an organism. This will explain the fact that though evolutionarily, there are
similarities in the gene sequences of several proteins in a variety of living organisms, the final forms of
these organisms are quite different.
Modular biology offers an intellectually demanding perception of metabolism and will amalgamate
the conventional biochemical studies and the recent high-throughput technologies to understand the
hierarchy of networks and molecular architectures and their dynamism in biological systems. This will
test the limits of old and new technologies in biology. A leap in faith in imagination and technology
would be required to conceptualize the modular molecular architectures in vivo for precise functions and
regulation of metabolic pathways.
Jayashree K. Sainis, Rachna Agarwal and Michael Melzer 35
Fig. 2.3 Modular organization. A shows modules of different metabolic pathways requiring energy inputs generated from
electron transport. A module is a discrete structural and functional entity, an ensemble of physically interacting proteins, which
deal with a specific metabolic process. The composition, location and function of a module would vary depending on cellular
requirements. Modules thus represent a dynamic entity of a group of molecules that occur and function together. The modules
may exist as isolated structure or may be interconnected in vivo, forming a network (Hartwell et al., 1999) (Figure 2.3 B).
Functional properties of modules would arise from the properties of the underlying components and their spatial interactions. The
modules would involve only a small fraction of the total molecules. Different molecules of same protein may belong to different
modules, at different time and cellular locations. Small structural differences in the enzyme molecules will be significant for their
involvement in different modules. Alternately, same protein may exhibit difference in function depending on the environment in
the module. Change in orientation or activity of individual components would disturb the harmony in function of a module.
Jayashree K. Sainis, Rachna Agarwal and Michael Melzer 37
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