Excited-State Properties of Protochlorophyllide Analogues and Implications for Light-Driven Synthesis of Chlorophyll.

Protochlorophyllide (Pchlide), an intermediate in the biosynthesis of chlorophyll, is the substrate for the light-driven enzyme protochlorophyllide oxidoreductase. Pchlide has excited-state properties that allow it to initiate photochemistry in the enzyme active site, which involves reduction of Pchlide by sequential hydride and proton transfer. The basis of this photochemical behavior has been investigated here using a combination of time-resolved spectroscopies and density functional theory calculations of a number of Pchlide analogues with modifications to various substituent groups. A keto group on ring E is essential for excited-state charge separation in the molecule, which is the driving force for the photoreactivity of the pigment. Vibrational "fingerprints" of specific regions of the Pchlide chromophore have been assigned, allowing identification of the modes that are crucial for excited-state chemistry in the enzyme. This work provides an understanding of the structural determinants of Pchlide that are important for harnessing light energy.

Chlorophyll c(CS-170) isolated from Ostreococcus sp. is [7-methoxycarbonyl-8-vinyl]protochlorophyllide a.

The controversial molecular identification of the so-called chlorophyll cCS-170 has been settled. Despite its relevance as a potential biomarker in the study of eukaryotic picophytoplankton, the structure of this chlorophyll remained so far uncertain. A full characterization by NMR, UV-vis, and ESI-MS is reported, revealing this chlorophyll as [7-methoxycarbonyl-8-vinyl]-protochlorophyllide a.

slr1923 of Synechocystis sp. PCC6803 is essential for conversion of 3,8-divinyl(proto)chlorophyll(ide) to 3-monovinyl(proto)chlorophyll(ide).

The deduced amino acid sequence of an slr1923 gene of Synechocystis sp. PCC6803 is homologous to archaean F(420)H(2) dehydrogenase, which acts as a soluble subcomplex of reduced nicotinamide adenine dinucleotide dehydrogenase complex I. In this study, the gene was inactivated and characteristics of the mutant were analyzed. The mutant grew slower than the wild type under 100 microE m(-2) s(-1) but did not grow under high light intensity (300 microE m(-2) s(-1)). The cellular content of chlorophyll was lower in the mutant, and the absorption spectrum showed a shift in the absorption peak of the Soret band to a longer wavelength by about 10 nm compared with the wild type. It was found, by high-performance liquid chromatography analysis, that the retention time of chlorophyll of the mutant is shorter than that of the wild type and that the peak wavelength of the Soret band was also shifted to a longer wavelength by 11 nm. Proton nuclear magnetic resonance analysis of the chlorophyll of the mutant revealed that the ethyl group of position 8 of ring B is replaced with a vinyl group. The spectrum indicates that the chlorophyll of the mutant is not a normal (3-vinyl)chlorophyll a but a 3,8-divinylchlorophyll a. These results strongly suggest that the Slr1923 protein is essential for the conversion from divinylchlorophyll(ide) to normal chlorophyll(ide). We thus designate this gene cvrA (a gene indispensable for cyanobacterial vinyl reductase).

Spectroscopic and kinetic characterization of the light-dependent enzyme protochlorophyllide oxidoreductase (POR) using monovinyl and divinyl substrates.

The enzyme POR [Pchlide (protochlorophyllide) oxidoreductase] catalyses the reduction of Pchlide to chlorophyllide, which is a key step in the chlorophyll biosynthesis pathway. This light-dependent reaction has previously been studied in great detail but recent reports suggest that a mixture of MV (monovinyl) and DV (divinyl) Pchlides may have influenced some of these properties of the reaction. Low-temperature absorbance and fluorescence spectroscopy have revealed several spectral differences between MV and DV Pchlides, which were purified from a Rhodobacter capsulatus strain that was shown to contain a mixture of the two pigments. A thorough steady-state kinetic characterization using both Pchlide forms demonstrates that neither pigment appears to affect the kinetic properties of the enzyme. The reaction has also been monitored following illumination at low temperatures and was shown to consist of an initial photochemical step followed by four 'dark' steps for both pigments. However, minor differences were observed in the spectral properties of some of the intermediates, although the temperature dependency of each step was nearly identical for the two pigments. This work provides the first detailed kinetic and spectroscopic study of this unique enzyme using biologically important MV and DV substrate analogues. It also has significant implications for the DV reductase enzyme, which is responsible for converting DV pigments into their MV counterparts, and its position in the sequence of reactions that comprise the chlorophyll biosynthesis pathway.

Characterization of the Arabidopsis thaliana mutant pcb2 which accumulates divinyl chlorophylls.

We characterized the pcb2 (pale-green and chlorophyll b reduced 2) mutant. We found through electron microscopic observation that chloroplasts of pcb2 mesophyll cells lacked distinctive grana stacks. High-performance liquid chromatography (HPLC) analysis showed that the pcb2 mutant accumulated divinyl chlorophylls, and the relative amount of divinyl chlorophyll b was remarkably less than that of divinyl chlorophyll a. The responsible gene was mapped in an area of 190 kb length at the upper arm of the 5th chromosome, and comparison of DNA sequences revealed a single nucleotide substitution causing a nonsense mutation in At5g18660. Complementation analysis confirmed that the wild-type of this gene suppressed the phenotypes of the mutation. Antisense transformants of the gene also accumulated divinyl chlorophylls. The genes homologous to At5g18660 are conserved in a broad range of species in the plant kingdom, and have similarity to reductases. Our results suggest that the PCB2 product is divinyl protochlorophyllide 8-vinyl reductase.

Identification of a vinyl reductase gene for chlorophyll synthesis in Arabidopsis thaliana and implications for the evolution of Prochlorococcus species.

Chlorophyll metabolism has been extensively studied with various organisms, and almost all of the chlorophyll biosynthetic genes have been identified in higher plants. However, only the gene for 3,8-divinyl protochlorophyllide a 8-vinyl reductase (DVR), which is indispensable for monovinyl chlorophyll synthesis, has not been identified yet. In this study, we isolated an Arabidopsis thaliana mutant that accumulated divinyl chlorophyll instead of monovinyl chlorophyll by ethyl methanesulfonate mutagenesis. Map-based cloning of this mutant resulted in the identification of a gene (AT5G18660) that shows sequence similarity with isoflavone reductase genes. The mutant phenotype was complemented by the transformation with the wild-type gene. A recombinant protein encoded by AT5G18660 was expressed in Escherichia coli and found to catalyze the conversion of divinyl chlorophyllide to monovinyl chlorophyllide, thereby demonstrating that the gene encodes a functional DVR. DVR is encoded by a single copy gene in the A. thaliana genome. With the identification of DVR, finally all genes required for chlorophyll biosynthesis have been identified in higher plants. Analysis of the complete genome of A. thaliana showed that it has 15 enzymes encoded by 27 genes for chlorophyll biosynthesis from glutamyl-tRNA(glu) to chlorophyll b. Furthermore, identification of the DVR gene helped understanding the evolution of Prochlorococcus marinus, a marine cyanobacterium that is dominant in the open ocean and is uncommon in using divinyl chlorophylls. A DVR homolog was not found in the genome of P. marinus but found in the Synechococcus sp WH8102 genome, which is consistent with the distribution of divinyl chlorophyll in marine cyanobacteria of the genera Prochlorococcus and Synechococcus.

Interacting regulatory circuits involved in orderly control of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1.

FnrL, the homolog of the global anaerobic regulator Fnr, is required for the induction of the photosynthetic apparatus in Rhodobacter sphaeroides 2.4.1. Thus, the precise role of FnrL in photosynthesis (PS) gene expression and its interaction(s) with other regulators of PS gene expression are of considerable importance to our understanding of the regulatory circuitry governing spectral complex formation. Using a CcoP and FnrL double mutant strain, we obtained results which suggested that FnrL is not involved in the transduction of the inhibitory signal, by which PS gene expression is "silenced," emanating from the cbb(3) oxidase encoded by the ccoNOQP operon under aerobic conditions. The dominant effect of the ccoP mutation in the FnrL mutant strain with respect to spectral complex formation under aerobic conditions and restoration of a PS-positive phenotype suggested that inactivation of the cbb(3) oxidase to some extent bypasses the requirement for FnrL in the formation of spectral complexes. Additional analyses revealed that anaerobic induction of the bchE, hemN, and hemZ genes, which are involved in the tetrapyrrole biosynthetic pathways, requires FnrL. Thus, FnrL appears to be involved at multiple loci involved in the regulation of PS gene expression. Additionally, bchE was also shown to be regulated by the PrrBA two-component system, in conjunction with hemN and hemZ. These and other results to be discussed permit us to more accurately describe the role of FnrL as well as the interactions between the FnrL, PrrBA, and other regulatory circuits in the regulation of PS gene expression.

Pigment-free NADPH:protochlorophyllide oxidoreductase from Avena sativa L. Purification and substrate specificity.

The enzyme NADPH:protochlorophyllide oxidoreductase (POR) is the key enzyme for light-dependent chlorophyll biosynthesis. It accumulates in dark-grown plants as the ternary enzyme-substrate complex POR-protochlorophyllide a-NADPH. Here, we describe a simple procedure for purification of pigment-free POR from etioplasts of Avena sativa seedlings. The procedure implies differential solubilization with n-octyl-beta-D-glucoside and one chromatographic step with DEAE-cellulose. We show, using pigment and protein analysis, that etioplasts contain a one-to-one complex of POR and protochlorophyllide a. The preparation of 13 analogues of protochlorophyllide a is described. The analogues differ in the side chains of the macrocycle and in part contain zinc instead of the central magnesium. Six analogues with different side chains at rings A or B are active substrates, seven analogues with different side chains at rings D or E are not accepted as substrates by POR. The kinetics of the light-dependent reaction reveals three groups of substrate analogues with a fast, medium and slow reaction. To evaluate the kinetic data, the molar extinction coefficients in the reaction buffer had to be determined. At concentrations above 2 mole substrate/mole enzyme, inhibition was found for protochlorophyllide a and for the analogues.

Altered monovinyl and divinyl protochlorophyllide pools in bchJ mutants of Rhodobacter capsulatus. Possible monovinyl substrate discrimination of light-independent protochlorophyllide reductase.

In land plants in particular, it has been well established that chlorophyll intermediates, Mg-protoporphyrin, Mg-protoporphyrin monomethylester, protochlorophyllide, and chlorophyllide occur as monovinyl and divinyl forms. The pool of monovinyl and divinyl intermediates differ according to species, age of tissue, and light regime. In this study, we investigated the monovinyl and divinyl characteristics of protochlorophyllide and chlorophyllide in the purple non-sulfur photosynthetic eubacterium Rhodobacter capsulatus. Our results indicate that mutations in genes known to completely block the reduction of protochlorophyllide to chlorophyllide (such as bchN, bchB, and bchL mutants), accumulate a pool of monovinyl and divinyl forms of protochlorophyllide just as observed in plants. However, we also observed that directed insertion and deletion mutations in bchJ, a gene located in the photosynthesis gene cluster, affected the ratio of monovinyl and divinyl protochlorophyllide. Specifically, bchJ-disrupted strains accumulate reduced levels of bacteriochlorophyll concomitant with the accumulation of divinyl protochlorophyllide. Mutants of bchJ in combination with a second mutation in bchL still produce a mixed pool of monovinyl and divinyl protochlorophyllide; however, the ratio of monovinyl to divinyl protochlorophyllide is skewed in favor of divinyl protochlorophyllide. These results thus identify bchJ as the first sequenced gene that affects the divinyl to monovinyl ratio of photopigment intermediates in any photosynthetic organism. In addition, the results of our study also suggest that light-independent protochlorophyllide reductase is discriminatory for a monovinyl substrate.

Phototransformation of monovinyl and divinyl protochlorophyllide by NADPH:protochlorophyllide oxidoreductase of barley expressed in Escherichia coli.

The chlorophyll precursors monovinyl protochlorophyllide (MV-PChlide) and divinyl protochlorophyllide (DV-PChlide) were extracted from mutant C-2A' of the unicellular green alga Scenedesmus obliquus which accumulates both protochlorophyllide derivatives in the dark. The two pigments were characterized by absorption and fluorescence spectroscopy and by plasma desorption mass spectrometry. The molecular masses of MV-PChlide and DV-PChlide were determined as 612 and 610 atomic mass units (amu) respectively. Both MV-PChlide and DV-PChlide were accepted as substrates and photoconverted to chlorophyllides in vitro by NADPH:protochlorophyllide oxidoreductase of barley expressed in Escherichia coli.

Chloroplast biogenesis: [4-vinyl] chlorophyllide a reductase is a divinyl chlorophyllide a-specific, NADPH-dependent enzyme.

Some properties of [4-vinyl] chlorophyllide a reductase are described. This enzyme converts divinyl chlorophyllide a to monovinyl chlorophyllide a. The latter is the immediate precursor of monovinyl chlorophyll a, the main chlorophyll in green plants. [4-Vinyl] chlorophyllide a reductase plays an important role in daylight during the conversion of divinyl protochlorophyllide a to monovinyl chlorophyll a. [4-Vinyl] chlorophyllide a reductase was detected in isolated plastid membranes. Its activity is strictly dependent on the availability of NADPH. Other reductants such as NADH and GSH were ineffective. The enzyme appears to be specific for divinyl chlorophyllide a, and it does not reduce divinyl protochlorophyllide a to monovinyl protochlorophyllide a. The conversion of divinyl protochlorophyllide a to monovinyl protochlorophyllide a has been demonstrated in barley and cucumber etiochloroplasts and appears to be catalyzed by a [4-vinyl] protochlorophyllide a reductase [Tripathy, B.C., & Rebeiz, C.A. (1988) Plant Physiol. 87, 89-94]. On the basis of reductant requirements and substrate specificity, it is possible that two different 4-vinyl reductases may be involved in the reduction of divinyl protochlorophyllide a and divinyl chlorophyllide a to their respective 4-ethyl analogues.

Chloroplast biogenesis. Detection of monovinyl protochlorophyll(ide) b in plants.

The occurrence of protochlorophyllide b and protochlorophyllide b phytyl ester in green plants is described. The chemical structure of protochlorophyllide b phytyl ester was established by proton nuclear magnetic resonance, fast atom bombardment mass spectroscopic analysis, and chemical derivatization coupled to electronic spectroscopic analysis. The macrocycles of protochlorophyll(ide) b are identical to those of conventional protochlorophyll(ide) except for the presence of a formyl group instead of a methyl group at position 3 of the macrocycles. They differ from chlorophyll(ide) b by the presence of an oxidized double bond at positions 7 and 8 of the macrocycles. The trivial name protochlorophyll(ide) b is proposed to differentiate these two tetrapyrroles from conventional protochlorophyll(ide), which in turn will be referred to as protochlorophyll(ide) a. Protochlorophyll(ide) b appears to be widely distributed in green plants. Its molar extinction coefficients in 80% acetone and diethyl ether are reported. The impact of this discovery on the heterogeneity of the chlorophyll a and b biosynthetic pathways is discussed.

Synthesis of divinyl protochlorophyllide. Enzymological properties of the Mg-protoporphyrin IX monomethyl ester oxidative cyclase system.

The resolution and reconstitution of the Mg-protoporphyrin IX monomethyl ester oxidative cyclase system into a supernatant and a pellet fraction was accomplished by a procedure involving salt treatment followed by osmotic shock. Recombination of pellet and supernatant fractions was required for cyclase activity. This restoration effect could be demonstrated using either Mg-protoporphyrin IX or Mg-protoporphyrin IX monomethyl ester as the cyclase substrate in the presence or absence of S-adenosylmethionine. Pretreatment of the pellet fraction with either 8-hydroxyquinoline or desferal mesylate inhibited cyclase activity, indicating that there is a heavy-metal-ion requirement in this fraction. The cyclase supernatant protein(s) was not internalized by Sephadex G-50 and did not bind to Blue Sepharose, suggesting that it has a molecular mass of over 30 kDa and that it does not bind the cofactor NADPH. The cyclase supernatant protein did bind to MgProtoMe2-bound Sepharose and could be eluted by raising the pH to 9.7 in the presence of 4 mM-n-octyl glucoside. The pH optimum of the cyclase was 9.0. About a 40-fold purification of the cyclase supernatant protein was achieved by a combination of (NH4)2SO4 fractionation and phenyl-Sepharose chromatography.

Incorporation of atmospheric oxygen into the carbonyl functionality of the protochlorophyllide isocyclic ring.

Detached cucumber (Cucumis sativus L. var. Beit Alpha) cotyledons incubated in darkness with 5-aminolaevulinic acid and either 16O2 air (control) or 18O2 in N2 accumulated protochlorophyllide. This was converted into methyl phaeoporphyrin alpha 5 and analysed by mass spectrometry. The molecular ion of the methyl phaeoporphyrin alpha 5 derived from the 18O2 incubation was 2 mass units greater than that of the control, establishing that the oxo oxygen atom of the isocyclic ring is derived from atmospheric oxygen.

Selective inhibition of chlorophyll biosynthesis by nicotinamide.

Rhodobacter sphaeroides grown in the presence of nicotinamide excreted bacteriochlorophyll precursors, 2,4-divinyl protochlorophyllide (DV-Pchlide) and a small amount of 2-monovinyl protochlorophyllide (MV-Pchlide). Accumulation of these pigments indicates that nicotinamide inhibited the bacteriochlorophyll biosynthetic pathway site-specifically between DV-Pchlide and MV-Pchlide. This phenomenon is also observed in an aerobic photosynthetic bacterium, Erythrobacter sp. OCh 114. Among 12 nicotinamide derivatives and isomers tested, only nicotinamide was effective, indicating that in addition to the completeness of the pyridine ring skeleton at positions 1 to 3, the carboxylic acid amide group is essential for this inhibition. The technique described in this report permits the simple preparation of large quantities of DV-Pchlide.

The magnesium-protoporphyrin IX (oxidative) cyclase system. Studies on the mechanism and specificity of the reaction sequence.

Mg-protoporphyrin IX monomethyl ester cyclase activity was assayed in isolated developing cucumber (Cucumis sativus L. var. Beit Alpha) chloroplasts [Chereskin, Wong & Castelfranco (1982) Plant Physiol. 70, 987-993]. The presence of both 6- and 7-methyl esterase activities was detected, which permitted the use of diester porphyrins in a substrate-specificity study. It was found that: (1) the 6-methyl acrylate derivative of Mg-protoporphyrin monomethyl ester was inactive as a substrate for cyclization; (2) only one of the two enantiomers of 6-beta-hydroxy-Mg-protoporphyrin dimethyl ester had detectable activity as a substrate for the cyclase; (3) the 2-vinyl-4-ethyl-6-beta-oxopropionate derivatives of Mg-protoporphyrin mono- or di-methyl ester were approx. 4 times more active as substrates for cyclization than the corresponding divinyl forms; (4) at the level of Mg-protoporphyrin there was no difference in cyclase activity between the 4-vinyl and 4-ethyl substrates; (5) reduction of the side chain of Mg-protoporphyrin in the 2-position from a vinyl to an ethyl resulted in a partial loss of cyclase activity. This work suggests that the original scheme for cyclization proposed by Granick [(1950) Harvey Lect. 44, 220-245] should now be modified by the omission of the 6-methyl acrylate derivative of Mg-protoporphyrin monomethyl ester and the introduction of stereo-specificity at the level of the hydroxylated intermediate.

Chloroplast biogenesis. Demonstration of the monovinyl and divinyl monocarboxylic routes of chlorophyll biosynthesis in higher plants.

It is shown that barley (Hordeum vulgare), a dark monovinyl/light divinyl plant species, and cucumber (Cucumis sativus L.) a dark divinyl/light divinyl plant species synthesize monovinyl and divinyl protochlorophyllide in darkness from monovinyl and divinyl protoporphyrin IX via two distinct monovinyl and divinyl monocarboxylic chlorophyll biosynthetic routes. Evidence for the operation of monovinyl monocarboxylic biosynthetic routes consisted (a) in demonstrating the conversion of delta-aminolevulinic acid to monovinyl protoporphyrin and to monovinyl Mg-protoporphyrins, and (b) in demonstrating the conversion of these tetrapyrroles to monovinyl protochlorophyllide by both isolated barley and cucumber etiochloroplasts. Likewise, evidence for the operation of divinyl monocarboxylic chlorophyll biosynthetic routes consisted (a) in demonstrating the biosynthesis of divinyl protoporphyrin and divinyl Mg-protoporphyrins from delta-aminolevulinic acid, and (b) in demonstrating the conversion of the latter tetrapyrroles to divinyl protochlorophyllide. Finally, it was shown that the divinyl tetrapyrrole substrates were metabolized differently by barley and cucumber. For example, divinyl protoporphyrin, divinyl Mg-protoporphyrin, and divinyl Mg-protoporphyrin monoester were converted predominantly to monovinyl protochlorophyllide and to smaller amounts of divinyl protochlorophyllide by barley etiochloroplasts. In contrast, cucumber etiochloroplasts converted the above substrates predominantly to divinyl protochlorophyllide, although smaller amounts of monovinyl protochlorophyllide were also formed. Furthermore, it was shown that monovinyl protochlorophyllide was not formed from divinyl protochlorophyllide either in barley or in cucumber etiochloroplasts. These metabolic differences are explained by the presence of strong biosynthetic interconnections between the divinyl and monovinyl monocarboxylic routes, prior to divinyl protochlorophyllide formation, in barley but not in cucumber.

Chloroplast biogenesis: quantitative determination of monovinyl and divinyl Mg-protoporphyrins and protochlorophyll(ides) by spectrofluorometry.

General equations which permit the determination of the amounts of any two closely related fluorescent compounds which can be distinguished by 77 degrees K but not by 293 degrees K spectrofluorometry have been described. This was achieved in the presence or absence of a third interfering compound, without prior separation of the fluorescent species. The adaptation of the generalized equations to the determination of the amounts of monovinyl (MV) and divinyl (DV) Mg-protoporphyrins or of MV and DV protochlorophyll(ides) in the presence or absence of Mg-Protos [Mg-protoporphyrin IX (Mg-Proto), Mg-Proto monoester, Mg-Proto diester or a mixture of those three tetrapyrroles] interference, was then demonstrated over a wide range of MV/DV tetrapyrrole proportions. These equations are likely to be very useful for the study of the intermediary metabolism of the monovinyl and divinyl chlorophyll biosynthetic routes in plants.

Mg-2,4-divinyl pheoporphyrin a5: the product of a reaction catalyzed in vitro by developing chloroplasts.

The major product of an aerobic reaction mixture containing developing chloroplasts, Mg-protoporphyrin IX, S-adenosylmethionine, and other cofactors was isolated and purified. Structural studies using nuclear magnetic resonance confirmed earlier reports, based on fluorescence and absorption spectra, that this compound is Mg-2,4-divinyl pheoporphyrin a5. The molecular weight determined by secondary-ion mass spectroscopy further confirmed the assigned structure. Absorption and fluorescence spectra indicate that this compound is identical to that reported previously by various workers in less-purified biological extracts. The nuclear magnetic resonance spectrum of the Mg-free base also supports the assigned structure.