VI. The Chl a Carboxylic Biosynthetic Routes: Protochlorophyll(ide) [(Pchl(ide)] a-Protein Complexes [(Pch(ide) a Holochromes (Hs)]
All metabolic pools in the plastid, between ALA and Chl are either loosely or tightly bound to the plastid membranes (Smith and Rebeiz, 1979; Lee et al, 1992). Upon binding to apoproteins, the spectroscopic properties of pigment chromophores change drastically (see below). If a pigment chromophore occurs in low amounts its presence in the plastid membranes is usually masked by more abundant pigment-protein such as Chl-protein complexes. In etiolated tissues, Pchlide a is the most abundant tetrapyrrole (90-95%) followed by less abundant Pchlide a phytyl ester (5-10%) (Rebeiz et al, 1970). That made it possible early on, to detect Pchlide a and Pchlide a phytyl esters-apoprotein complexes by various electronic spectroscopic techniques (Smith, 1952; Smith and Kupke, 1956) . Since these complexes are a mixture of Pchlide a (90-95%) and small amounts of Pchlide a phytyl ester (5-10%), they will be referred to collectively as Pchl(ide) a] holochromes (Hs).
A. Spectroscopic Properties of Various [Pchl(ide) a] Holochromes
The existence of at least two spectroscopically different [Pchl(ide) a] H was first reported by Hill et al (1953). Using a Zeiss microspectroscope they observed that in etiolated barley leaves, a band absorbing at 650 nm disappeared (was phototransformed i. e. photoconverted to a Chl-like compound) as the light was turned on and was replaced by the appearance of two new absorbance bands: one near 670 nm which corresponded to newly formed Chl a, and one at 635 nm, which did not appear to be convertible to Chl. These results gave rise to the notion that etiolated tissues contained two spectroscopically different Pchl(ide) a H complexes. A longer wavelength, phototransformable (t) complex absorbing at 650 nm, and a shorter wavelength, non-phototransformable complex (nt), absorbing at 635 nm.
To explain the difference between the long and short wavelength Pchl(ide) a] holochrome species, Butler and Briggs (1966), on the basis of freezing and thawing treatments of plant tissues, proposed that aggregation of pigment molecules in etioplasts shifts the absorption maximum to longer wavelengths, while disaggregation of pigment molecules shifts the absorption maximum to shorter wavelengths. Using freezing and thawing as well as extraction, heat and acid treatments, Dujardin and Sironval (1970) suggested the presence of three universal Pchl(ide) a H in plants, namely: an aggregated, phototransformable species absorbing at 647-648 nm that involves pigment-protein and pigment-pigment interactions, a second phototransformable species absorbing at 639-640 nm which involves only pigment-protein interactions, and a non-phototransformable species absorbing at 627-628 nm, which is loosely bound to proteins. They also proposed that pigment-pigment interaction is not required for phototransformation while binding to a specific protein is required.
Using absorption, fluorescence emission and fluorescence excitation spectroscopy at 77 K, Kahn et al, (1970), further characterized the three Pchl(ide) a H species as consisting of (a) a non-phototransformable fluorescent species with a red excitation maximum at 628 nm and a red fluorescence emission maximum at 630 nm [an (E628 F630) species], (b) a phototransformable, nonfluorescent species with a red excitation maximum at 639 nm [(E639 F00)] which transfers its excitation energy to a Pchl(ide) a H with a red excitation maximum at 650 nm and a red fluorescence emission maximum at 655 nm [(E650 F655) species]. The latter is the predominant Pchl(ide) a H in etiolated tissues.
Using high resolution 77 K spectrofluorometry, Cohen and Rebeiz (1981) carried out a detailed studies of the Pchl(ide) a H species that accumulate in etiolated cucumber and bean. The various Pchl(ide) a species were assigned Soret excitation maxima (E), and fluorescence emission maxima (F) using matrix analysis. The following Pchl(ide) a H species were detected in etiolated cucumber cotyledons: non-phototransformable (nt) short wavelength (SW) Pchl(ide) a H (E440 F630), phototransformable (t) SW Pchl(ide) a H (E443 F633), (E444 F636) and (E445 F640), and t-long wavelength (LW) Pchl(ide) a (E450 F657), which was the predominant species in etiolated cucumber. In red-kidney bean, the following Pchl(ide) a H species were detected nt-SW Pchl(ide) a H (E440 F630), t-SW Pchl(ide) a H (E441 F633), (E442 F636) and (E443 F640), and t-long wavelength Pchl(ide) a (E447 F657), which was the predominant species in etiolated bean.
The contribution of SW and LW Pchl(ide) a Hs to the natural greening process was assessed during photoperiodic greening, i. e. during greening under alternating light/dark photoperiods (Cohen and Rebeiz, 1978). The following observations were made (a) SW Pchl(ide) H species appeared within the first 24 h of germination of cucumber seedlings, (b) subsequently, LW Pchl(ide) H species appeared then disappeared, (c) The ratio of LW/SW Pchl(ide) H species reached a maximum of 3:1 by the end of the second dark cycle and reached a value of zero by the end of the 6 th dark cycle, (d) SW Pchl(ide) H species were continuously present during the dark and light cycles and appeared to contribute actively to the greening process, (e) primary corn and bean leaves exhibited a similar pattern of Pchl(ide) H formation.
B. Nature of the Pigment-Protein Association of [Pchl(ide) a] Holochromes
Various Pchl(ide) a chromophores are bound to the different holochrome apoproteins by non-covalent forces. This is evidenced by the ready extraction of the Pchl(ide) a chromophores by organic solvents such as acetone. Association of the pigment chromophores with apoproteins, probably involve (a) axial coordination of the pigment central Mg-atom to nucleophyllic amino acid side chains, and (b) hydrogen bonding between the keto group of the cyclopentanone ring of the Pchl(ide) a chromophore and appropriate amino acid side chains. Pigment-pigment interaction may involve axial coordination of the keto group of the cyclopentanone ring of one Pchl(ide) a chromophore to the central Mg-atom of another Pchl(ide) a chromophore as suggested by Katz et al (1966) for Chl-Chl association in hydrophobic environments, as well as Pi-Pi interactions (Boucher and Katz, 1967). Axial coordination of the histidine nitrogen of apoproteins to the central Mg-atom (Deisenhofer and Michel, 1991) of Pchl(ide) a has not been established for various Pchl(ide) a H.
Early work dealing with the purification of Pchl(ide) a] H was described by Boardman (1966). The partially purified Pchl(ide) a] holochrome (MW = 600,000) exhibited a red absorption maximum at 637.5 nm. Upon illumination part of the Pchl(ide) a was converted into Chlide a with a red absorption maximum at 681 nm which after two minutes shifted to 675 nm. This preparation, however, did not preserve the heterogeneous spectral properties observed in vivo. A purer preparation from etiolated bean leaves (MW = 300,000) was described by Schopfer and Siegelman (1968). The purified Pchl(ide) a H exhibited a red absorption maximum at 639 nm, which did not reflect the spectral heterogeneity observed in vivo. In the light the red absorption maximum shifted to a Chlide a red absorption maximum at 678 nm, which drifted to 672 nm in darkness. More purified Pchl(ide) a Hs were prepared from etiolated barley (MW 63,000) and bean (MW 100,000) by Henningsen and Kahn (1971). Photoconversion yielded a Chl(ide) a complex with a red absorption maximum at 678 nm. In this case too, the spectral properties of the purified Pchl(ide) a holochrome did not reflect the spectral heterogeneity observed in vivo.