Heme and Chlorophyll a Biosynthetic Pathways
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Heme and chlorophyll (Chl) are porphyrins. Porphyrins (also referred to as tetrapyrroles) are essential for life in the biosphere). Chlorophyll catalyzes the conversion of solar energy to chemical energy via the process of photosynthesis (see below). Organic life in the biosphere is made possible by consumption of the chemical energy generated by photosynthesis.
Hemes are the prosthetic groups of cytochromes which are involved in electron transport during oxidative phosphorylation and photosynthetic phosphorylation which generate ATP and NADPH. The latters are essential for many cellular functions.
A synopsis of the biosynthetic pathways responsible for the formation of heme and Chl is given below. Discussion of various topics is accompanied by appropriate structural formulas that can be viewed in 2 or 3 dimensions (2D or 3D), and can be manipulated on the screen. Proper viewing and manipulation of these formulas require the installation of MDL Chemscape Chime as a browser plug in. Chime can be DOWNLOADED from the MDL homepage. At this stage the chemical structures in this document are best viewed with Netscape Communicator, version 4.03
The world population of about 6 billion is expected to increase by about 3 billion by the year 2030. It may reach 18 billion by the end of the century. Worldwide, there has been a progressive decline in cereal yield, and at present the annual rate of yield increase is below the rate of population increase (Somerville and Briscoe, 2001). Since it will be difficult to increase the land area under cultivation without serious environmental consequences, the increased demand for food and fiber will have to be met by higher agricultural plant productivity. Plant productivity depends in turn on photosynthetic efficiency. Indeed, life in the biosphere depends on the process of photosynthesis which converts light energy, carbon dioxide and water into the chemical energy, required for the formation of food and fiber. Photosynthetic efficiency is controlled by extrinsic factors such as the availability of water, CO2, inorganic nutrients, ambient temperature and the metabolic and developmental state of the plant, as well as by intrinsic factors (Lien and San Pietro, 1975)). The most important intrinsic factor is the efficiency of the photosynthetic electron transport system (PETS).
PETS is driven by two photochemical reactions that take place in membrane-bound photosystem I (PSI) and PSII Chl-protein complexes. Under natural conditions, Chl concentration in photosynthetic membranes is high enough to result in near total absorption of all incident photosynthetically active photons between 400 and 700 nm. Under normal weather conditions, these photons represent about 44.5 % of the total incident radiation. If PSI and PSII operate at the maximal quantum efficiency of 1, the maximum possible overall photosynthetic energy conversion efficiency would amount to about 12% (Lien and San Pietro, 1975)). Yet the average net photosynthetic productivity in conventional agriculture is in the range of 2-8 tons of dry organic matter per acre per year. This corresponds to a photosynthetic conversion efficiency of about 0.1-0.4% of the total incident radiation. Therefore the discrepancy between the maximal theoretical efficiency of 12% and observed efficiencies, range from 3000-12000%. This discrepancy is due to rate limiting extrinsic factors, and to intrinsic limitations of PETS.
According to Lien and San Pietro (Lien and San Pietro, 1975), under moderate light intensities of a shady sky (1/10 full sunlight), and using the figure of 200 light harvesting Chl molecules per reaction center per photosystem (PS), each reaction center receives about 200 hits (excitons) s-1. Under these conditions, in order to maintain a quantum efficiency of 1, the slowest dark reaction of the entire photosystem must have a turnover rate of 200 s-1. On the other hand, under full sunlight, the turnover rate of the limiting dark reaction is about 2000 s-1. This turnover rate corresponds to a rate of O2 evolution of about 9000 mmoles of O2 evolved per mg Chl h-1. The maximal rate of O2 evolution observed during a Hill reaction under optimal light conditions and saturating light intensities rarely exceeds 5-10% of the maximal turnover rate of O2 evolution observed under full sunlight. Extensive kinetic studies have demonstrated that the rate limiting step of the PETS resides within the redox-carriers connecting the two PS, rather than in the photochemical reactions. The discrepancy between the capacities of the photon gathering apparatus (i. e. antenna Chl-protein complexes) and the capacity of the rate-limiting dark reactions has been referred to as the antenna/PS mismatch (Lien and San Pietro, 1975).
The first recognized effect of the antenna/PS mismatch is one of reduced quantum conversion efficiencies at light intensities above shade levels. The second effect has been identified with the destructive effects of the excess photons collected by antenna Chl, but not used in the photosynthetic photochemical act. It is recognized that the energy of these unused photons lead to Photodestruction of the PETS. Lien and San Pietro concluded that one way of correcting the antenna/PS mismatch is by reducing the size of the photosynthetic unit (PSU). One way of achieving this goal is by inducing the formation of more PSI and PSII Chl-Protein units and less antenna Chl per photosynthetic membrane area surface unit. Research performed in the 1970s failed however in its efforts to alter significantly the size of the PSU in algal cell cultures (Lien and San Pietro, 1975).
On the basis of recent advances in the understanding of the chemistry and biochemistry of the greening process and significant advances in the molecular biology of greening, we have reason to believe that alteration of the PSU size has become a realistic possibility. In our opinion, the bioengineering of reduced PSU size would require (a) thorough knowledge of the biosynthesis of thylakoid components such as porphyrin, Chl, carotenoids and lipids (b) thorough knowledge of photosynthetic membrane (thylakoid) apoprotein biosynthesis and (c) deeper understanding of the biosynthesis and regulation of the assembly of pigment-protein complexes. In this context, this overview is devoted to an in depth discussion of our present knowledge of the Chl biosynthetic pathway. It is also hoped that this overview will trigger closer cooperation between porphyrin, pigment, lipid and thylakoid apoprotein biochemists, molecular biologists of the greening process and functional scientists involved in photosynthesis research.
In this overview, the complexity and biochemical heterogeneity of the Chl biosynthetic pathway and the relationship of this complexity
to the structural and biosynthetic complexity of photosynthetic membranes will be emphasized. We will also emphasize in historical perspective,
key stages in our understanding of the Chl biosynthetic heterogeneity. The reader should keep in mind that a complex biosynthetic process
is only fully understood when it becomes possible to reconstitute in vitro every step of the process. We are not yet at this
stage of understanding of thylakoid membrane biogenesis. Considerable progress has been achieved however, in the understanding of numerous
facets of the Chl biosynthetic pathway, namely (a) detection and identification of various major and minor metabolic intermediates
(b) precursor-product relationships between various intermediates, (c) structure and regulation of many enzymes of the pathway, and
(d) the relationship of the Chl biosynthetic heterogeneity to the structural and functional heterogeneity of thylakoid membranes.
Since the 1963 seminal review of Smith and French, our understanding of the Chl biosynthetic pathway has changed dramatically. Several factors have contributed to this phenomenon, among which: (a) development of systems capable of Chl and thylakoid membrane biosynthesis in organello and in vitro, (Rebeiz and Castelfranco, 1971a, 1971b; Daniell and Rebeiz, 1982; Rebeiz et al, 1984, Kolossov et al, 1999, (b) powerful analytical techniques that allowed the qualitative and quantitative determination of various intermediates of the pathway (Rebeiz, 2001), (c) recognition that the greening process proceeds differently in etiolated and green tissues, in darkness and in the light and in plants belonging to different greening groups (Carey and Rebeiz, 1985; Ioannides et al, 1994, Abd-El- Mageed et al, 1997), and (d) recognition of the probability that the structural and functional complexity of thylakoid membranes is rooted in a multibranched, heterogeneous Chl biosynthetic pathway (Rebeiz, et al, 1999).
Chlorophyll biosynthetic heterogeneity (Rebeiz et al, 1981, 1983 , 1994) refers either (a) to spatial biosynthetic heterogeneity, (b) to chemical biosynthetic heterogeneity, or (c) to a combination of spatial and chemical biosynthetic heterogeneities. Spatial biosynthetic heterogeneity refers to the biosynthesis of an anabolic tetrapyrrole or end product by identical sets of enzymes, at several different locations of the thylakoid membranes. On the other hand, chemical biosynthetic heterogeneity refers to the biosynthesis of an anabolic tetrapyrrole or end product at several different locations of the thylakoid membranes, via different biosynthetic routes, each involving at least one different enzyme.
Figure 1 (15 biosynthetic routes) and Figure 2 (2 biosynthetic routes)
organize all known Chl biosynthetic reactions into a logical scheme made up of 17 different biosynthetic routes. Each route consists of
one or more biosynthetic reactions that will be discussed in some details in ensuing sections. Figure 2 is depicted below Figure 1.
Fig. 1. Integrated Chl a/b biosynthetic pathway depicting 15 carboxylic routes. To facilitate
understanding of the text, various biosynthetic routes are designated by the numbers 1-14. Black boxes with yellow lettering joined by black
arrows and numbers refer to biosynthetic routes that occur in etiolated tissues in darkness, or in greening tissues at the beginning of the
light phase of the photoperiod. White boxes with green lettering joined by yllowish-brown arrows and numbers refer to biosynthetic routes
that occur in green tissues, during the light phases of the photoperiod. Blue frames refer to routes that occurin DDV-LDDV greening groups.
Red frames refer to routes that occurin DMV-LDMV greening groups.
Fig. 2. Integrated Chl a/b biosynthetic pathway depicting the two fully esterified biosynthetic
routes. To facilitate understanding of the text, the biosynthetic routes are designated by the numbers 1, 16, and 17. Biosynthetic
routes 16 and 17 branch from biosynthetic routes 1 and 12 of Fig. 1. Black boxes with yellow lettering joined by black arrows and numbers
refer to biosynthetic routes that occur in etiolated tissues in darkness, or in greening tissues at the beginning of the light phase of
the photoperiod.
Fig. 3. Schematics of the Single-Branched and Multibranched Chl-Thylakoid Apoprotein
Biosynthesis Models in a Chl-Protein Biosynthesis Center i. e. in a Photosynthetic Unit. A: single-branched single location model;
B: single-branched multi-location model; C: multi-branched-sublocation model. SBP: single-branched Chl biosynthetic pathway;
MBP: multi-branched Chl biosynthetic pathway. LHCI: light harvesting Chl-protein complex I; PSII: photosystem II; LHCII: Chl a/b light
harvesting Chl-protein complex. Curved lines indicate energy transfer between a tetrapyrrole and a Chl-protein complex.
The models take into account the dimension of the PSU Bassi et al, 1990, the biochemical heterogeneity of the Chl biosynthetic pathway Rebeiz et al, 1994, 1999 , 2001, and the biosynthetic and structural complexity of thylakoid membranes Sundqvist and Ryberg, 1993. Within a PSU, the three putative Chl-apoprotein thylakoid biosynthesis models are referred to as: (a) the single branched biosynthetic pathway (SBP)-single location model, (b) the SBP-multilocation model and (c) the multibranched biosynthetic pathway (MBP)-sublocation model (Fig. 3).
Within the PSU, the SBP-single location model (Fig. 3A) was considered to accommodate only one Chl-apoprotein
thylakoid biosynthesis center and no Chl-apoprotein thylakoid biosynthesis subcenters. Within the Chl-apoprotein thylakoid biosynthesis
center, Chl a and b are formed via the conventional single-branched Chl biosynthetic pathway (Fig. 4) at a
single location accessible to all Chl-binding apoproteins. An apoprotein moves to that location in the unfolded state, picks up a complement
of MV Chl a and/or MV Chl b, and undergoes appropriate folding. Then the folded Chl-apoprotein complex moves from the Chl biosynthesis location
to a specific PSI, PSII, or LHC location within the Chl-apoprotein biosynthesis center.
In the SBP-multilocation model (Fig. 3B), every location within the photosynthetic unit is considered to be a Chl-apoprotein thylakoid biosynthesis subcenter. In every Chl-apoprotein biosynthesis subcenter, a complete single-branched Chl a/b biosynthetic pathway is active. Association of Chl a and/or Chl b with specific PSI, PSII, or LHC apoproteins at any location is random. In every Chl-apoprotein biosynthesis subcenter, distances separating metabolic tetrapyrroles from the Chl-protein complexes are shorter than in the SBP-single-location model.
In the MBP-sublocation model (Fig. 3C), the unified multi-branched Chl a/b biosynthetic pathway is visualized
as the template of a Chl-protein biosynthesis center where the assembly of PSI, PSII and LHC take place (
Rebeiz et al, 1999). The multiple Chl biosynthetic routes are visualized, individually or in groups of one or several adjacent
routes, as Chl-apoprotein thylakoid biosynthesis subcenters earmarked for the coordinated assembly of individual Chl-apoprotein complexes.
Apoproteins destined to some of the biosynthesis subcenters may possess specific signals for specific Chl biosynthetic enzymes peculiar
to that subcenter, such as 4-vinyl reductases, formyl synthetases or Chl a and Chl b synthetases. Once an apoprotein formed in the cytoplasm
or in the plastid reaches its biosynthesis subcenter destination and its signal is split off, it binds nascent Chl formed via one or more
biosynthetic routes. During Chl binding, the apoprotein folds properly and act at that location, while folding or after folding, as a template
for the assembly of other apoproteins. In this case too, shorter distances separate the accumulated tetrapyrroles from the Chl-protein complexes.
Fluorescence resonance energy transfer involves the transfer of excited state energy from an excited donor D* to an unexcited acceptor A (Calverts and Pitts, 1967; Lakowicz, 1999; Turro, 1965). The transfer is the result of dipole-dipole interaction between donor and acceptor and does not involve the exchange of a photon. The rate of energy transfer depends upon (a) the extent of overlap of the emission spectrum of the donor and the absorption spectrum of the acceptor, (b) the relative orientation of the donor and acceptor transition dipoles, and (c) the distance between donor and acceptor molecules. As soon as the excited donor D* and unexcited acceptor A states are coupled by an appropriate interaction, they become degenerate if there is an excited state of the acceptor A, which requires exactly the same excitation energy available in D*. When such a condition exists, excitation of one of the degenerate states leads to a finite probability that the same excitation will appear in the other degenerate state (Turro, 1965). This probability increases with time but is inversely proportional to the sixth power of the fixed distance separating the centers of the donor and acceptor molecules. It has been estimated that dipole-dipole energy transfer between donor and acceptor molecules may occur up to a separation distance of 50 to 100 ( Calverts and Pitts, 1967).
Resonance excitation energy transfer from three tetrapyrrole donors to the Chl a of Chl-protein complexes were monitored, namely: from protoporphyrin IX (Proto), divinyl (DV) Mg-Proto and its methyl ester and monovinyl (MV) and DV Pchlide a. DV Proto is a common precursor of heme and Chl (section 4.7). It is the immediate precursor of DV Mg-Proto. As such, it is an early intermediate along the Chl biosynthetic chain. Biosynthetically, it is several steps removed from the Chl end product. Mg-Proto is a mixed MV-DV, dicarboxylic tetrapyrrole pool, consisting of DV and MV Mg-Proto (section 7). It is the precursor of DV and MV Pchlide a. The protochlorophyll(ide) [(Pchl(ide)] of higher plants (section 10, 12) consists of about 95% protochlorophyllide (Pchlide) a and about 5% Pchlide a ester (Pchlide a E). The latter is esterified with long chain fatty alcohols (LCFAs) at position 7 of the macrocycle. While Pchlide a ester consists mainly of MV Pchlide a ester, Pchlide a consists of DV and MV Pchlide a. The latter are the immediate precursors of DV and MV chlorophyllide (Chlide) a. Accumulation of the various tetrapyrrole donors was induced by incubation of green tissues with delta-aminolevulinic acid (ALA) and/or 2,2-dipyridyl (Rebeiz et al, 1988).
The task of selecting appropriate Chl a-protein acceptors was facilitated by the fluorescence properties of green plastids. At 77 K, emission spectra of isolated chloroplasts exhibit maxima at 683-686 nm (~F685), 693-696 nm (~F695), and 735-740 nm (~F735). It is believed that the fluorescence emitted at ~F685 nm arises from the Chl a of LHCII, the major thylakoid LHC antenna, and LHCI-680, one of the LHC antennae of PSI (Bassi et al, 1990). That emitted at ~F695 nm is believed to originate mainly from the Chl a of CP47 and CP29, two PSII antennae Bassi et al, 1990. That emitted at ~F735 nm is believed to originate primarily from the Chl a of LHCI-730, a PSI antenna Bassi et al, 1990. Since these emission maxima are readily observed in the fluorescence emission spectra of green tissues and are associated with definite thylakoid Chl a-protein complexes, it was conjectured that they would constitute a meaningful resource for monitoring excitation resonance energy transfer between anabolic tetrapyrroles and representative Chl a-protein complexes. To monitor the possible occurrence of resonance energy transfer between the accumulated anabolic tetrapyrroles and Chl a-protein complexes, excitation spectra were recorded at 77K at the respective emission maxima of the selected Chl a acceptors, namely at ~685, ~695, and ~735nm. It was conjectured that if resonance energy transfers were to be observed between the tetrapyrrole donors and the selected Chl a acceptors, definite excitation maxima would be observed. These excitation maxima would correspond to absorbance maxima of the various tetrapyrrole donors, and would represent the peaks of the excitation resonance energy transfer bands.
Pronounced excitation resonance energy transfer bands from Proto, Mp(e), and Pchl(ide) a to Chl a ~F685, ~F695, and ~F735 were detected (Table 1).
Table 1
Assignment of in situ excitation maxima to various metabolic tetrapyrroles was unambiguous except for a few cases at the short wavelength and long wavelength extremes of excitation bands. Contrary to previous believes, it was surprising to observe a significant diversity in various intra-membrane environments of Proto, Mp(e), and Pchl(ide) a. This diversity was manifested by a differential donation of resonance excitation energy transfer to the different Chl a-apoprotein complexes from multiple Proto, Mp(e) and Pchl(ide) a sites, and is highly compatible with the biosynthetic heterogeneity of the Chl biosynthetic pathway. Thus, the multi-branched Chl biosynthetic pathway reported in Figs 1, 2 accounts for the existence of multiple Proto, Mp(e) and Pchl(ide) a donor sites by depicting multiple Biosynthetic routes that originate in multiple ALA, Proto, Mg-Proto and Pchlide a sites.
Since resonance energy transfer is insignificant at distances larger than 100 (Calvert and Pitts, 1967), The detection of pronounced resonance excitation energy transfer from Proto, Mp(e), and Pchl(ide) a to Chl a ~F685, ~F695, and ~F735 (Table 1) indicates that these anabolic tetrapyrroles are within distances of 100 or less of the Chl a acceptors. This in turn is incompatible with the functionality of the SBP-single location Chl-thylakoid biogenesis model. Indeed, it can be estimated from published data that the size of the PSU which includes the two PS, LHC, as well as the CF0-CF1 ATP synthase is about 130 x 450 (Bassi et al, 1990). Most PSU models depict a central cyt b6 complex flanked on one side by PSI and coupling factor CF1, and on the other side by PSII and LHCPII. With this configuration, the shortest distance between the single-branched Chl biosynthetic pathway and PSI, PSII, and LHCII, in the SBP-single location model would be achieved if the SBP occupied a central location within the PSU. In that case it can be calculated from the PSU model proposed by Bassi (1990) that the core of PSII including CP29, would be located about 126 away from the SBP. On the other hand, LHCI-730 would be located about 159 on the other side of the SBP. The centers of the inner and outer halves of LHCII surrounding the PSII core would be located about 156 (outer half) and 82 (inner half) from the SBP. The detection of pronounced excitation resonance energy transfer from Proto, Mp(e), and Pchl(ide) a to Chl a ~F685, ~F695, and ~F735 indicates that these anabolic tetrapyrroles are within distances of 100 or less of the Chl a acceptors. In view of the above considerations it was concluded that the detection of resonance excitation energy transfer between anabolic tetrapyrroles and Chl a of the various thylakoid Chl-protein complexes was not compatible with the functionality of the SBP-single location Chl-thylakoid biogenesis model. Further calculations of resonance excitation energy transfer rates, of distances separating tetrapyrrole donors from Chl a acceptors and other considerations favored the operation of the MBP-sublocation Chl biosynthesis-thylakoid biogenesis model (Kopetz, 2000).
The remaining sections of this overview will therefore be devoted to a discussion of the Chl biosynthetic heterogeneity of the multibranched
Chl biosynthetic pathway in the context of the PSU size.