Measurement of ferrochelatase activity using a novel assay suggests that plastids are the major site of haem biosynthesis in both photosynthetic and non-photosynthetic cells of pea (Pisum sativum L.)

2002 ◽  
Vol 362 (2) ◽  
pp. 423-432 ◽  
Author(s):  
Johanna E. CORNAH ◽  
Jennifer M. ROPER ◽  
Davinder Pal SINGH ◽  
Alison G. SMITH
Keyword(s):  
Ferrous Iron ◽  
Specific Activity ◽  
Higher Plants ◽  
Protoporphyrin Ix ◽  
Chlorophyll Synthesis ◽  
Marker Enzyme ◽  
Hexane Extraction ◽  
Higher Plant ◽  
Major Site ◽  
Haem Biosynthesis

Ferrochelatase is the terminal enzyme of haem biosynthesis, catalysing the insertion of ferrous iron into the macrocycle of protoporphyrin IX, the last common intermediate of haem and chlorophyll synthesis. Its activity has been reported in both plastids and mitochondria of higher plants, but the relative amounts of the enzyme in the two organelles are unknown. Ferrochelatase is difficult to assay since ferrous iron requires strict anaerobic conditions to prevent oxidation, and in photosynthetic tissues chlorophyll interferes with the quantification of the product. Accordingly, we developed a sensitive fluorimetric assay for ferrochelatase that employs Co2+ and deuteroporphyrin in place of the natural substrates, and measures the decrease in deuteroporphyrin fluorescence. A hexane-extraction step to remove chlorophyll is included for green tissue. The assay is linear over a range of chloroplast protein concentrations, with an average specific activity of 0.68nmol·min−1·mg of protein−1, the highest yet reported. The corresponding value for mitochondria is 0.19nmol·min−1·mg of protein−1. The enzyme is inhibited by N-methylprotoporphyrin, with an estimated IC50 value of ≈ 1nM. Using this assay we have quantified ferrochelatase activity in plastids and mitochondria from green pea leaves, etiolated pea leaves and pea roots to determine the relative amounts in the two organelles. We found that, in all three tissues, greater than 90% of the activity was associated with plastids, but ferrochelatase was reproducibly detected in mitochondria, at levels greater than the contaminating plastid marker enzyme, and was latent. Our results indicate that plastids are the major site of haem biosynthesis in higher plant cells, but that mitochondria also have the capacity for haem production.

Terminal steps of haem biosynthesis

2002 ◽  
Vol 30 (4) ◽  
pp. 590-595 ◽  
Author(s):  
H. A. Dailey
Keyword(s):  
Crystal Structure ◽  
Bacillus Subtilis ◽  
Ferrous Iron ◽  
Protoporphyrin Ix ◽  
Oxidative Decarboxylation ◽  
Protoporphyrinogen Oxidase ◽  
Coproporphyrinogen Oxidase ◽  
Cellular Location ◽  
Haem Biosynthesis ◽  
Small Set

The terminal three steps in haem biosynthesis are the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX, followed by the six-electron oxidation of protoporphyrinogen to protoporphyrin IX, and finally the insertion of ferrous iron to form haem. Interestingly, Nature has evolved distinct enzymic machinery to deal with the antepenultimate (co-proporphyrinogen oxidase) and penultimate (protoporphyrinogen oxidase) steps for aerobic compared with anaerobic organisms. The terminal step is catalysed by the enzyme ferrochelatase. This enzyme is clearly conserved with regard to a small set of essential catalytic residues, but varies significantly with regard to size, subunit composition, cellular location and the presence or absence of a [2Fe-2S] cluster. Coproporphyrinogen oxidase and protoporphyrinogen oxidase are reviewed with regard to their enzymic and physical characteristics. Ferrochelatase, which is the best characterized of these three enzymes, will be described with particular emphasis paid to what has been learned from the crystal structure of the Bacillus subtilis and human enzymes.

scholarly journals Protochlorophyllide reductase in photosynthetic prokaryotes and its role in chlorophyll synthesis

1995 ◽  
Vol 311 (2) ◽  
pp. 417-424 ◽  
Author(s):  
J D Rowe ◽  
W T Griffiths
Keyword(s):  
Dna Sequences ◽  
Specific Activity ◽  
Higher Plants ◽  
Red Light ◽  
Protein A ◽  
Chlorophyll Synthesis ◽  
Proteolytic Fragment ◽  
A Value ◽  
Protochlorophyllide Reductase ◽  
Reductase Gene

1. DNA sequences hybridizing with the wheat protochlorophyllide reductase gene have been detected during genomic Southern blots of various cyanobacterial DNA samples. No such hybridization was observed with DNA from photosynthetic bacteria. 2. A fragment amplified from Phormidium laminosum DNA has been characterized and shown to be 73% similar to the corresponding wheat sequence. At the protein level the similarity is 91%. When used as a probe for Southern blotting the Phormidium DNA fragment confirmed the authenticity of some of the original signals obtained with the wheat probe. 3. Peptides of molecular mass 36, 30 and 60 kDa are immunodetected by a wheat reductase antibody during Western blotting of Phormidium preparations. These are purported to correspond to the cyanobacterial mature reductase protein, a stable proteolytic fragment and soluble dimeric forms of the latter respectively. 4. Adaptation of Phormidium to growth in red light (delta > 670 nm) or darkness led to no significant changes in the total level of immunodetected peptides or protochlorophyllide within the cells. 5. The specific activity of the reductase in Phormidium membranes has been tentatively estimated as 0.5 unit/mg of protein, a value comparable with that found in preparations from mature chloroplasts of higher plants.

scholarly journals The coproporphyrin ferrochelatase of Staphylococcus aureus: mechanistic insights into a regulatory iron-binding site

2017 ◽  
Vol 474 (20) ◽  
pp. 3513-3522 ◽  
Author(s):  
Charlie Hobbs ◽  
James D. Reid ◽  
Mark Shepherd
Keyword(s):  
Staphylococcus Aureus ◽  
Ferrous Iron ◽  
Substrate Inhibition ◽  
Iron Concentration ◽  
Protoporphyrin Ix ◽  
Site Directed Mutagenesis ◽  
Bacterial Phyla ◽  
Haem Biosynthesis ◽  
And Staphylococcus Aureus ◽  
Dependent Pathway

The majority of characterised ferrochelatase enzymes catalyse the final step of classical haem synthesis, inserting ferrous iron into protoporphyrin IX. However, for the recently discovered coproporphyrin-dependent pathway, ferrochelatase catalyses the penultimate reaction where ferrous iron is inserted into coproporphyrin III. Ferrochelatase enzymes from the bacterial phyla Firmicutes and Actinobacteria have previously been shown to insert iron into coproporphyrin, and those from Bacillus subtilis and Staphylococcus aureus are known to be inhibited by elevated iron concentrations. The work herein reports a Km (coproporphyrin III) for S. aureus ferrochelatase of 1.5 µM and it is shown that elevating the iron concentration increases the Km for coproporphyrin III, providing a potential explanation for the observed iron-mediated substrate inhibition. Together, structural modelling, site-directed mutagenesis, and kinetic analyses confirm residue Glu271 as being essential for the binding of iron to the inhibitory regulatory site on S. aureus ferrochelatase, providing a molecular explanation for the observed substrate inhibition patterns. This work therefore has implications for how haem biosynthesis in S. aureus is regulated by iron availability.

scholarly journals Haem synthesis during cytochrome P-450 induction in higher plants. 5-Aminolaevulinic acid synthesis through a five-carbon pathway in Helianthus tuberosus tuber tissues aged in the dark

1988 ◽  
Vol 249 (2) ◽  
pp. 473-480 ◽  
Author(s):  
D Werck-Reichhart ◽  
O T G Jones ◽  
F Durst
Keyword(s):  
Pyridoxal Phosphate ◽  
Higher Plants ◽  
Jerusalem Artichoke ◽  
Acid Synthesis ◽  
Chlorophyll Synthesis ◽  
Higher Plant ◽  
Aminolaevulinic Acid ◽  
C5 Pathway ◽  
Suicide Inhibitors ◽  
Cytochrome P 450

Chlorophyll and haem synthesis in illuminated Jerusalem artichoke tuber tissues were very efficiently inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid). This inhibition seems to be due specifically to a blockade of the pathway for 5-aminolaevulinate biosynthesis which used glutamate as a substrate (the so-called C5 pathway) since we could not detect any inhibition of protein synthesis in the treated tissues and there was no effect of gabaculine on the glycine-dependent yeast 5-aminolaevulinate synthase used as a model. In dark-aged artichoke tissues, gabaculine also effectively blocked cytochrome P-450 induction, peroxidase activity and 5-aminolaevulinic acid synthesis, thus suggesting the involvement of a C5 pathway in cytoplasmic and microsomal haemoprotein synthesis in this higher plant. Allylglycine and (2-amino-ethyloxyvinyl)glycine, two olefinic glycine analogues which are potential suicide inhibitors of pyridoxal phosphate enzymes, were also demonstrated to be effective blockers of chlorophyll synthesis in artichoke tuber and Euglena cells exposed to light.

scholarly journals Electronic Structure of Tyrosyl D Radical of Photosystem II, as Revealed by 2D-Hyperfine Sublevel Correlation Spectroscopy

2021 ◽  
Vol 7 (9) ◽  
pp. 131
Author(s):  
Maria Chrysina ◽  
Georgia Zahariou ◽  
Nikolaos Ioannidis ◽  
Yiannis Sanakis ◽  
George Mitrikas
Keyword(s):  
Electronic Structure ◽  
Photosystem Ii ◽  
Water Oxidation ◽  
Spinacia Oleracea ◽  
Higher Plants ◽  
Correlation Spectroscopy ◽  
Oxygen Evolving Complex ◽  
Higher Plant ◽  
Proton Coupled Electron Transfer ◽  
S Oxidation

The biological water oxidation takes place in Photosystem II (PSII), a multi-subunit protein located in thylakoid membranes of higher plant chloroplasts and cyanobacteria. The catalytic site of PSII is a Mn4Ca cluster and is known as the oxygen evolving complex (OEC) of PSII. Two tyrosine residues D1-Tyr161 (YZ) and D2-Tyr160 (YD) are symmetrically placed in the two core subunits D1 and D2 and participate in proton coupled electron transfer reactions. YZ of PSII is near the OEC and mediates electron coupled proton transfer from Mn4Ca to the photooxidizable chlorophyll species P680+. YD does not directly interact with OEC, but is crucial for modulating the various S oxidation states of the OEC. In PSII from higher plants the environment of YD• radical has been extensively characterized only in spinach (Spinacia oleracea) Mn- depleted non functional PSII membranes. Here, we present a 2D-HYSCORE investigation in functional PSII of spinach to determine the electronic structure of YD• radical. The hyperfine couplings of the protons that interact with the YD• radical are determined and the relevant assignment is provided. A discussion on the similarities and differences between the present results and the results from studies performed in non functional PSII membranes from higher plants and PSII preparations from other organisms is given.

scholarly journals Purification and characterization of a thermostable ADP-glucose pyrophosphorylase from Thermus caldophilus GK-24

1996 ◽  
Vol 319 (3) ◽  
pp. 977-983 ◽  
Author(s):  
Jeong Heon KO ◽  
Cheorl Ho KIM ◽  
Dae-Sil LEE ◽  
Yu Sam KIM
Keyword(s):  
Molecular Mass ◽  
Specific Activity ◽  
Gel Filtration ◽  
Higher Plant ◽  
Sds Page ◽  
Thermus Caldophilus ◽  
Internal Peptide ◽  
Adp Glucose Pyrophosphorylase ◽  
Fructose 1,6 Bisphosphate

An extremely thermostable ADP-glucose pyrophosphorylase (AGPase) has been purified from Thermus caldophilus GK-24 to homogeneity by chromatographic methods, including gel filtration and ion-exchange and affinity chromatography. The specific activity of the enzyme was enriched 134.8-fold with a recovery of 10.5%. The purified enzyme was a single band by SDS/PAGE with a molecular mass of 52 kDa. The homotetrameric structure of the native enzyme was determined by gel filtration analysis, which showed a molecular mass of 230 kDa on a Superose-12 column, indicating that the structure of the enzyme is different from the heterotetrameric structures of higher-plant AGPases. The enzyme was most active at pH 6.0. The activity was maximal at 73–78 °C and its half-life was 30 min at 95 °C. Kinetic and regulatory properties were characterized. It was found that AGPase activity could be stimulated by a number of glycolytic intermediates. Fructose 6-phosphate, fructose 1,6-bisphosphate, phenylglyoxal and glucose 6-phosphate were effective activators, of which fructose 1,6-bisphosphate was the most effective. The enzyme was inhibited by phosphate, AMP or ADP. ATP and glucose 1-phosphate gave hyperbolic-shaped rate-concentration curves in the presence or absence of activator. A remarkable aspect of the amino acid composition was the existence of the hydrophobic and Ala+Gly residues. The N-terminal and internal peptide sequences were determined and compared with known sequences of various sources. It was apparently similar to those of AGPases from other bacterial and plant sources, suggesting that the enzymes are structurally related.

scholarly journals Genetic fine-structure of the GA-1 locus in the higher plant Arabidopsis thaliana (L.) Heynh

1983 ◽  
Vol 41 (1) ◽  
pp. 57-68 ◽  
Author(s):  
M. Koornneef ◽  
J. Van Eden ◽  
C. J. Hanhart ◽  
A. M. M. De Jongh
Keyword(s):  
Arabidopsis Thaliana ◽  
Fine Structure ◽  
Higher Plants ◽  
Wild Type ◽  
Higher Plant ◽  
Selfed Progeny ◽  
Intragenic Deletion ◽  
Genetic Length ◽  
Genetic Fine Structure ◽  
Half Diallel

SUMMARYNon-germinating gibberellin (GA) responsive mutants are a powerful tool to study genetic fine structure in higher plants. Nine alleles (EMS-and fast neutron-induced) of the ga-1 locus of Arabidopsis thaliana were tested in a complete half-diallel. No wild type ‘recombinants’ were found in the selfed progeny of 9 homoallelic combinations (in total 3 × 105 plants); in the progenies from the 36 selfed hetero allelics the wild type frequency ranged from zero to 6·6 × 10−4. These frequencies allowed the construction of an internally consistent map for five different sites representing eight alleles. The ninth allele covered three sites and thus behaved like an intragenic deletion. The estimate of the total genetic length of the ga-1 locus was 0·07 cM. The order of the sites was also clearly reflected by the association with proximal outside markers. On the assumption that wild type gametes predominantly arise from reciprocal events, it was shown that a cross-over within the ga-1 locus leads to positive interference in the adjacent region.The results are discussed with respect to the mutagen used, the frequencies found in other plant and Drosophila genes, and the possible occurrence of gene conversion.

The higher plant PII signal transduction protein: structure, function and properties

2007 ◽  
Vol 85 (6) ◽  
pp. 533-537 ◽  
Author(s):  
Greg B.G. Moorhead ◽  
Tony S. Ferrar ◽  
Yan M. Chen ◽  
Yutaka Mizuno ◽  
Catherine S. Smith ◽  
...  
Keyword(s):  
Higher Plants ◽  
Structural Features ◽  
Signaling Proteins ◽  
Higher Plant ◽  
Binding Partner ◽  
Signal Transduction Protein ◽  
Metabolic Sensor ◽  
Nitrogen Sensing ◽  
Over 40 Years

The PII carbon/nitrogen sensing protein was discovered in Escherichia coli (Migula 1895) Castellani and Chalmers 1919, over 40 years ago. Orthologues have been discovered in three kingdoms of life making it one of the most ancient and conserved signaling proteins known. Recent advances in the field have established its primary binding partner in plants as N-acetyl glutamate kinase and the crystal structure has revealed features unique to plants that likely contribute to its function in vivo. Here, we review the properties, function, and novel structural features of this chloroplast-localized metabolic sensor of higher plants.

Photoreduction of NADP+ in Photosystem II of Higher Plants: Requirement for Manganese

1992 ◽  
Vol 47 (1-2) ◽  
pp. 57-62 ◽  
Author(s):  
Suleyman I. Allakhverdiev ◽  
Vyacheslav V. Klimov
Keyword(s):  
Photosystem Ii ◽  
Higher Plants ◽  
Complete Removal ◽  
Reaction Centers ◽  
Higher Plant ◽  
Ps Ii ◽  
Alternate Pathway ◽  
Manganese Extraction ◽  
Quinone Acceptor ◽  
Reduced State

Abstract The effects of reversible manganese extraction on NADP+ photoreduction were studied with higher plant subchloroplast preparations of photosystem II (PS II). Under anaerobic conditions, when the reaction centers (RCs) of PS II are “closed” (i.e. in the state [P680 Pheo] QA), and in the presence of ferredoxin-ferredoxin-NADP+ reductase, NADP+ reduction is observed at a rate of 0.8 -1.1 nmol/mg × chlorophyll × h. After complete removal of manganese from PS II, the rate of NADP+ reduction is reduced 40 - 50-fold. Upon the addition of Mn at a concentration of approx. 4 Mn atoms per reaction center, the NADP+ reduction is restored up to 85 -90% of the initial value. When half of this amount of Mn is combined with about 40 times of the equivalent concentration of other divalent ions (Ca2+, Sr2+, Mg2+ etc.) the reaction is also reactivated. Dinoseb (10-6 m) an inhibitor of electron transfer in PS II prevents NADP+ photoreduction. It is concluded that under conditions when the first quinone acceptor, QA, is in its reduced state (QA-) electrons are transferred from reduced pheophytin (Pheo·̅) to NADP+, indicating that PS II can reduce NADP+ without the participation of PS I. On the basis of these and literature data, an alternate pathway for electron phototransfer in PS II reaction centers of higher plants is suggested. Some problems concerning the Z-scheme are discussed.

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