Insertion Of Mg Research Articles

The ATPase activity of the ChlI subunit of magnesium chelatase and formation of a heptameric AAA+ ring.

The AAA(+) ATPase component of magnesium chelatase (ChlI) drives the insertion of Mg(2+) into protoporphyrin IX; this is the first step in chlorophyll biosynthesis. We describe the ATPase activity, nucleotide binding kinetics, and structural organization of the ChlI protein. A consistent reaction scheme arises from our detailed steady state description of the ATPase activity of the ChlI subunit and from transient kinetic analysis of nucleotide binding. We provide the first demonstration of metal ion binding to a specific subunit of any of the multimeric chelatases and characterize binding of Mg(2+) to the free and MgATP(2)(-) bound forms of ChlI. Transient kinetic studies with the fluorescent substrate analogue TNP-ATP show that there are two forms of monomeric enzyme, which have distinct magnesium binding properties. Additionally, we describe the self-association properties of the subunit and provide a structural analysis of the multimeric ring formed by this enzyme in the presence of nucleotide. This single particle analysis demonstrates that this species has a 7-fold rotational symmetry, which is in marked contrast to most members of the AAA(+) family that tend to form hexamers.

Chlorophyll biosynthesis. Expression of a second chl I gene of magnesium chelatase in Arabidopsis supports only limited chlorophyll synthesis.

Magnesium (Mg) chelatase is a heterotrimeric enzyme complex that catalyzes a key regulatory and enzymatic reaction in chlorophyll biosynthesis, the insertion of Mg(2+) into protoporphyrin IX. Studies of the enzyme complex reconstituted in vitro have shown that all three of its subunits, CHL I, CHL D, and CHL H, are required for enzymatic activity. However, a new T-DNA knockout mutant of the chlorina locus, ch42-3 (Chl I), in Arabidopsis is still able to accumulate some chlorophyll despite the absence of Chl I mRNA and protein. In barley (Hordeum vulgare), CHL I is encoded by a single gene. We have identified an open reading frame that apparently encodes a second Chl I gene, Chl I2. Chl I1 and Chl I2 mRNA accumulate to similar levels in wild type, yet CHL I2 protein is not detectable in wild type or ch42-3, although the protein is translated and stromally processed as shown by in vivo pulse labeling and in vitro chloroplast imports. It is surprising that CHL D accumulates to wild-type levels in ch42-3, which is in contrast to reports that CHL D is unstable in CHL I-deficient backgrounds of barley. Our results show that limited Mg chelatase activity and CHL D accumulation can occur without detectable CHL I, despite its obligate requirement in vitro and its proposed chaperone-like stabilization and activation of CHL D. Thus, the unusual post-translational regulation of the CHL I2 protein provides an opportunity to study the different steps involved in stabilization and activation of the heterotrimeric Mg chelatase in vivo.

Tetrapyrrole regulation of nuclear gene expression.

Tetrapyrroles are the structural backbone of chlorophyll and heme, and are essential for primary photochemistry, light harvesting, and electron transport. The biochemistry of their synthesis has been studied extensively, and it has been suggested that some of the tetrapyrrole biochemical intermediates can affect nuclear gene expression. In this review, tetrapyrrole biosynthesis, which occurs in the chloroplast, and its regulation will be covered. An analysis of the intracellular location of tetrapyrrole intermediates will also be included. The focus will be on tetrapyrrole intermediates that have been suggested to affect gene expression. These include Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester. Recent evidence also suggests a specific signaling role for the H subunit of Mg-chelatase, an enzyme that catalyzes the insertion of Mg into the tetrapyrrole ring. Since gene expression studies have been done in plants and green algae, our discussion will be limited to these organisms.

Possible role for molecular chaperones in assembly and repair of photosystem II.

Genes of the HSP70 chaperone family are induced by light. In Chlamydomonas reinhardtii, the induction of HSP70 (70 kDa heat shock protein) chaperones by light results in a partial protection of photosystem II against damage by photoinhibitory conditions. Underexpression of a chloroplast-localized HSP70 protein caused an increased sensitivity of photosystem II to light. Overexpression of this protein had a protective effect. Fluorescence measurements and studies of the turnover of photosystem II core components suggest that this HSP70 might function in both the protection and the regeneration of photosystem II. This concept is supported by fractionation studies in which the plastid HSP70 was found associated with chloroplast membranes. Because the light-induced elevation of HSP70 levels provides protection for photosystem II, we examined whether the chloroplast is involved in this regulation and found that mutants defective in plastid-localized chlorophyll synthesis, i.e. the insertion of Mg(2+) into protoporphyrin IX are impaired in the induction of HSP70 by light. Exogenous addition of Mg-protoporphyrin in the dark induced the genes. The combined results support a model in which chlorophyll precursors are essential in the signalling from chloroplast to nucleus that regulates the chaperone genes.

Interplay between an AAA module and an integrin I domain may regulate the function of magnesium chelatase

In chlorophyll biosynthesis, insertion of Mg 2+ into protoporphyrin IX is catalysed in an ATP-dependent reaction by a three-subunit (BchI, BchD and BchH) enzyme magnesium chelatase. In this work we present the three-dimensional structure of the ATP-binding subunit BchI. The structure has been solved by the multiple wavelength anomalous dispersion method and refined at 2.1 Å resolution to the crystallographic R-factor of 22.2 % ( R free = 24.5 %). It belongs to the chaperone-like “ATPase associated with a variety of cellular activities” (AAA) family of ATPases, with a novel arrangement of domains: the C-terminal helical domain is located behind the nucleotide-binding site, while in other known AAA module structures it is located on the top. Examination by electron microscopy of BchI solutions in the presence of ATP demonstrated that BchI, like other AAA proteins, forms oligomeric ring structures. Analysis of the amino acid sequence of subunit BchD revealed an AAA module at the N-terminal portion of the sequence and an integrin I domain at the C terminus. An acidic, proline-rich region linking these two domains is suggested to contribute to the association of BchI and BchD by binding to a positively charged cleft at the surface of the nucleotide-binding domain of BchI. Analysis of the amino acid sequences of BchI and BchH revealed integrin I domain-binding sequence motifs. These are proposed to bind the integrin I domain of BchD during the functional cycle of magnesium chelatase, linking porphyrin metallation by BchH to ATP hydrolysis by BchI. An integrin I domain and an acidic and proline-rich region have been identified in subunit CobT of cobalt chelatase, clearly demonstrating its homology to BchD. These findings, for the first time, provide an insight into the subunit organisation of magnesium chelatase and the homologous colbalt chelatase.

ATPase activity associated with the magnesium-protoporphyrin IX chelatase enzyme of Synechocystis PCC6803: evidence for ATP hydrolysis during Mg2+ insertion, and the MgATP-dependent interaction of the ChlI and ChlD subunits

Insertion of Mg2+ into protoporphyrin IX catalysed by the three-subunit enzyme magnesium-protoporphyrin IX chelatase (Mg chelatase) is thought to be a two-step reaction, consisting of activation followed by Mg2+ chelation. The activation step requires ATP and two of the subunits, ChlI and ChlD (I and D respectively), and it has been speculated that this step results in the formation of an I–D–ATP complex. The subsequent step, in which Mg2+ is inserted into protoporphyrin, also requires ATP and the third subunit, H, in addition to ATP-activated I–D complex. In the present study, we examine the interaction of the I and D subunits of the Mg chelatase from the cyanobacterium Synechocystis PCC 6803. We demonstrate the purification of an I–D complex, and show that ATP and Mg2+ are absolute requirements for the formation of this complex, probably as MgATP. However, ATP may be replaced by the slowly hydrolysable analogue, adenosine 5´-[γ-thio]triphosphate, and, to a minor extent, by ADP and the non-hydrolysable ATP analogue, adenosine 5´-[β,γ-imido]triphosphate, all of which suggests that ATP hydrolysis is not necessary for the formation of the ChlI–ChlD complex. A sensitive continuous assay was used to detect ATPase activity during Mg2+ chelation, and it was found that the maximum rate of ATP hydrolysis coincided with the maximum rate of Mg2+ insertion. The rate of ATP hydrolysis depended on factors that determined the rate of Mg2+ chelation, such as increasing the concentration of the H subunit and the concentration of protoporphyrin. Thus ATP hydrolysis has been identified as an absolute requirement for the chelation step. The I subunit possessed strong ATPase activity when assayed on its own, whereas the D subunit had no detectable activity, and when the I and D subunits were assayed in combination, the ATPase activity of the I subunit was repressed.

ATPase activity associated with the magnesium-protoporphyrin IX chelatase enzyme of Synechocystis PCC6803: evidence for ATP hydrolysis during Mg2+ insertion, and the MgATP-dependent interaction of the ChlI and ChlD subunits

Insertion of Mg2+ into protoporphyrin IX catalysed by the three-subunit enzyme magnesium-protoporphyrin IX chelatase (Mg chelatase) is thought to be a two-step reaction, consisting of activation followed by Mg2+ chelation. The activation step requires ATP and two of the subunits, ChlI and ChlD (I and D respectively), and it has been speculated that this step results in the formation of an I-D-ATP complex. The subsequent step, in which Mg2+ is inserted into protoporphyrin, also requires ATP and the third subunit, H, in addition to ATP-activated I-D complex. In the present study, we examine the interaction of the I and D subunits of the Mg chelatase from the cyanobacterium Synechocystis PCC 6803. We demonstrate the purification of an I-D complex, and show that ATP and Mg2+ are absolute requirements for the formation of this complex, probably as MgATP. However, ATP may be replaced by the slowly hydrolysable analogue, adenosine 5'-[gamma-thio]triphosphate, and, to a minor extent, by ADP and the non-hydrolysable ATP analogue, adenosine 5'-[beta,gamma-imido]triphosphate, all of which suggests that ATP hydrolysis is not necessary for the formation of the ChlI-ChlD complex. A sensitive continuous assay was used to detect ATPase activity during Mg2+ chelation, and it was found that the maximum rate of ATP hydrolysis coincided with the maximum rate of Mg2+ insertion. The rate of ATP hydrolysis depended on factors that determined the rate of Mg2+ chelation, such as increasing the concentration of the H subunit and the concentration of protoporphyrin. Thus ATP hydrolysis has been identified as an absolute requirement for the chelation step. The I subunit possessed strong ATPase activity when assayed on its own, whereas the D subunit had no detectable activity, and when the I and D subunits were assayed in combination, the ATPase activity of the I subunit was repressed.

Theory of magnesium/Alq3 interaction in organic light emitting devices

The interaction between the magnesium (Mg) atom and Alq3 molecule in organic light emitting devices has been theoretically studied using the PM3 method of the molecular orbital theory. It has been shown that various interactions can happen when the Mg atom approaches the Alq3 molecule, leading to significant changes in the molecular geometry and electronic structure of Alq3. The resulting configurations with insertion of Mg into the central part of the Alq3 molecule are found to be energetically more favorable than those with Mg forming bond(s) with a benzene ring of Alq3. In agreement with recent experiments, this result shows that the interaction between Mg and the Alq3 molecule depends on the order of deposition. Interestingly, the density of states calculated for these systems reveals that the creation of the new electronic gap states observed in the experiments is mainly relating to the aluminum atomic orbital due to interaction, instead of a direct contribution of Mg.

Magnesium chelatase from Rhodobacter sphaeroides: initial characterization of the enzyme using purified subunits and evidence for a BchI–BchD complex

The enzyme magnesium-protoporphyrin IX chelatase (Mg chelatase) catalyses the insertion of Mg into protoporphyrin IX, the first committed step in (bacterio)chlorophyll biosynthesis. In the photosynthetic bacterium Rhodobacter sphaeroides, this reaction is catalysed by the products of the bchI, bchD and bchH genes. These genes have been expressed in Escherichia coli so that the BchI, BchD and BchH proteins are produced with N-terminal His6 affinity tags, which has led to the production of large amounts of highly purified, highly active Mg chelatase subunits from a single chromatography step. Furthermore, BchD has been purifed free of contamination with the chaperone GroEL, which had proven to be a problem in the past. BchD, present largely as an insoluble protein in E. coli, was purified in 6 M urea and refolded by addition of BchI, MgCl2 and ATP, yielding highly active protein. BchI/BchD mixtures prepared in this way were used in conjunction with BchH to determine the kinetic parameters of R. sphaeroides Mg chelatase for its natural substrates. We have been able to demonstrate for the first time that BchI and BchD form a complex, and that Mg2+ and ATP are required to establish and maintain this complex. Gel filtration data suggest that BchI and BchD form a complex of molecular mass 200 kDa in the presence of Mg2+ and ATP. Our data suggest that, in vivo, BchD is only folded correctly and maintained in its correct conformation in the presence of BchI, Mg2+ and ATP.

Magnesium chelatase subunit D from pea: characterization of the cDNA, heterologous expression of an enzymatically active protein and immunoassay of the native protein.

Mg-chelatase catalyzes the insertion of Mg into protoporphyrin and lies at the branchpoint of heme and (bacterio)chlorophyll synthesis. In prokaryotes, three genes--BchI, D and H--encode subunits for Mg-chelatase. In higher plants, homologous cDNAs for the I, D and H subunits have been characterized. Since the N-terminal half of the D subunit is homologous to the I subunit, the C-terminal portion of the pea D was used for antigen production. The antibody recognized the chloroplast D subunit and was used to demonstrate that this subunit associated with the membranes in the presence of MgCl2. The antibody immunoprecipitated the native protein and inhibited Mg-chelatase activity. Expression in Escherichia coli with a construct for the full-length protein (minus the putative transit peptide) resulted in induction of 24.5 kDa (major) and 89 kDa (minor) proteins which could only be solubilized in 6 M urea. However, when host cells were co-transformed with expression vectors for the full-length D subunit and for the 70 kDa HSP chaperonin protein, a substantial portion of the 89 kDa protein was expressed in a soluble form which was active in a Mg-chelatase reconstitution assay.

Synthesis and Characterization of Dimeric Mutually Coordinated Magnesium meso-2-Pyridylporphyrins.

The synthesis and characterization of a series of meso-2-pyridylporphyrins and their Mg(2+) complexes are reported. Condensation of 4-alkylbenzyl-2,2'-dipyrromethanes (alkyl = Me, n-Pr, or n-Bu) with 2-pyridinecarboxaldehyde yielded a series of free-base meso-2-pyridylporphyrins. Insertion of Mg(2+) into the free-base porphyrins yielded the respective magnesium complexes. These compounds were characterized using 1D ((1)H and (13)C) and 2D ((1)H-(1)H COSY) NMR methods, UV-visible absorption spectroscopy, fluorescence spectroscopy, and mass spectrometry. The interplanar spacing of the dimers is sufficiently small that there is excitonic coupling of the constituent chromophores. The overall dissociation constant of these dimers is estimated at 2 x 10(-)(6) M. Addition of donor ligands such as acetone, DMF, DMSO, or pyridine converts the dimeric species to their respective constituent monomers. Titration of the dimeric complex with pyridine-d(5)() shows that disaggregation requires coordination of two pyridine molecules at independent binding sites. Tracking of the pyridine coordination by (1)H NMR spectroscopy allowed for determination of the equilibrium constant for the pyridine-induced disaggregation reaction (2.1 x 10(-)(3) M(2)). Both the spontaneous dissociation and the pyridine-induced disaggregation reactions occur by two steps.

XANES investigations and catalytic properties of coprecipitated AlF3/MgF2 phases

Stepwise replacement of aluminium by magnesium in β-AlF3 phases leads to considerable alterations in the structure and surface properties of the calcination products of co-precipitates. This is accompanied by significant changes in the catalytic activity for heterogeneous chlorine/fluorine exchange reactions. The maximum catalytic activity is reached between 10 and 20 atom% magnesium doping. Due to the decrease of the strength of Lewis acid sites with further increasing the Mg content, the catalytic activity is more and more diminished. The XRD patterns indicate a loss of crystallinity for β-AlF3 with increasing Mg content. From Mg K-edge XANES and Al K-edge XANES spectra of Mg added β-AlF3 phases one can conclude a partial incorporation of Mg into the β-AlF3 lattice. Furthermore, a partial electron transfer from magnesium to the β-AlF3 lattice resulting in an increased electron deficiency (increased Lewis acidity) at low Mg concentrations can be stated, a result which correlates well with the increased catalytic activity observed for co-precipitated phases with lower Mg contents. The insertion of Mg into the β-AlF3 lattice is also supported by XPS studies on the same samples which show an increase of the modified Auger parameter for magnesium of about 0.4 eV, which indicates a change of the chemical environment of the ionized magnesium atom.

Mechanism and regulation of Mg-chelatase.

Mg-chelatase catalyses the insertion of Mg into protoporphyrin IX (Proto). This seemingly simple reaction also is potentially one of the most interesting and crucial steps in the (bacterio)chlorophyll (Bchl/Chl)-synthesis pathway, owing to its position at the branch-point between haem and Bchl/Chl synthesis. Up until the level of Proto, haem and Bchl/Chl synthesis share a common pathway. However, at the point of metal-ion insertion there are two choices: Mg2+ insertion to make Bchl/Chl (catalysed by Mg-chelatase) or Fe2+ insertion to make haem (catalysed by ferrochelatase). Thus the relative activities of Mg-chelatase and ferrochelatase must be regulated with respect to the organism's requirements for these end products. How is this regulation achieved? For Mg-chelatase, the recent design of an in vitro assay combined with the identification of Bchl-biosynthetic enzyme genes has now made it possible to address this question. In all photosynthetic organisms studied to date, Mg-chelatase is a three-component enzyme, and in several species these proteins have been cloned and expressed in an active form. The reaction takes place in two steps, with an ATP-dependent activation followed by an ATP-dependent chelation step. The activation step may be the key to regulation, although variations in subunit levels during diurnal growth may also play a role in determining the flux through the Bchl/Chl and haem branches of the pathway.

Magnesium-containing carbonate apatites

Hydroxyapatites precipitated at pH 7.0 and 9.0 with and without carbonate and with different amounts of magnesium were studied. Mg uptake, Ca/P ratios, and lattice constant data indicate that Mg is incorporated into the apatite lattice. IR spectra demonstrate the formation of B-type carbonate apatites with carbonate substituting for phosphate. Decomposition of carbonate-containing apatites at elevated temperatures up to 1000°C is more gradual for apatites prepared at pH 9.0 than for those prepared at pH 7.0 for which an abrupt loss of carbonate occurs after 600°C. Compounds synthesized without added carbonate partially transform to β Ca 3 (PO 4) 2 (TCP) at about 700°C. Greater transformation to TCP occurs as the Mg incorporation is increased, indicating the insertion of Mg into TCP and consequent stabilization of the TCP. SEM micrographs show increases in the size of crystallites when apatites are precipitated with Mg (in the 0.2–1.5% range), providing further evidence for Mg incorporation into the apatite structure.

Crystal Structure of the Product of Mg2+Insertion into V2O5Single Crystals

Chemical (by interaction with a dibutylmagnesium solution) and electrochemical (in acetonitrile solution of magnesium perchlorate) insertion of Mg2+into the single crystals of V2O5was performed. The morphology change of V2O5crystals as a result of the Mg2+insertion was studied by scanning electron microscopy. The wavelength dispersive electron probe microanalysis clearly showed the presence of Mg (at least) at the surface of intercalated V2O5. Based on the single crystal X-ray diffraction study of intercalated V2O5(orthorhombic,Pmn21,a= 11.544(6),b= 4.383(3),c= 3.574(2) Å,Z= 4) the location of a small amount of Mg (∼1%) in the bulk V2O5may be suggested, with [6 + 4] oxygen atoms surrounding Mg. The resulting Mg–O separations essentially exceed the accepted values for the Mg–O distances in crystals with hexacoordinated Mg atoms, which may be correlated with the structural and electrochemical properties of Mg2+-inserted V2O5.

Magnesium-protoporphyrin chelatase of Rhodobacter sphaeroides: reconstitution of activity by combining the products of the bchH, -I, and -D genes expressed in Escherichia coli.

Magnesium-protoporphyrin chelatase lies at the branch point of the heme and (bacterio)chlorophyll biosynthetic pathways. In this work, the photosynthetic bacterium Rhodobacter sphaeroides has been used as a model system for the study of this reaction. The bchH and the bchI and -D genes from R. sphaeroides were expressed in Escherichia coli. When cell-free extracts from strains expressing BchH, BchI, and BchD were combined, the mixture was able to catalyze the insertion of Mg into protoporphyrin IX in an ATP-dependent manner. This was possible only when all three genes were expressed. The bchH, -I, and -D gene products are therefore assigned to the Mg chelatase step in bacteriochlorophyll biosynthesis. The mechanism of the Mg chelation reaction and the implications for chlorophyll biosynthesis in plants are discussed.

The magnesium-insertion step of chlorophyll biosynthesis is a two-stage reaction.

Mg(2+)-chelatase catalyses the first step unique to chlorophyll synthesis, namely the insertion of Mg2+ into protoporphyrin IX. When pea (Pisum sativum L., cv. Spring) chloroplasts are lysed in a buffer lacking Mg2+ and the thylakoids removed by centrifugation, the remaining mixture of light membranes and soluble proteins (LM/S) has high Mg(2+)-chelatase activity. Several lines of evidence are presented to show that the Mg2+ insertion catalysed by this preparation is a two-step reaction consisting of activation followed by Mg2+ chelation. An activated state of Mg(2+)-chelatase is achieved by preincubating LM/S with ATP. The activated state is observed as the elimination of the approx. 6 min lag in the rate of Mg2+ chelation on addition of the porphyrin substrate. The activity of LM/S assayed at low protein concentrations can be greatly enhanced by preincubating at high protein concentrations (12 mg/ml is optimal). This activation effect requires the presence of both LM and S fractions, as well as ATP. Both steps require ATP, but at different concentrations; the first step is optimal at > 0.5 mM (EC50 = 0.3 mM) and the second step is optimal at 0.3 mM (EC50 < 0.2 mM). ATP in the first step could be replaced by ATP[S]; this analogue could not sustain activity in the second step. This activated state was stable for at least 30 min at room temperature, but chilling of preincubated LM/S on ice for 30 min caused an almost complete loss of the activated state.

Further Characterization of the Magnesium Chelatase in Isolated Developing Cucumber Chloroplasts

Mg-chelatase catalyzes the first step unique to the chlorophyll branch of tetrapyrrole biosynthesis, namely the insertion of Mg into protoporphyrin IX (Proto). Mg-chelatase was assayed in intact chloroplasts from semi-green cucumber (Cucumis sativus, cv Sumter) cotyledons. In the presence of Proto and MgATP, enzyme activity was linear for 50 minutes. Plastid intactness was directly related to (and necessary for) Mg-chelatase activity. Uncouplers and ionophores did not inhibit Mg-Chelatase in the presence of ATP. The nonhydrolyzable ATP analogs, beta,gamma-methylene ATP and adenylylimidodiphosphate, could not sustain Mg-chelatase activity alone and were inhibitory in the presence of ATP (I(50) 10 and 3 millimolar, respectively). Mg-chelatase was also inhibited by N-ethylmaleimide (I(50), 50 micromolar) and the metal ion chelators 2,2'-dipyridyl and 1, 10 phenanthroline (but not to the same degree by their nonchelating analogs). In addition to Proto, the following porphyrins acted as Mg-chelatase substrates, giving comparable specific activities: deuteroporphyrin, mesoporphyrin, 2-ethyl, 4-vinyl Proto and 2-vinyl, 4-ethyl Proto. Mg-chelatase activity and freely exchangeable heme levels increased steadily with greening, reaching a maximum and leveling off after 15 hours in the light. Exogenous protochlorophyllide, chlorophyllide, heme, and Mg-Proto had no measurable effect on Mg-chelatase activity. The potent ferrochelatase inhibitors, N-methylmesoporphyrin and N-methylprotoporphyrin, inhibited Mg-chelatase at micromolar concentrations.

Intercalation of Mg in V2O5

Abstract The intercalation produced by the insertion of Mg2+ into V2O5 was investigated by XRD. It was found that there were three phases similar closely to that of Li in V2O5 and one of them was a more stable state.

Dynamics of the reactions of Mg(3s3p 1p 1) with H 2, HD, and D 2: Rotational quantum state distributions of MgH (MgD) products

The initial bimodal distributions of product MgH(ν = 0) rotational states in the reaction of Mg( 1P 1) with H 2 or HD are found to be identical. Similar results were found for MgD (ν = 0) produced from D 2 or HD. From these as well as other recent results, it is proposed that both the predominant “high- N” as well as the minor “low- N” components of the distributions result from insertion of Mg( 1P 1) into the H-H bond, and that there are two different primary modes of decomposition of the isotopic H-Mg-H intermediates.