Chloroplast biogenesis: quantitative determination of monovinyl and divinyl chlorophyll(ide) a and b by spectrofluorometry

Simultaneous equations for the determination of monovinyl (MV) and divinyl (DV) Chl a and b by spectrofluorometry in unsegregated mixtures of these tetrapyrroles were derived. The same equations can also be used for the quantitative determination of MV and DV chlorophyllide (Chlide) a and b in unpurified mixtures, after extraction of the Chls in hexane. The equations used differences in the Soret excitation maxima of these tetrapyrroles in ether at 77 degrees K, in order to correct the Soret excitation overlap between MV and DV Chl(ide) a and between MV and DV Chl(ide) b.     

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 92: In situ screening for divinyl chlorophyll(ide) a reductase mutants by spectrofluorometry

Chlorophyll biosynthetic heterogeneity is rooted mainly in parallel divinyl (DV) and monovinyl (MV) biosynthetic routes interconnected by 4-vinyl reductases (4VRs) that convert DV tetrapyrroles to MV tetrapyrroles by conversion of the vinyl group at position 4 of the macrocycle to ethyl. What is not clear at this stage is whether the various 4VR activities are catalyzed by one enzyme of broad specificity or by a family of enzymes encoded by one gene or multiple genes with each enzyme having narrow specificity. Additional research is needed to identify the various regulatory components of 4-vinyl reduction. In this undertaking, Arabidopsis mutants that accumulate DV chlorophyllide a and/or DV chlorophyll [Chl(ide)] a are likely to provide an appropriate resource. Because the Arabidopsis genome has been completely sequenced, the best strategy for identifying 4VR and/or putative regulatory 4VR genes is to screen Arabidopsis Chl mutants for DV Chl(ide) a accumulation. In wild-type Arabidopsis, a DV plant species, only MV chlorophyllide (Chlide) a is detectable. However in Chl mutants lacking 4VR activity, DV Chl(ide) a may accumulate in addition to MV Chl(ide) a. In the current work, an in situ assay of DV Chl(ide) a accumulation, suitable for screening a large number of mutants lacking 4-vinyl Chlide a reductase activity with minimal experimental handling, is described. The assay involves homogenization of the tissues in Tris-HCl:glycerol buffer and the recording of Soret excitation spectra at 77K. DV Chlide a formation is detected by a Soret excitation shoulder at 459 nm over a wide range of DV Chlide a/MV Chl a ratios. The DV Chlide a shoulder became undetectable at DV Chlide a/MV Chl a ratios less than 0.049, that is, at a DV Chlide a content of less than 5%.     

Monovinyl and divinyl protochlorophyllide pools in etiolated tissues of higher plants

The composition of chlorophyll-precursor pigments, particularly the contents of monovinyl (MV) and divinyl (DV) protochlorophyllides (Pchlides), in etiolated tissues of higher plants were determined by polyethylene-column HPLC (Y. Shioi, S. I. Beale [1987] Anal Biochem 162: 493-499), which enables the complete separation of these pigments. DV-Pchlide was ubiquitous in etiolated tissue of higher plants. From the analyses of 24 plant species belonging to 17 different families, it was shown that the concentration of DV-Pchlide was strongly dependent on the plant species and the age of the plants. The ratio of DV-Pchlide to MV-Pchlide in high DV-Pchlide plants such as cucumber and leaf mustard decreased sharply with increasing age. Levels of DV-Pchlide in Gramineae plants were considerably lower at all ages compared with those of other plants. Etiolated tissues of higher plants such as barley and corn were, therefore, good sources of MV-Pchlide. Absorption spectra of the purified MV- and DV-Pchlides in ether are presented and compared.     

Chloroplast biogenesis 76. Regulation of 4-vinyl reduction during conversion of divinyl Mg-protoporphyrin IX to monovinyl protochlorophyllide a is controlled by plastid membrane and stromal factors

Most of the chlorophyll (Chl) a of green plants is formed via two biosynthetic routes, namely the carboxylic divinyl and monovinyl chlorophyll biosynthetic routes. These two routes are linked by (4-vinyl) reductases that convert divinyl tetrapyrroles to monovinyl tetrapyrroles by reduction of the vinyl group at position four of the macrocycle to ethyl. The activities of these two routes are very sensitive to cell disruption. For example in barley leaves, cell disruption, a mandatory step during plastid isolation, results in partial inactivation of the carboxylic divinyl route. Investigations with subplastidic fractions revealed that the carboxylic divinyl and monovinyl biosynthetic routes were regulated by a delicate interaction that involved plastid membranes, stroma, and reduced pyridine nucleotides. While the monovinyl biosynthetic route was very active in isolated plastid membranes, activation of the divinyl biosynthetic route required the joint presence of plastid membranes and stroma. Contrary to expectation, activity of the carboxylic divinyl biosynthetic route was greatly enhanced by addition of NADPH to the lysing buffer used during plastid membranes and stroma preparation. NADPH in cooperation with the plastid stroma may play an important regulatory role during the biosynthesis of divinyl and monovinyl protochlorophyllide a.     

Chloroplast biogenesis 76. Regulation of 4-vinyl reduction during conversion of divinyl Mg-protoporphyrin IX to monovinyl protochlorophyllide a is controlled by plastid membrane and stromal factors

Most of the chlorophyll (Chl) a of green plants is formed via two biosynthetic routes, namely the carboxylic divinyl and monovinyl chlorophyll biosynthetic routes. These two routes are linked by (4-vinyl) reductases that convert divinyl tetrapyrroles to monovinyl tetrapyrroles by reduction of the vinyl group at position four of the macrocycle to ethyl. The activities of these two routes are very sensitive to cell disruption. For example in barley leaves, cell disruption, a mandatory step during plastid isolation, results in partial inactivation of the carboxylic divinyl route. Investigations with subplastidic fractions revealed that the carboxylic divinyl and monovinyl biosynthetic routes were regulated by a delicate interaction that involved plastid membranes, stroma, and reduced pyridine nucleotides. While the monovinyl biosynthetic route was very active in isolated plastid membranes, activation of the divinyl biosynthetic route required the joint presence of plastid membranes and stroma. Contrary to expectation, activity of the carboxylic divinyl biosynthetic route was greatly enhanced by addition of NADPH to the lysing buffer used during plastid membranes and stroma preparation. NADPH in cooperation with the plastid stroma may play an important regulatory role during the biosynthesis of divinyl and monovinyl protochlorophyllide a. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chloroplast Biogenesis 60 : Conversion of Divinyl Protochlorophyllide to Monovinyl Protochlorophyllide in Green(ing) Barley, a Dark Monovinyl/Light Divinyl Plant Species

In higher plants, most of the chlorophyll a is formed via the divinyl and monovinyl chlorophyll monocarboxylic biosynthetic routes. These two routes are strongly interconnected prior to protochlorophyllide formation in barley (Hordeum vulgare L. cv Morex), a dark monovinyl-light divinyl plant species, but not in cucumber (Cucumis sativus L. cv Beit Alpha MR), a dark divinyl-light divinyl plant species (BC Tripathy, CA Rebeiz, 1986 J Biol Chem 261: 13556-13564). It is shown that in dark monovinyl-light divinyl plant species such as barley, the divinyl and monovinyl monocarboxylic routes become interconnected at the level of protochlorophyllide during transition from the divinyl to the monovinyl protochlorophyllide biosynthetic mode. In cucumber, a dark divinyl-light divinyl plant species, in which the monovinyl monocarboxylic biosynthetic route becomes preponderant only after an abnormally long sojourn in darkness, the conversion of divinyl to monovinyl protochlorophyllide does not take place on the barley time-scale of incubation.     

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.     

Molecular environments of divinyl chlorophylls in Prochlorococcus and Synechocystis: differences in fluorescence properties with chlorophyll replacement

A marine cyanobacterium, Prochlorococcus, is a unique oxygenic photosynthetic organism, which accumulates divinyl chlorophylls instead of the monovinyl chlorophylls. To investigate the molecular environment of pigments after pigment replacement but before optimization of the protein moiety in photosynthetic organisms, we compared the fluorescence properties of the divinyl Chl a-containing cyanobacteria, Prochlorococcus marinus (CCMP 1986, CCMP 2773 and CCMP 1375), by a Synechocystis sp. PCC 6803 (Synechocystis) mutant in which monovinyl Chl a was replaced with divinyl Chl a. P. marinus showed a single fluorescence band for photosystem (PS) II at 687nm at 77K; this was accompanied with change in pigment, because the Synechocystis mutant showed the identical shift. No fluorescence bands corresponding to the PS II 696-nm component and PS I longer-wavelength component were detected in P. marinus, although the presence of the former was suggested using time-resolved fluorescence spectra. Delayed fluorescence (DF) was detected at approximately 688nm with a lifetime of approximately 29ns. In striking contrast, the Synechocystis mutant showed three fluorescence bands at 687, 696, and 727nm, but suppressed DF. These differences in fluorescence behaviors might not only reflect differences in the molecular structure of pigments but also differences in molecular environments of pigments, including pigment-pigment and/or pigment-protein interactions, in the antenna and electron transfer systems.     

Chloroplast Biogenesis 49 : Differences among Angiosperms in the Biosynthesis and Accumulation of Monovinyl and Divinyl Protochlorophyllide during Photoperiodic Greening

Various angiosperms differed in their monovinyl and divinyl protochlorophyllide biosynthetic capabilities during the dark and light phases of photoperiodic growth. Some plant species such as Cucumis sativus L., Brassica juncea (L.) Coss., Brassica kaber (DC.) Wheeler, and Portulaca oleracea L. accumulated mainly divinyl protochlorophyllide at night. Monocotyledonous species such as Avena sativa L., Hordeum vulgare L., Triticum secale L., Zea mays L., and some dicotyledonous species such as Phaseolus vulgaris L., Glycine max (L.) Merr., Chenopodium album L., and Lycopersicon esculentum L. accumulated mainly monovinyl protochlorophyllide at night.

Under low light intensities meant to simulate the first 60 to 80 minutes following daybreak divinyl protochlorophyllide appeared to contribute much more to chlorophyll formation than monovinyl protochlorophyllide in species such as Cucumis sativus L. Under the same light conditions, species which accumulated mainly monovinyl protochlorophyllide at night appeared to form chlorophyll preferably via monovinyl protochlorophyllide.

These results were interpreted in terms of: (a) a differential contribution of monovinyl and divinyl protochlorophyllide to chlorophyll formation at daybreak in various plant species; and (b) a differential regulation of the monovinyl and divinyl protochlorophyllide biosynthetic routes by light and darkness.

     

Alterations in photosynthetic pigments and amino acid composition of D1 protein change energy distribution in photosystem II

The marine cyanobacterium Prochlorococcus marinus accumulates divinyl chlorophylls instead of monovinyl chlorophylls to harvest light energy. As well as this difference in its chromophore composition, some amino acid residues in its photosystem II D1 protein were different from the conserved amino acid residues in other photosynthetic organisms. We examined PSII complexes isolated from mutants of Synechocystis sp. PCC 6803, in which chromophore and D1 protein were altered (Hisashi Ito and Ayumi Tanaka, 2011) to clarify the effects of chromophores/D1 protein composition on the excitation energy distribution. We prepared the mutants accumulating divinyl chlorophyll (DV mutant). The amino acid residues of V205 and G282 in the D1 protein were substituted with M205 and C282 in the DV mutant to mimic Prochlorococcus D1 protein (DV-V205M/G282C mutant). Isolated PSII complexes were analyzed by time-resolved fluorescence spectroscopy. Energy transfer in CP47 was interrupted in PSII containing divinyl chlorophylls. The V205M/G282C mutation did not recover the energy transfer pathway in CP47, instead, the mutation allowed the excitation energy transfer from CP43 to CP47, which neighbors in the PSII dimer. Mutual orientation of the subcomplexes of PSII might be affected by the substitution. The changes of the energy transfer pathways would reduce energy transfer from antennae to the PSII reaction center, and allow Prochlorococcus to acquire light tolerance.     

Separation of monovinyl and divinyl protochlorophyllides and chlorophyllides from etiolated and phototransformed cucumber cotyledons

A method was developed to separate the monovinyl and divinyl forms of protochlorophyllide and chlorophyllide by high pressure liquid chromatography using a silicic acid column coated with dodecyl residues and a moving phase containing the lipophilic cation, tetrabutyl ammonium. The solvent was 70% methyl alcohol containing varying amounts of methyl ethyl ketone. The separation was carried out at 0°C. This method was used to test and confirm a previous report that, in cucumber cotyledons, divinyl protochlorophyllide is phototransformed to give divinyl chlorophyllide, which is biologically unstable and disappears rapidly in the dark.     

Chloroplast biogenesis 51 : modulation of monovinyl and divinyl protochlorophyllide biosynthesis by light and darkness in vitro

It is shown that the monovinyl and divinyl protochlorophyllide biosynthetic patterns of etiolated maize (Zea mays L.), and cucumber (Cucumis sativus L.) seedlings and of their isolated etiochloroplasts can be modulated by light and darkness as was shown for green photoperiodically grown plants (E. E. Carey, C. A. Rebeiz 1985 Plant Physiol. 79: 1-6). In etiolated corn and cucumber seedlings and isolated etiochloroplasts poised in the divinyl protochlorophyllide biosynthetic mode by a 2 hour light pretreatment, darkness induced predominantly the biosynthesis of monovinyl protochlorophyllide in maize and of divinyl protochlorophyllide in cucumber. When etiolated seedlings and their isolated etiochloroplasts were poised in the monovinyl protochlorophyllide biosynthetic mode by a prolonged dark-pretreatment, light induced mainly the biosynthesis of divinyl protochlorophyllide in both maize and cucumber.     

Role of aspartate 400, arginine 262, and arginine 401 in the catalytic mechanism of human coproporphyrinogen oxidase

Coproporphyrinogen oxidase (CPO) is the sixth enzyme in the heme biosynthetic pathway, catalyzing two sequential oxidative decarboxylations of propionate moieties on coproporphyrinogen-III forming protoporphyrinogen-IX through a monovinyl intermediate, harderoporphyrinogen. Site-directed mutagenesis studies were carried out on three invariant amino acids, aspartate 400, arginine 262, and arginine 401, to determine residue contribution to substrate binding and/or catalysis by human recombinant CPO. Kinetic analyses were performed on mutant enzymes incubated with three substrates, coproporphyrinogen-III, harderoporphyrinogen, or mesoporphyrinogen-VI, in order to determine catalytic ability to perform the first and/or second oxidative decarboxylation. When Asp400 was mutated to alanine no divinyl product was detected, but the production of a small amount of monovinyl product suggested the K(m) value for coproporphyrinogen-III did not change significantly compared to the wild-type enzyme. Upon mutation of Arg262 to alanine, CPO was again a poor catalyst for the production of a divinyl product, with a catalytic efficiency <0.01% compared to wild-type, including a 15-fold higher K(m) for coproporphyrinogen-III. The efficiency of divinyl product formation for mutant enzyme Arg401Ala was approximately 3% compared to wild-type CPO, with a threefold increase in the K(m) value for coproporphyrinogen-III. These data suggest Asp400, Arg262, and Arg401 are active site amino acids critical for substrate binding and/or catalysis. Possible roles for arginine 262 and 401 include coordination of carboxylate groups of coproporphyrinogen-III, while aspartate 400 may initiate deprotonation of substrate, resulting in an oxidative decarboxylation.     

Detection of protoporphyrin IX in envelope membranes of pea chloroplasts

Envelope membranes were prepared from mature pea chloroplasts. The tetrapyrrole contents of envelope membranes were analysed. The envelope membranes of pea chloroplasts contained substantial amounts of protoporphyrin IX and trace amounts of Mg-protoporphyrin IX and its monoester in addition to protochlorophyllide. The protoporphyrin IX content of envelope membranes was 89.25 pmol (mg protein)(-1). Its content in pea envelope membrane was higher than that of protochlorophyllide. The proportion of monovinyl and divinyl forms of protochlorophyllide present in pea chloroplast envelope membrane was 3:7. The significance of the presence of protoporphyrin IX in the envelope membrane is discussed in relation to plastidic Chl biosynthesis.     

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.     

Regulation of etioplast pigment-protein complexes, inner membrane architecture, and protochlorophyllide a chemical heterogeneity by light-dependent NADPH:protochlorophyllide oxidoreductases A and B

The etioplast of dark-grown angiosperms is characterized by the prolamellar body (PLB) inner membrane, the absence of chlorophyll, and the accumulation of divinyl and monovinyl derivatives of protochlorophyll(ide) a [Pchl(ide) a]. Either of two structurally related, but differentially expressed light-dependent NADPH:Pchlide oxidoreductases (PORs), PORA and PORB, can assemble the PLB and form dark-stable ternary complexes containing enzymatically photoactive Pchlide-F655. Here we have examined in detail whether these polypeptides play redundant roles in etioplast differentiation by manipulating the total POR content and the PORA-to-PORB ratio of etiolated Arabidopsis seedlings using antisense and overexpression approaches. POR content correlates closely with PLB formation, the amounts, spectroscopic properties, and photoreduction kinetics of photoactive Pchlide, the ratio of photoactive Pchlide-F655 to non-photoactive Pchl(ide)-F632, and the ratio of divinyl- to monovinyl-Pchl(ide). This last result defines POR as the first endogenous protein factor demonstrated to influence the chemical heterogeneity of Pchl(ide) in angiosperms. It is intriguing that excitation energy transfer between different spectroscopic forms of Pchl(ide) in etiolated cotyledons remains largely independent of POR content. We therefore propose that the PLB contains a minimal structural unit with defined pigment stoichiometries, within which a small amount of non-photoactive Pchl(ide) transfers excitation energy to a large excess of photoactive Pchlide-F655. In addition, our data suggests that POR may bind not only stoichiometric amounts of photoactive Pchlide, but also substoichiometric amounts of non-photoactive Pchl(ide). We conclude that the typical characteristics of etioplasts are closely related to total POR content, but not obviously to the specific presence of PORA or PORB.     

Characterization of protochlorophyllide and protochlorophyllide esters in roots of dark-grown plants

The protochlorophyll pools of roots of dark-grown wheat ( Triticum aestivum L. cv. Walde), maize ( Zea mays L. cv. Goldcrest) and wrinkledseeded pea ( Pisum sativum L. ssp. sativurh cv. Kelvedon Wonder) were investigated by high performance liquid chromatography (HPLC) and low temperature fluorescence spectroscopy. All roots contained protochlorophyllide and esterified protochlorophyllides (protochlorophylls) but with considerably larger relative amounts of the latter compared with etiolated leaves. The alcohol moieties of the 4 detected protochlorophylls were geranylgeraniol (GG), dihydrogeranylgeraniol (DHGG), tetrahydrogeranylgeraniol (THGG) and phytol. The relative amounts of the different protochlorophylls varied between the species. Protochlorophyllide and the 4 protochlorophylls all contained monovinyl forms. The divinyl forms could not be detected by our instruments. Wrinkledseeded pea contained in addition chlorophyll a , some unidentified chlorophylls and negligible amounts of chlorophyllide. Small amounts of carotenoids were found in roots of all investigated species. The carotenoids were the same as those found in green or etiolated leaves, but present in different relative amounts.     

Photoreduction of monovinyl- and divinyl-protochlorophyllide in vitro and in vivo in the light-dependent greening mutant C-2A' of Scenedesmus obliquus

Dark-grown cells of mutant C-2A' of Scenedesmus obliquus accumulate monovinyl-and mainly divinyl-protochlorophyllide (PChlide). Both PChlide-forms are equally well photoconverted in vitro by the NADPH-protochlorophyllide oxidoreductase of the light-dependent greening mutant C-2A'of Scenedesmus obliquus. The chlorophyllide (Chlide) resulting from this photoconversion in vivo has a predominantly monovinyl character. Only small traces of a transient Chlide-form with divinyl fluorescence could be detected.     

DNA helicase RepA: cooperative ATPase activity and binding of nucleotides

The steady-state kinetic parameters of the ATPase activity of the homohexameric DNA helicase RepA and the binding of the fluorescent analogue epsilonADP to RepA have been studied. ssDNA stimulates RepA ATPase activity optimally at acidic pH 5.3-6.0. The sigmoidal kinetic curves in both the absence and presence of ssDNA show strong positive cooperativity for ATP hydrolysis, with oligonucleotides longer than 10mer optimal for ssDNA-stimulated ATPase activity. Fluorescence titrations show that, at 25 degrees C and in the absence of DNA, the binding of epsilonADP to RepA is biphasic with three high (K(1) = 1.54 x 10(6) M(-1)) and three low (K(2) = 4.71 x 10(4) M(-)(1)) affinity binding sites differing by 30-40-fold in binding constants. In the absence of cofactors, RepA melts cooperatively at T(m) = 65.8 +/- 0.1 degrees C and is more stable in the presence of ATPgammaS, T(m) = 68.1 +/- 0.2 degrees C (DeltaDeltaG 0.95 kcal/mol), than in the presence of ADP, T(m) = 66. 5 +/- 0.1 degrees C (DeltaDeltaG 0.29 kcal/mol), indicating that the additional phosphate group in ATPgammaS has a significant influence on RepA structure. A model is proposed in which individual subunits of RepA sequentially and cooperatively perform a multistep ATP hydrolytic cycle.