Regulation of 5-Aminolevulinic Acid (ALA) Synthesis in Developing Chloroplasts : III. Evidence for Functional Heterogeneity of the ALA Pool

Gabaculine and 4-amino-5-hexynoic acid (AHA) up to 3.0 millimolar concentration strongly inhibited 5-aminolevulinic acid (ALA) synthesis in developing cucumber (Cucumis sativus L. var Beit Alpha) chloroplasts, while they hardly affected protochlorophyllide (Pchlide) synthesis. Exogenous protoheme up to 1.0 micromolar had a similar effect. Exogenous glutathione also exhibited a strong inhibitory effect on ALA synthesis in organello but hardly inhibited Pchlide synthesis. Pchlide synthesis in organello was highly sensitive to inhibition by levulinic acid, both in the presence and in the absence of gabaculine, indicating that the Pchlide was indeed formed from precursor(s) before the ALA dehydratase step. The synthesis of Pchlide in the presence of saturating concentrations of glutamate was stimulated by exogenous ALA, confirming that Pchlide synthesis was limited at the formation of ALA. The gabaculine inhibition of ALA accumulation occurred whether levulinic acid or 4,6-dioxohepatonic acid was used in the ALA assay system. ALA overproduction was also observed in the absence of added glutamate and was noticeable after 10-minute incubation. These observations suggest that although Pchlide synthesis in organello is limited by ALA formation, it does not utilize all the ALA that is made in the in organello assay system. Gabaculine, AHA, and probably also protoheme, inhibit preferentially the formation of that portion of ALA that is not destined for Pchlide. A model proposing a heterogenous ALA pool is described.     

Gabaculine Inhibition of Chlorophyll Biosynthesis and Nodulation in Phaseolus lunatus L

Gabaculine (3-amino-2,3-dihydrobenzoic acid) was an inhibitor of in vivo chlorophyll biosynthesis in lima bean (Phaseolus lunatus L. cv Henderson). When applied to roots of 9-day-old plants, 10 micromolar gabaculine was sufficient to terminate biosynthesis of new chlorophyll. The trifoliolate leaves which emerged after gabaculine treatment were yellow. Gabaculine-treated plants had slightly lower dry weights; yet, overall plant size showed very little change. Chlorophyll fluorescence induction kinetics and CO₂ exchange measurements were used to monitor both immediate and long-term effects of gabaculine on photosynthesis. A lowered rate of the decline from the maximum level of fluorescence was observed after 10 hours for nitrate-supplemented plants, and all treated plants showed a slightly increased level of original fluorescence after 6 days. No change was observed in the rate of photosynthesis by unifoliolate leaves. The trifoliolate leaves, though not able to photosynthesize, were able to continue respiration. This suggested that heme biosynthesis for mitochondrial cytochromes was not abolished. In untreated lima bean, root nodules were induced by Rhizobium sp. 127E15. Following gabaculine treatment, root nodules formed, but were largely ineffective in nitrogen fixation. Nodule dry weight, nitrogen fixation activity, and leghemoglobin content were decreased by gabaculine.     

Inhibition of phytochrome synthesis by gabaculine

Gabaculine (5-amino-1,3-cyclohexadienylcarboxylic acid), a transaminase inhibitor, also inhibits chlorophyll formation in plants, and the effect of this compound can be counteracted by 5-aminolevulinic acid (ALA) (Flint, personal communication, 1984). Since it is probable that ALA also serves as a precursor to phytochrome, the effects of gabaculine on phytochrome synthesis in developing etiolated seedlings were examined using in vivo spectrophotometry. Preemergence treatment with gabaculine was found to inhibit initial phytochrome synthesis in peas (Pisum sativum L.), corn (Zea mays L.), and oats (Avena sativa L.). In general, reduction in phytochrome correlated with reduction in chlorophyll. However, the extent of inhibition of phytochrome synthesis was not as great as that of chlorophyll synthesis, perhaps due to preexisting phytochrome in the seed. Foliar treatment of etiolated pea seedlings prior to light-induced destruction of phytochrome inhibited subsequent phytochrome resynthesis in the dark. These results suggest that both initial synthesis and resynthesis of phytochrome require de novo synthesis of chromophore as well as apoprotein.     

Inhibition of phytochrome synthesis by the transaminase inhibitor, 4-amino-5-fluoropentanoic Acid

Gardner and Gorton (1985 Plant Physiol 77: 540-543) demonstrated that the transaminase inhibitor gabaculine (5-amino-1,3-cyclohexadienyl-carboxylic acid) inhibits the initial synthesis and resynthesis of spectrophotometrically detectable phytochrome in vivo. Another mechanism-based transaminase inhibitor, 4-amino-5-fluoropentanoic acid (AFPA), is examined in this report for its effects on phytochrome synthesis in developing etiolated seedlings. Preemergence treatment with AFPA was found to inhibit initial phytochrome synthesis in peas (Pisum sativum L.), corn (Zea mays L.), and oats (Avena sativa L.). In general, reduction in phytochrome correlated with reduction in chlorophyll. However, the extent of inhibition of phytochrome synthesis was not as great as that of chlorophyll synthesis. These results confirm those with gabaculine, indicating that both initial synthesis and resynthesis of phytochrome require de novo synthesis of chromophore as well as apoprotein. AFPA was a more effective inhibitor of both chlorophyll and phytochrome synthesis than was gabaculine, suggesting that AFPA may be the preferred tool with which to probe the physiological consequences of the inhibition of phytochrome biosynthesis.     

Influence of gabaculine on growth,chlorophyll synthesis,and δ-aminolevulinic acid synthase activity in Euglena gracilis

Euglena gracilis is capable of forming the heme and chlorophyll precursor δ-aminolevulinic acid (ALA) by two routes: from glutamate via the five-carbon path in the chloroplasts, and by ALA synthase-mediated condensation of succinyl-CoA and glycine, probably in the mitochondrion. 5-Amino-1,3-cyclohexadienyl carboxylic acid (gabaculine), a powerful inhibitor of ALA formation via the five-carbon path, was administered to E. gracilis Klebs strain Z Pringsheim cells growing in the light or dark, and its effects on growth, chlorophyll accumulation and extractable ALA synthase activity were measured. Gabaculine had no effect in vitro on ALA synthase or ALA dehydratase, even at 100 μM. Administration of 100 μM gabaculine to wild-type cells growing in the light slowed growth, inhibited chlorophyll accumulation, and induced an increase in extractable ALA synthase activity. Chlorophyll accumulation in the light was abolished by prior administration of the compound to growing cells for 6 h in the dark, whereas chlorophyll accumulation in cells without gabaculine began immediately after transfer to light. Extractable ALA synthase activity from gabaculine-pretreated dark-grown cells was initially lower than the activity from untreated cells, but it did not undergo a further decline after transfer of the cells to the light, whereas the activity from untreated cells dropped to less than one eighth the dark level after 2 h in the light, and by 4 h had fallen to a level five times lower than that extractable from gabaculine-treated cells. These results suggest that suppression of ALA synthase activity by light in untreated cells is related to light-induced activation of the five-carbon ALA biosynthetic pathway in the plastids, and may result from a contribution by a product of the five-carbon pathway to non-plastid tetrapyrrole pools in the light.     

Evidence for a hetero-oligomeric structure of the chloroplast cytochrome b-559

A second nonhomologous polypeptide in the thylakoid membrane cytochrome b-559 has been suggested by the finding of a smaller reading frame just slightly downstream from that corresponding to the 9 kDa cytochrome polypeptide that is dominant on a Coomassie-stained gel. This reading frame encoded a 39-residue polypeptide that was similar in having a central hydrophobic domain of 25–26 residues and a single His residue at the same position in the hydrophobic domain. The smallest polypeptide seen on SDS gels of the cytochrome was isolated by high-performance liquid chromatography (HPLC). The NH₂-terminal sequence matched that of the downstream gene. The stoichiometry of the 2 gene products separated by HPLC was approx. 1:1, based on the molecular masses of 9.16 and 4.27 kDa calculated from the nucleotide sequence. It is concluded that the cytochrome contains both the 9.16 kDa (α) and 4.27 kDa (β) polypeptides. These data, the single His residue on each polypeptide, and the previous finding of a bis-histidine coordination, imply that the unit heme binding structure of the cytochrome is a heme cross-linked dimer. If the cytochrome contains a single heme, the dimer structure would be (αβ). If there are 2 hemes/cytochrome, the more likely structure would be (αβ)₂, a tetramer consisting of 2 heme cross-linked hetero-dimers.     

Purification and characterization of highly purified cytochrome b from Complex III of baker's yeast

A simple, high-yield purification procedure for cytochrome b from yeast Complex III has been developed. This procedure involves solubilization using chemical modification of the lysine residues with 3,4,5,5-tetrahydrophthalic anhydride followed by hydroxyapatite column chromatography. This purified cytochrome b has a heme content of 37.0 nmol cytochrome b/mg and a molecular weight on SDS gels of 25000–26000. Amino acid analysis indicates high hydrophobicity and is very comparable to the composition deduced from the gene sequence (Nobrega, F.G. and Tzagoloff, A. (1980) J. Biol. Chem. 255, 9828–9837). The latter data indicate a molecular weight of 42000 for the polypeptide; our heme analyses thus imply the presence of two hemes per polypeptide chain. Optical and MCD spectra are typical of a low-spin b-type cytochrome. MCD-potentiometric titration indicates a one-electron carrier with a single midpoint potential of ?44 mV at pH 7.4 and 25°C. The EPR spectrum of isolated cytochrome b has only one g_z signal at 3.70, indicating that the ‘strained’ heme structure (Carter, K., T'sai, A. and Palmer, G. (1981) FEBS Lett. 132, 243–246) is still maintained. No indication of antimycin binding was demonstrated either by the direct-fluorescence method or binding-precipitation method although stoichiometric binding to the parent Complex III was readily demonstrated.     

Comparative analysis of plastocyanin–cytochrome f complex formation in higher plants,green algae and cyanobacteria

Mechanisms of the complex formation between plastocyanin and cytochrome f in higher plants (Spinacia oleracea and Brassica rapa), green microalgae Chlamydomonas reinhardtii and two species of cyanobacteria (Phormidium laminosum and Nostoc sp.) were investigated using combined Brownian and molecular dynamics simulations and hierarchical cluster analysis. In higher plants and green algae, electrostatic interactions force plastocyanin molecule close to the heme of cytochrome f. In the subsequent rotation of plastocyanin molecule around the point of electrostatic contact in the vicinity of cytochrome f, copper (Cu) atom approaches cytochrome heme forming a stable configuration where cytochrome f molecule behaves as a rather rigid body without conformational changes. In Nostoc plastocyanin molecule approaches cytochrome f in a different orientation (head‐on) where the stabilization of the plastocyanin–cytochrome f complex is accompanied by the conformational changes of the G188E189D190 loop that stabilizes the whole complex. In cyanobacterium P. laminosum, electrostatic preorientation of the approaching molecules was not detected, thus indicating that random motions rather than long‐range electrostatic interactions are responsible for the proper mutual orientation. We demonstrated that despite the structural similarity of the investigated electron transport proteins in different photosynthetic organisms, the complexity of molecular mechanisms of the complex formation increases in the following sequence: non‐heterocystous cyanobacteria – heterocystous cyanobacteria – green algae – flowering plants.     

Subunit II of Bacillus subtilis Cytochrome c Oxidase Is a Lipoprotein

The sequence of the N-terminal end of the deduced ctaC gene product of Bacillus species has the features of a bacterial lipoprotein. CtaC is the subunit II of cytochrome caa₃, which is a cytochrome c oxidase. Using Bacillus subtilis mutants blocked in lipoprotein synthesis, we show that CtaC is a lipoprotein and that synthesis of the membrane-bound protein and covalent binding of heme to the cytochrome c domain is not dependent on processing at the N-terminal part of the protein. Mutants blocked in prolipoprotein diacylglyceryl transferase (Lgt) or signal peptidase type II (Lsp) are, however, deficient in cytochrome caa₃ enzyme activity. Removal of the signal peptide from the CtaC polypeptide, but not lipid modification, is seemingly required for formation of functional enzyme.     

Effects of gabaculine,a new potent inhibitor of gamma-aminobutyrate transaminase,on the brain gamma-aminobutyrate content and convulsions in mice

Effects of gabaculine (5-amino cyclohexa-1,3-dienyl carboxylic acid), a new potent inhibitor of gamma-aminobutyrate (GABA) transaminase, on the brain GABA content and convulsions were studied in mice. Gabaculine (0.12–2.0 ug) injected intraventricularly elevated brain GABA content for a long period. Three hrs after treatment with gabaculine, picrotoxin- and thiosemicarbazide-induced convulsions were prevented, but not electroschock-induced convulsions.Intraperitoneal injection of gabaculine also increased brain GABA content.Thus gabaculine seems to be a useful tool for investigating the GABA function in the brain.     

A suicide vector for allelic recombination involving the gene for glutamate 1-semialdehyde aminotransferase in the cyanobacterium Synechococcus PCC 7942

Gabaculine (2,3-dihydro 3-amino benzoic acid) is a potent inhibitor of tetrapyrrole biosynthesis in organisms that use the C₅ pathway for the synthesis of δ-aminolaevulinic acid. Glutamate semialdehyde aminotransferase (GSA-AT), the enzyme catalysing the formation of this key precursor of tetrapyrroles, is normally inhibited by concentrations of gabaculine in the order of 5?μM. However, in Synechococcus 6301 strain GR6, a cyanobacterium that is resistant to 100?μM gabaculine, this enzyme has undergone two changes in structure: a deletion of three amino acids from positions 5 to 7 and the substitution of isoleucine for methionine at position 248. To establish the effect in vivo of these specific changes in the gene for GSA-AT (hemL), a suicide vector (pHS7) containing an antibiotic cassette was constructed to achieve the replacement, by homologous recombination, of the wild-type hemL gene in the chromosome by a modified form of the gene. Recombinant strains of Synechococcus 7942 obtained using pHS7-hemL ^GR6 were indistinguishable from Synechococcus 6301 GR6 in terms of the resistance of growth and of chlorophyll accumulation to high concentrations of gabaculine, while a wild-type recombinant produced using pHS7-hemL ^WT had retained its sensitivity. Southern hybridisation using gene probes for hemL, amp ^r and cm ^r confirmed that chromosomal integration of the plasmids had occurred in both WT and GR6 recombinants. Growth and chlorophyll accumulation in equivalent strains with the hemL gene containing either the deletion or the transition characteristic of Synechococcus 6301 GR6 were inhibited by 10?μM gabaculine. Consequently, resistance in vivo to high concentrations of this compound is dependent on both the changes in gene/enzyme structure. This investigation has established the effectiveness of the suicide vector pHS7 for studying the effect in vivo of specific changes in the hemL gene. It has also demonstrated that replacement of the wild-type gene by that from Synechococcus 6301 GR6 is sufficient to confer resistance in vivo to high concentrations of gabaculine.     

Incorporation of radioactive- -aminolevulinic acid into microsomal cytochrome P 450 : selective breakdown of the hemoprotein by allylisopropylacetamide and carbon tetrachloride

Administration of allylisopropylacetamide (AIA) or CCl₄ to rats previously treated with phenobarbital leads to a rapid decrease in cytochrome P₄₅₀ within 1 hr. The amount of cytochrome b₅ and NADPH cytochrome c reductase in liver microsomes remains unchanged following AIA treatment. In contrast, CCl₄ administration causes a decrease in total microsomal protein thus leading to a net loss in cytochrome b₅ and NADPH cytochrome c reductase. By using ³H-δ-aminolevulinic acid to label microsomal cytochrome P₄₅₀ heme, the effect of AIA and CCl₄ on this cytochrome was shown to be caused by destruction of preexisting CO-binding pigment and not from inhibition of synthesis. In addition, the breakdown products of cytochrome P₄₅₀ heme accumulate in the liver after AIA or CCl₄ treatment.     

The Rieske/cytochrome b complex of Heliobacteria

Heliobacteria have a Rieske/cytochrome b complex composed of a Rieske protein, a cytochrome b_6, a subunit IV and a di-heme cytochrome c. The overall structure of the complex seems close to the b₆f complex from cyanobacteria and chloroplasts to the exception of the di-heme cytochrome. We show here by biochemical and biophysical studies that a heme c_i is covalently attached to the Rieske/cytochrome b complex from Heliobacteria. We studied the EPR signature of this heme in two different species, Heliobacterium modesticaldum and Heliobacillus mobilis. In contrast to the case of b₆f complex, a strong axial ligand to the heme is present, most probably a protonatable amino acid residue.     

cis/trans Isomerase of Unsaturated Fatty Acids of Pseudomonas putida P8: Evidence for a Heme Protein of the Cytochrome c Type

From a pool of 600 temperature-sensitive transposon mutants of Pseudomonas putida P8, 1 strain was isolated that carries a mini-Tn5 insertion within the cytochrome c operon. As a result, genes involved in the attachment of heme to cytochrome c-type proteins are turned off. Accordingly, cytochrome c could not be detected spectrophotometrically. The mutant also exhibited a remarkable reduction of cis-trans isomerization capability for unsaturated fatty acids. Consistent with the genetic and physiological data is the detection of a cytochrome c-type heme-binding motif close to the N terminus of the predicted polypeptide of the cis/trans isomerase (cti) gene (CVACH; conserved amino acids in italics). The functional significance of this motif was proven by site-directed mutagenesis. A possible mechanism of heme-catalyzed cis-trans isomerization of unsaturated fatty acids is discussed.     

Studies on the cytochrome b6/f complex. I. Characterization of the complex subunits in Chlamydomonas reinhardtii

We have analyzed the heme-associated peroxidase activity in thylakoid membranes from the green algae Chlamydomonas reinhardtii after electrophoresis in the presence of sodium dodecyl sulfate. Besides cytochrome f and cytochrome b₆, we observed peroxidase activity in two other bands, of 34 and 11 kDa, of unknown origin. Characterization of the b₆/f complex subunits was undertaken by means of a comparison of the polypeptide deficiencies in several b₆/f mutants with the polypeptide content of preparations enriched in b₆/f complexes. We conclude that the b₆/f complex consists of five subunits. Using site-specific translation inhibitors, we show that cytochrome f, cytochrome b₆ and subunit IV are of chloroplast origin, whereas the Rieske protein and probably subunit V are translated on cytoplasmic ribosomes. A model of assembly of the complex is proposed: a cytochrome moiety, comprising the subunits of chloroplast origin, is assembled in the thylakoid membranes prior to the insertion and assembly of the subunits encoded in the nuclear genome.     

CCS5, a Thioredoxin-like Protein Involved in the Assembly of Plastid c-Type Cytochromes

The c-type cytochromes are metalloproteins with a heme molecule covalently linked to the sulfhydryls of a CXXCH heme-binding site. In plastids, at least six assembly factors are required for heme attachment to the apo-forms of cytochrome f and cytochrome c₆ in the thylakoid lumen. CCS5, controlling plastid cytochrome c assembly, was identified through insertional mutagenesis in the unicellular green alga Chlamydomonas reinhardtii. The complementing gene encodes a protein with similarity to Arabidopsis thaliana HCF164, which is a thylakoid membrane-anchored protein with a lumen-facing thioredoxin-like domain. HCF164 is required for cytochrome b₆f biogenesis, but its activity and site of action in the assembly process has so far remained undeciphered. We show that CCS5 is a component of a trans-thylakoid redox pathway and operates by reducing the CXXCH heme-binding site of apocytochrome c prior to the heme ligation reaction. The proposal is based on the following findings: 1) the ccs5 mutant is rescued by exogenous thiols; 2) CCS5 interacts with apocytochrome f and c₆ in a yeast two-hybrid assay; and 3) recombinant CCS5 is able to reduce a disulfide in the CXXCH heme-binding site of apocytochrome f.     

Cytochrome distribution across chloroplast thylakoid membranes. Controlled proteolysis of inside-out and right-side-out vesicles

The transverse distribution of chloroplast cytochromes b-559 (high and low potentials), b-563 and f in pea thylakoid membranes was studied by the effects of trypsin and pronase on inside-out and right-side-out thylakoid vesicles. The high potential (HP) form of cytochrome b-559 was degraded to a low potential (LP) form most rapidly in right-side-out vesicles. In either type of vesicle there was no overall loss of the cytochrome from the membrane. This suggests that the haem group is buried in the membrane but that the cytochrome environment is most labile at the outer surface. Cytochrome b-563 was unaffected by trypsin and only slightly degraded by pronase in inverted vesicles. However, pronase caused the loss of an M_r 1000, non-haem fraction from the cytochrome f polypeptide in inside-out vesices only. The total cytochrome f content (measured spectrophotometrically and by staining polyacrylamide gels for haem associated peroxidase activity) decayed only slightly in either type of vesicle. These observations suggest that cytochrome f is, in part, exposed to the intrathylakoid lumen, whilst its haem group is retained in a more hydrophobic region.     

Leaf Development in Lolium temulentum: Formation of the Photosynthetic Apparatus in the Presence of the Porphyrin Synthesis Inhibitor Gabaculine

Gabaculine, a potent inhibitor of the biosynthesis of aminolaevulinicacid in plants, was supplied via the roots to seedlings of Loliumtemulentum and chloroplast development along the age gradientof the emergent leaf was followed. Exposure to the inhibitorprevented the development of an organized photosynthetic membranesystem and a noticeable accompanying feature was the appearanceof aggregates of osmiophilic globules even in young plastids.In untreated leaves these were only seen to any extent in chloroplastsundergoing senescence. Though chlorophyll formation was inhibitedby gabaculine, levels of carotenoid in treated tissue were somewhatelevated. However, there was a selective depletion ofß-carotenewhich, in situ, is closely associated with the photosyntheticreaction centre. Amounts of two plastid-localized tetrapyrrole-bindingproteins, light-harvesting chlorophyll protein of photosystem-2(LHCP-2) and cytochrome f, were determined. There was a closecorrespondence in inhibition between LHCP-2 and its prostheticgroup with the greatest decrease, compared to leaves of untreatedplants, being at the leaf base. In contrast, formation of theapoprotein and haem components of cytochrome f was apparentlynot so tightly coupled. Amounts of apoprotein were similar tothose of untreated controls, but haem was concentrated in theregion proximal to the base and declined along the age gradient,being absent from the leaf tip. Appearance of novel haem-peroxidaseactivities in the basal region of treated leaves was observed. Key words: Lolium temulentum, gabaculine, chlorophyll, LHCP-2, cytochrome f.     

Participation of ornithine aminotransferase in the synthesis and catabolism of ornithine in mice. Studies using gabaculine and arginine deprivation.

Gabaculine, a potent suicide inhibitor of ornithine aminotransferase (OAT), at a dose of 50 mg/kg inhibited this enzyme in mouse tissues and dramatically increased tissue ornithine concentrations, whether or not arginine was present in the diet. Thus even under arginine deprivation there is catabolism of ornithine which involves OAT. This was confirmed by administration of [14C]ornithine to arginine-deprived mice. Gabaculine (3-amino-2,3-dihydrobenzoic acid) drastically decreased the release of 14CO2 and increased the radioactivity in the basic amino acids in the tissues. When [1-14C]glutamate was injected into mice deprived of arginine, a significant amount of radioactivity was recovered in tissue ornithine and arginine, and gabaculine decreased this labelling by about two-thirds, indicating that ornithine was synthesized in vivo from glutamate via OAT. In addition, we failed to detect in liver and small intestine alpha-N-acetylornithine, N-acetylglutamate kinase or N-acetylornithine aminotransferase, which are obligatory components of a potential route of ornithine synthesis from N-acetylglutamate. Our results indicate that at least 45 mumol of ornithine was synthesized and catabolized daily via OAT in the mouse deprived of arginine.