In contrast to porphyrins and chlorins, the direct metalation of bacteriochlorins is difficult. Nevertheless, Cu2+ and Zn2+ can be introduced into bacteriopheophytin in acetic acid, whereas Cd2+ can be inserted in dimethylformamide. The former reactions depend on the substituents of the isocyclic ring:  they are facilitated if enolization of the β-ketoester system is inhibited. Starting with [Cd]-bacteriochlorophyll-a or its 132-hydroxy derivative, a series of metallo-bacteriochlorins with central divalent ions Pd2+, Co2+, Ni2+, Cu2+, Zn2+, and Mn2+ have been obtained by transmetalation. Like in the parent Mg complex, the four principal optical transitions are well-separated in these complexes, and their responses to changes in the central metal and its coordination state can be followed in detail. The energies of the Q y and B x transitions are almost independent of the central metal, whereas the Q x and B y transition energies change significantly, depending on the central metal as well as the presence of additional axial ligands. If the complexes are grouped by their coordination number, empirical linear correlations exist between these shifts and the ratio / , where is Pauling's electronegativity value and is the ionic radius of the metal. A similar correlation was found for those 1H NMR signals influenced mainly by the ring current and for the redox potentials. This observation was in contrast with the linear relationships with alone, found for metal-substituted porphyrins. The spectral variations influenced by the central metal and its state of ligation are attributed, within the framework of the four-orbital model, to the electrostatic interaction of the electron densities in the four orbitals with the effective charge of the central metal ions, which is most pronounced for the a2u orbital (HOMO-1). Ligation studies have revealed that addition of the first axial ligand decreases the effective charge of the central metal by approximately 50% and addition of the second axial ligand by another 20% with respect to the absence of axial ligands. The singlet−triplet splitting deduced from fluorescence and phosphorescence measurements is similar for [Pd]-, [Cu]-, [Zn]-, and [Mg]-BChl (4550 ± 100 cm-1).

Chromopeptides were prepared by proteolytic digestion of phytochrome (far-red absorbing form, Pfr) and of phycocyanin. The phycocyanobilin peptide, the chromophore of which is Z,Z,Z-configurated, was modified to the Z,ZE isomeric chromophore. It has been demonstrated earlier that the Pfr chromopeptide and the Z,Z,E-configurated phycocyanin chromopeptide behave similarly with regard to spectral and chromatographic properties and reactivity. We present evidence here, obtained by high-resolution 'H NMR spectroscopy, that both the modified phycocyanobilin chromophore and the phytochrome chromophore obtained directly from Pfr are 15E-configurated.Plant development is influenced by light in many ways. An important photoreceptor of higher plants is the chromoprotein phytochrome (1-3), which can mediate light-dependent irreversible differentiation (e.g., seed germination, flowering, and stem and leaf growth) and reversible modulations (e.g., leaflet or chloroplast movement, root tip adhesion, and transmembrane potentials).A characteristic property of phytochrome in vivo (in the plant cell) and in vitro is its photoreversibility. The physiologically inactive (red-absorbing) Pr form (Ama. = 660 nm) is transformed by red light to the physiologically active Pfr form (Amak = 730 nm), which in turn is reconverted by far-red light to the Pr form. nm Pr = Pfr 730 nmThe chemical structure of the Pr chromophore (structure la), including its linkage to the protein, was elucidated by combination of oxidative degradation and UV/visible spectroscopy (4-6), by comparison of the cleaved chromophore with the product obtained by total synthesis (7,8), and by high-resolution NMR spectroscopy of a chromopeptide (9). It is closely related to the structure of the phycocyanin chromophore

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Chlorophyll synthetase catalyzes the last step of chlorophyll biosynthesis, namely prenylation (esterification) of chlorophyllide with phytyl diphosphate or geranylgeranyl diphosphate. During investigation of various chlorophyllide derivatives as potential substrates we observed lower esterification with increasing percentages of chlorophyllide a' in epimeric mixtures of chlorophyllides a and a: To avoid epimerization during esterification, we studied the reaction in detail with model compounds [zinc-1 32(R)-methoxy-pheophorbide a and zinc-1 3'(S)-rnethoxy-pheophorbide a, zinc-1 32(R)-methoxy-pyropheophorbide a and zinc-chlorin e,-l3', 1 5'-dimethylester]. We conclude that compounds which have the 13'-carbomethoxy group at the same side of the macrocycle as the propionic side chain of ring D are neither substrates nor competitive inhibitors. Only compounds having the 132-carbomethoxy group at the opposite site are substrates for the enzyme. Naturally occuring chlorophyll a ' must be formed by epimerization after esterification.Chlorophyll a' [a-'prime', 13Z(S)-chlorophyll a ] has been known since 1942 (Strain and Manning, 1942) as a byproduct of isolation of chlorophyll a [13'(R)-chlorophyll a]. Due to the easy epimerization of chlorophyll at C-13' (Hynninen, 1991) it is generally believed that it is formed from chlorophyll a during the extraction procedure. However, increasing evidence has accumulated during the last decade that chlorophyll a' is a natural constituent of higher plants and cyanobacteria (Watanabe et al., 1985 a,b;Kobayashi et al., 1988). Investigations on pigment composition of Chlamydomonas reinhardtii (Maroc and Tremolieres, 1990) and of P700-enriched chloroplasts of higher plants (Maeda et al., 1992) revealed that two chlorophyll a' molecules are situated in the core of photosystem I. Furthermore, the presence of two bacteriochlorophyll g 'molecules in the reaction center of heliobacteria was also described (Kobayashi et al., 1990(Kobayashi et al., , 1991. The question now arises, at which stage of the biosynthetic pathway of the chlorophylls is the prime pigment synthesized, especially whether it is formed before or after esterification of chlorophyllide a.Chlorophyll synthetase catalyzes prenylation of chlorophyllides with geranylgeranyl diphosphate (GerGerP,) or phytyl diphosphate (PhyP,), the last step of chlorophyll biosynthesis (Rudiger et al., 1980). This step is essential for translation and accumulation of chlorophyll a apoproteins (Eichacker et al., 1990(Eichacker et al., , 1992 and probably for stable assembly also for other components of the thylakoid membrane (Paulsen et al., 1990; Rudiger 1992 Rudiger , 1993. Chlorophyll synthetase catalyzes prenylation not only of chlorophyllide a, but also of chlorophyllide b and some modified derivatives (Benz and Rudiger, 1981 and Rudiger, 1992;Vezitskii and Sherbakov, 1987). During our studies on the substrate specificity of chlorophyll synthetase, we observed fractions of chlorophyllide a with a greatly reduced ability for esterificat...

The mechanism of formation of the formyl group of chlorophyll b has long been obscure but, in this paper, the origin of the 7-formyl-group oxygen of chlorophyll b in higher plants was determined by greening etiolated maize leaves, excised from dark-grown plants, by illumination under white light in the presence of either H,"0 or I8O2 and examining the newly synthesized chlorophylls by mass spectroscopy. To minimize the possible loss of label from the 7-formyl substituent by reversible formation of chlorophyll b-7l-gem-diol (hydrate) with unlabelled water in the cell, the formyl group was reduced to a hydroxymethyl group during extraction with methanol containing NaBH, : chlorophyll a remained unchanged during this rapid reductive extraction process.Mass spectra of chlorophyll a and [7-hydroxymethyl] -chlorophyll b extracted from leaves greened in the presence of either H,' *O or 1802 revealed that "0 was incorporated only from molecular oxygen but into both chlorophylls : the mass spectra were consistent with molecular oxygen providing an oxygen atom not only for incorporation into the 7-formyl group of chlorophyll b but also for the well-documented incorporation into the 131-oxo group of both chlorophylls a and b [see Walker, C. J., Mansfield, K. E., Smith, K. M. & Castelfranco, P. A. (1989) Biochem. J. 257, 599-6021. The incorporation of isotope led to as much as 77% enrichment of the 131-oxo group of chlorophyll a : assuming identical incorporation into the 13' oxygen of chlorophyll b, then enrichment of the 7-formyl oxygen was as much as 93%. Isotope dilution by re-incorporation of photosynthetically produced oxygen from unlabelled water was negligible as shown by a greening experiment in the presence of 3-(3,4-dichlorophenyl)-l ,l-dimethylurea.The high enrichment using '*02, and the absence of labelling by H,"0, unequivocally demonstrates that molecular oxygen is the sole precursor of the 7-formyl oxygen of chlorophyll b in higher plants and strongly suggests a single pathway for the formation of the chlorophyll b formyl group involving the participation of an oxygenase-type enzyme.The biosynthesis of chlorophylls (Chls) a (I) and b (111), the two major chlorophylls of higher plants, green algae and some prochlorophytes, is now largely understood [l-31, but the mechanism of the formation of the 7-formyl group of Chl b has been an outstanding gap in our current knowledge.

Using mass spectroscopy, we demonstrate as much as 93% enrichment of the 7-formyl group oxygen of chlorophyll b when dark-grown, etiolated maize leaves are greened under white light in the presence of "OZ. This suggests that a mono-oxygenase is mvolved in the oxidation of tts methyl group precursor.The concomitant enrichment of about 75% of the 13'-oxygen confirms the well-documented finding that this 0x0 group, in both chlorophyll (I and b, also arises from 0,. High "0 enrichment into the 7-formyl oxygen relative to the substrate "0, was achieved by optimization of the greenmg conditions in combination with a reductive extraction procedure. It indicates not only a single pathway for Chl b formyl group formation, but also unequivocally demonstrates that molecular oxygen is the sole precursor of the 'I-formyl oxygen.

[3-Vinyl]-bacteriochlorophyll a and related pigments modified at C-3 and/or C-13 z havc been synthesized from bactcriochlorophyll a. The reactivity at C-3 is strongly influenced by the C-132 substituent, and vice versa. Spcctroscopical data and comparison among derivatives modified at the isocyclic ring indicate that this interaction is related to formation of an intcrmcdiatc cnol(atc) structure. The possible role of enol(ate) formation in (bacterio)chlorophylls in nature is discussed. IntroductionThe chemistry of plant chlorophylls (Chl a, b) is a well-studied part of porphyrin chemistry. A large amount of data has been accumulated on the chemical reactivity of side-groups and their physical and spectroscopic properties [1][2][3]. Much of that work is motivated by the impor: 1nee of these chlorophylls in oxygenic photosynthesis. While most of these data originate from in vitro work in organic or micellar solution, much less is known on the structural and functional details in their native protein environment. In the case of bacteriochlorophylls, among which BChl a and b are most prominent, the situation is reversed. Due to the possibility to isolate bacterial light-harvesting complexes [4] and purple bacterial reaction centers [5] for more than 15 years, and to crystallize some of them, high precision structural data in their native protein environment are available [6][7][8][9][10]. However, their chemistry is relatively seldom investigated. In connection with the recently introduced methods for exchanging modified (bacterio)chlorophylls and -pheophytins into bacterial reaction centers [11][12][13][14] To understand the structure-function relationships of bacteriochlorophylls (and chlorophylls) in more detail, it is important to obtain structural links between the different naturally occurring (bacterio)chlorophyll structures. One such link is [3-acetyl]-chlorophyll a [18], bearing the 3-acetyl group characteristic of BChl a and b, and the macrocycle characteristic of the green plant Chl a and b. The complementary link is [3-vinyl]-Bchl a, which differs vice versa from Chl a by the macrocycle, and from BChl a by the presence ~l a C-3 vinyl instead of an acetyl group. Here, we wish to report a procedure to synthesize [3-vinyl]-BChl a and some related pigments, and discuss some physical properties of these pigments in vitro. Attention is given to a pronounced, and hitherto unreported, long-range interaction between substituents at the positions 3 and 13 2 , which was observed during these studies. The reactivity at C-3 is strongly influenced by the nature of the C-13 2 substitucnt, and vice versa. The data suggesl, that this 'connection' is related to f,-.~rmation of enol(ateJ structures at the isocyclic ring. There has been considerable interest before it~ the enolisation and the epimerisation at C-13 2, and their possible involvement in photosynthesis [19][20][21][22][23]. The results are discussed in this context. Material and Methods General conditionsAll chemicals and solvents used were reagent grade. eigl...

Monomeric bacteriochlorophylls BA and Ba in photosynthetic reaction centers from Rhodobacter sphueroides R26 were exchanged with (132-hydroxy-)bacteriochlorophylls containing a 3-vinyl-or 3-(a-hydroxyethyl)-substituent instead of the 3-acetyl group. The corresponding binding sites must be tolerant to the introduction of the polar residue at C-13* andmodifications of the 3-acetyl group. According to HPLC analysis, the exchange with both pigments amounts to S$ 50% of the total BChl contained in the complex, corresponding to 6 100% of the monomeric BChl aBA,a. The absorption spectra show significant changes in the Qx and Qv-region of the monomeric bacteriochlorophylls. By contrast, the absorption of the primary donor (P870) and reversible photobleaching is retained. The circular dichroism is also unchanged in the 870 nm region. The positive cd band located at around 800 nm in native reaction centers, shifts with the (blue-shifted) QY absorption(s) of BA and/or Ba, whereas the position of the negative one remains nearly unaffected. The data indicate that the latter is the upper excitonic band of the primary donor, and that there is little interaction of the monomeric BA/Bs with the primary donor.