Alienware Computers
Chlorophyll biosynthetic heterogeneity (Rebeiz et al, 2003) refers either (a) to spatial biosynthetic heterogeneity, to (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 and Figure 2, organize all known Chl biosynthetic reactions into a logical scheme made up of 16 different biosynthetic routes. Each route consists of one or more biosynthetic reactions that has been discussed in some details in previous sections.
During the past few years a systematic research effort has been undertaken in the laboratory of plant biochemistry and photobiology (LPBP) to explore the relationship of Chl biosynthetic heterogeneity to the assembly of thylakoid membranes. In a departure from a conventional discussion of the Chl biosynthetic pathway as a standalone entity, this section will focus on a discussion of Chl biosynthetic heterogeneity in the context of a photosynthetic unit made up of PSI, PSII and light harvesting Chl-protein complexes (LHCs) (see below).
Fig. 8A. Schematics of the SBP-single location model in a PSU. As an example, the functionality of the model was illustrated with the use of three apoproteins namely CP29, LCHI-730 and CP47. Abbreviations: SBP = single-branched Chl biosynthetic pathway; PSII = photosystem II; LHCII, the major light-harvesting Chl-protein complex of PSII, LHCI, one of the LHC antennae of PSI, CP47 and CP29, two PSII antennae, LHCI-730, the LHC antenna of PSI. Curved lines indicate putative energy transfer between tetrapyrroles and a Chl-protein complex. Adapted from reference (Rebeiz et al, 2003).
Within the PSU, the SBP-single location model (Fig. 8 A) 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.
Fig. 9B. Schematics of the SBP-multilocation model in a PSU. All abbreviations and conventions are as in Fig. 8A.
In the SBP-multilocation model (Fig. 9 B), 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.
Fig. 10C. Schematics of the MPP-sublocation model in a PSU. All abbreviations and conventions are as in Fig. 8A.
In the MBP-sublocation model (Fig. 10 C), 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, 2003, Kolossove et al, 2003). 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 Å (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. 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. It is the precursor of DV and MV Pchlide a. The protochlorophyll(ide) [(Pchl(ide)] of higher plants 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 d-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 excitation energy transfer between the accumulated anabolic tetrapyrroles and Chl a-protein complexes, excitation spectra were recorded at 77°K at the respective emission maxima of the selected Chl a acceptors, namely at ~685, ~695, and ~735nm. It was conjectured that if excitation 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). Assignment of in situ excitation maxima to various metabolic tetrapyrroles was unambiguous except
for a few cases at theshort 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 biosynthetic heterogeneity of the Chl biosynthetic pathway. Thus, the multi-branched Chl biosynthetic pathway reported in Figs 1, 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 Å (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 Å 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 Å ( 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 (Bassi et al, 1990), that the core of PSII including CP29, would be located about 126 Å away from the SBP. On the other hand, LHCI-730 would be located about 159 Å 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) Å 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 Å 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, and distances separating tetrapyrrole donors from Chl a acceptors and other considerations favored the operation of the MBP-sublocation Chl biosynthesis-thylakoid biogenesis model as described below.
First analytical tools were developed to calculate the distances separating Proto, Mp(e), and DV and MV Pchlide a from Chl a acceptors ( Kopetz et al, 2004). The calculated distances were next compared to current concepts of the photosynthetic unit structure (Allen and Forsberg, 2001 ; Anderson, 2002; Staehelin, 2003), and the Chl-thylakoid biogenesis models proposed in Figs. 8A, 9B, and 10C (see above). The calculated distances separating Proto, Mp(e) and DV and MV Pchlide a from various Chl a acceptors in situ are reported in Table 10.
Table 2. Exerpted from Kopetz et al, 2004
The early concept of a PSU consisting of about 500 antenna Chl per reaction center has evolved into two pigment systems each with its own reaction center and antenna
Chl (Allen and Forsberg, 2001; Anderson, 2002; Staehelin, 2003). The early visualization
of the two photosystems consisted of various pigment-protein complexes arrayed into a linear PSU (the continuous array model), about 450 Å in length and 130 Å in width
(Bassi, et al, 1990). In the PSU, the LHCII is shared between the two photosystems. More recent models favor the concept of a laterally
heterogeneous PSU (Allen and Forsberg, 2001; Anderson, 2002; Staehelin, 2003).
In this model LHCII shuttles between PSI and PSII upon phosphorylation and dephosphorylation (11). Furthermore while PSII is mainly (but not exclusively) located in appressed
thylakoid domains, PSI is located in non-appressed stroma thylakoids, grana margins, and end membranes (13, 15). The calculated distances separating Proto, Mp(e) and DV and MV Pchlide a
from various Chl a acceptors in situ are reported in Table 10.
Distances separating anabolic tetrapyrroles from various Chl-protein complexes ranged from a low of 16.26 Å for Proto-Chl a separation in cumber, to a high of 40.91 Å for Proto-Chl a-F695 separation in barley (Table 10). The magnitude of these distances is certainly compatible with the observation of intense resonance excitation energy transfer reported in (2).
In cucumber, a DDV-LDDV plant species (4), the distances that separate Proto were shorter than those that separate Mp(e) and DV Pchlide a from the Chl a species (Table 10). Since Proto is an earlier intermediate of Chl a biosynthesis than Mp(e) and Pchlide a, it indicates that in cucumber, the Chl a-protein biosynthesis subcenter is a highly folded entity, where linear distances between intermediates and end products bear little meaning (see discussion). On the other hands, in barley, a DMV-LDMV plant species (4) distances separating Proto from various Chl a acceptors were generally longer than those separating Mp(e) and MV Pchlide a from the Chl a acceptors (Table 10). This in turn suggests that the tetrapyrrole-protein complex folding in cucumber (DV subcenters) is different than in barley (MV subcenters).
On the other hands the shorter distances separating anabolic tetrapyrroles from Chl-protein complexes (Table 10) are compatible with the SBP-multilocation and MBP-sublocation models. Since overwhelming experimental evidence argues against the operation of a single-branched Chl biosynthetic pathway in plants (1) that leaves us with the MBP-sublocation model alternative. In this model (1), the unified multibranched Chl a/b biosynthetic pathway, is visualized as the template of a Chl-protein biosynthesis center where the assembly of PSI, PSII and LHC takes place (17, 18). The multiple Chl biosynthetic routes are visualized, individually or in groups of one or several adjacent routes, as Chl-apoprotein biosynthesis subcenters earmarked for the coordinated assembly of individual Chl-apoprotein complexes. Apoproteins destined to some of the subcenters may possess specific polypeptide 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 subcenter destination and its signal is split off, it binds nascent Chl formed via one or more biosynthetic routes, as well as carotenoids. During pigment binding, the apoprotein folds properly and acts at that location, while folding or after folding, as a template for the assembly of other pigment-proteins. This model is certainly compatible with the lateral heterogeneity of the PSU and can account for the observed resonance excitation energy transfer and the short distances separating anabolic tetrapyrroles from Chl-protein complexes in the distinct PSI, PSII and shuttling LHCII entities that compose the PSU.
In all cases, it was observed that while distances separating metabolic tetrapyrroles from Chl a E670F685 and Chl a E677F695 were in the same range, those separating Chl a E704F735 from the anabolic tetrapyrroles were much shorter (Table 10). As may be recalled, it is believed that the fluorescence emitted at F685 nm arises from the Chl a of the light-harvesting Chl-protein complexes (LHCII and LHCI-680), that emitted at F695 nm originates mainly from the PSII antenna Chl a (CP47 and/or CP29), while that emitted at F735 nm originates primarily from the PSI antenna Chl a (LHCI-730) (Bassi et al., 1990). This in turn suggests that in the Chl a-protein biosynthesis subcenters, protein folding is such that the PSI antenna Chl a (LHCI-730) is much closer to the terminal steps of anabolic tetrapyrrole biosynthesis than the LHCII and LHCI-680 Chl-protein complexes or the CP47 and/or CP29 PSII antenna Chl a complexes.