Characterization of three homologs of the large subunit of the magnesium chelatase from Chlorobaculum tepidum and interaction with the magnesium protoporphyrin IX methyltransferase

Green bacteria synthesize several types of (bacterio)chlorophylls for the assembly of functional photosynthetic reaction centers and antenna complexes. A distinctive feature of green bacteria compared with other photosynthetic microbes is that their genomes contain multiple homologs of the large subunit (BchH) of the magnesium chelatase which is a three-subunit enzyme complex (BchH, BchD, and BchI) that inserts magnesium into protoporphyrin IX as the first committed step of (bacterio)chlorophyll biosynthesis. There is speculation that the additional BchH homologs may regulate the biosynthesis of each type of chlorophyll, although the biochemical properties of the different magnesium chelatase complexes from a single species of green bacteria have not yet been compared. In this study, we investigated the activities of all three chelatase complexes from the green sulfur bacterium Chlorobaculum tepidum and interactions with the next enzyme in the pathway, magnesium protoporphyrin IX methyltransferase (BchM). Although all three chelatase complexes insert magnesium into protoporphyrin IX, the activities range by a factor of 10(5). Further, there are differences in the interactions between the BchH homologs and BchM; two of the subunits increase the methyltransferase activity by 30-60%, and the third decreases it by 30%. Expression of the chelatase complexes alone and together with BchM in Escherichia coli overproducing protoporphyrin IX suggests that the chelatase is the rate-limiting enzyme. We observed that BchM uses protoporphyrin IX without bound metal as a substrate. Our results conflict with expectations generated by previous gene inactivation studies and suggest a complex regulation of chlorophyll biosynthesis in green bacteria.     

Control of leaf and chloroplast development by the Arabidopsis gene pale cress.

Leaf plastids of the Arabidopsis pale cress (pac) mutant do not develop beyond the initial stages of differentiation from proplastids or etioplasts and contain only low levels of chlorophylls and carotenoids. Early in development, the epidermis and mesophyll of pac leaves resemble those of wild-type plants. In later stages, mutant leaves have enlarged intercellular spaces, and the palisade layer of the mesophyll can no longer be distinguished. To study the molecular basis of this phenotype, we cloned PAC and determined that this gene is regulated by light and has the capacity to encode an acidic, predominantly alpha-helical protein. The PAC gene appears to be a novel component of a light-induced regulatory network that controls the development of leaves and chloroplasts.     

Knock-out of the genes coding for the Rieske protein and the ATP-synthase delta-subunit of Arabidopsis. Effects on photosynthesis,thylakoid protein composition,and nuclear chloroplast gene expression

In Arabidopsis, the nuclear genes PetC and AtpD code for the Rieske protein of the cytochrome b(6)/f (cyt b(6)/f) complex and the delta-subunit of the chloroplast ATP synthase (cpATPase), respectively. Knock-out alleles for each of these loci have been identified. Greenhouse-grown petc-2 and atpd-1 mutants are seedling lethal, whereas heterotrophically propagated plants display a high-chlorophyll (Chl)-fluorescence phenotype, indicating that the products of PetC and AtpD are essential for photosynthesis. Additional effects of the mutations in axenic culture include altered leaf coloration and increased photosensitivity. Lack of the Rieske protein affects the stability of cyt b(6)/f and influences the level of other thylakoid proteins, particularly those of photosystem II. In petc-2, linear electron flow is blocked, leading to an altered redox state of both the primary quinone acceptor Q(A) in photosystem II and the reaction center Chl P700 in photosystem I. Absence of cpATPase-delta destabilizes the entire cpATPase complex, whereas residual accumulation of cyt b(6)/f and of the photosystems still allows linear electron flow. In atpd-1, the increase in non-photochemical quenching of Chl fluorescence and a higher de-epoxidation state of xanthophyll cycle pigments under low light is compatible with a slower dissipation of the transthylakoid proton gradient. Further and clear differences between the two mutations are evident when mRNA expression profiles of nucleus-encoded chloroplast proteins are considered, suggesting that the physiological states conditioned by the two mutations trigger different modes of plastid signaling and nuclear response.     

Effects of photoproducts on the binding properties of protoporphyrin IX to proteins

Photosensitisers are the photoactive molecules used in photodynamic therapy (PDT) of cancer. Despite the importance of their interaction with polypeptides, only the binding to plasma proteins has been investigated in some detail. In our study we compared the binding of Protoporphyrin IX (a clinically useful photosensitiser) to an immunoglobulin G, with the binding to albumins. Binding to IgG is relevant because a possible method of increasing tumour specificity of photosensitisers is to bind them to tumour-specific antibodies. Binding constants to albumins and the immunoglobulin were comparable ( congruent with6 x 10(-6) M(-1)). The apparent number of PPIX molecules bound to each protein was also within a similar range (from 4 to 7). The absence of a shift in the emission spectrum of PPIX bound to IgG, however, indicates that either larger aggregates of PPIX bind to the immunoglobulin or that the binding site leaves PPIX exposed to the buffer. We observed that PPIX photoproducts compete with PPIX for the same binding sites. The number of PPIX molecules bound to each protein in the presence of photoproducts decreased by 50-80%. Due to the spectral overlap between PPIX and its photoproducts, the binding in the presence of photoproducts was investigated using Derivative Synchronous Fluorescence Spectroscopy (DSFS) to improve the spectral separation between chromophores in solution. We also concluded that fluorescence measurements underestimate the number of PPIX molecules binding each protein. In fact, non-linear Scatchard plots (in the case of albumin binding) by definition yield a minimum number of molecules attached to a protein. Moreover, the binding of large aggregates, formed by an unknown number of PPIX molecules, to IgG results in the underestimate of the number of molecules bound. The number of PPIX molecules bound to these proteins is also much larger than the number of sites estimated by protein fluorescence quenching.     

The plant S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase is located in both envelope and thylakoid chloroplast membranes.

Chlorophyll biosynthesis requires a metabolic dialog between the chloroplast envelope and thylakoids where biosynthetic activities are localized. Here, we report the first plant S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase (MgP(IX)MT) sequence identified in the Arabidopsis genome owing to its similarity with the Synechocystis sp. MgP(IX)MT gene. After expression in Escherichia coli, the recombinant Arabidopsis thaliana cDNA was shown to encode a protein having MgP(IX)MT activity. The full-length polypeptide exhibits a chloroplast transit peptide that is processed during import into the chloroplast. The mature protein contains two functional regions. The C-terminal part aligns with the Synechocystis full-length protein. The corresponding truncated region binds to Ado-met, as assayed by UV crosslinking, and is shown to harbor the MgP(IX)MT activity. Downstream of the cleaved transit peptide, the 40 N-terminal amino acids of the mature protein are very hydrophobic and enhance the association of the protein with the membrane. In A. thaliana and spinach, the MgP(IX)MT protein has a dual localization in chloroplast envelope membranes as well as in thylakoids. The protein is active in each membrane and has the same apparent size corresponding to the processed mature protein. The protein is very likely a monotopic membrane protein embedded within one leaflet of the membrane as indicated by ionic and alkaline extraction of each membrane. The rationale for a dual localization of the protein in the chloroplast is discussed.     

Introduction of a new branchpoint in tetrapyrrole biosynthesis in Escherichia coli by co-expression of genes encoding the chlorophyll-specific enzymes magnesium chelatase and magnesium protoporphyrin methyltransferase.

The genes encoding the three Mg chelatase subunits, ChlH, ChlI and ChlD, from the cyanobacterium Synechocystis PCC6803 were all cloned in the same pET9a-based Escherichia coli expression plasmid, forming an artificial chlH-I-D operon under the control of the strong T7 promoter. When a soluble extract from IPTG-induced E. coli cells containing the pET9a-ChlHID plasmid was assayed for Mg chelatase activity in vitro, a high activity was obtained, suggesting that all three subunits are present in a soluble and active form. The chlM gene of Synechocystis PCC6803 was also cloned in a pET-based E. coli expression vector. Soluble extract from an E. coli strain expressing chlM converted Mg-protoporphyrin IX to Mg-protoporphyrin monomethyl ester, demonstrating that chlM encodes the Mg-protoporphyrin methyltransferase of Synechocystis. Co-expression of the chlM gene together with the chlH-I-D construct yielded soluble protein extracts which converted protoporphyrin IX to Mg-protoporphyrin IX monomethyl ester without detectable accumulation of the Mg-protoporphyrin IX intermediate. Thus, active Mg chelatase and Mg-protoporphyrin IX methyltransferase can be coupled in E. coli extracts. Purified ChlI, -D and -H subunits in combination with purified ChlM protein were subsequently used to demonstrate in vitro that a molar ratio of ChlM to ChlH of 1 to 1 results in conversion of protoporphyrin IX to Mg-protoporphyrin monomethyl ester without significant accumulation of Mg-protoporphyrin.     

A mutation in the Arabidopsis DET3 gene uncouples photoregulated leaf development from gene expression and chloroplast biogenesis

The genetic and phenotypic characterization of a new Arabidopsis mutant, de-etiolated -3, ( det 3), involved in light-regulated seedling development is described. A recessive mutation in the DET 3 gene uncouples light signals from a subset of light-dependent processes. The det 3 mutation causes dark-grown Arabidopsis thaliana seedlings to have short hypocotyls, expanded cotyledons, and differentiated leaves, traits characteristic of light-grown seedlings. Despite these morphological changes, however, the det 3 mutant does not develop chloroplasts or show elevated expression of nuclear- and chloroplast-encoded light-regulated mRNAs. The det 3 mutation thus uncovers a downstream branch of the light transduction pathways that separates leaf development from chloroplast differentiation and light-regulated gene expression. In addition, light-grown det 3 plants have reduced stature and apical dominance, suggesting that DET3 functions during growth in normal light conditions as well. The genetic interactions between mutations in det 1, det 2, and det 3 are described. The phenotypes of doubly mutant strains suggest that there are at least two parallel pathways controlling light-mediated development in Arabidopsis .     

Mg-chelatase I subunit 1 and Mg-protoporphyrin IX methyltransferase affect the stomatal aperture in Arabidopsis thaliana

To elucidate the molecular mechanisms of stomatal opening and closure, we performed a genetic screen using infrared thermography to isolate stomatal aperture mutants. We identified a mutant designated low temperature with open-stomata 1 (lost1), which exhibited reduced leaf temperature, wider stomatal aperture, and a pale green phenotype. Map-based analysis of the LOST1 locus revealed that the lost1 mutant resulted from a missense mutation in the Mg-chelatase I subunit 1 (CHLI1) gene, which encodes a subunit of the Mg-chelatase complex involved in chlorophyll synthesis. Transformation of the wild-type CHLI1 gene into lost1 complemented all lost1 phenotypes. Stomata in lost1 exhibited a partial ABA-insensitive phenotype similar to that of rtl1, a Mg-chelatase H subunit missense mutant. The Mg-protoporphyrin IX methyltransferase (CHLM) gene encodes a subsequent enzyme in the chlorophyll synthesis pathway. We examined stomatal movement in a CHLM knockdown mutant, chlm, and found that it also exhibited an ABA-insensitive phenotype. However, lost1 and chlm seedlings all showed normal expression of ABA-induced genes, such as RAB18 and RD29B, in response to ABA. These results suggest that the chlorophyll synthesis enzymes, Mg-chelatase complex and CHLM, specifically affect ABA signaling in the control of stomatal aperture and have no effect on ABA-induced gene expression.     

The enzymic conversion of protoporphyrinogen IX to protoporphyrin IX in mammalian mitochondria.

Protoporphyrinogen oxidase, an enzyme which catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX in yeast cells, has been found in several mammalian tissues. It has been extracted from rat liver mitochondria by sonication in the presence of salt and detergent and partially purified. The enzyme is similar in many respects to yeast protoporphyrinogen oxidase. Based on its behavior on Sephadex G-200 the molecular weight of the enzyme is approximately 35,000. Catalysis by protoporphyrinogen oxidase was specific for proteoporphyrinogen IX (apparent Km of 11 muM) and proceeded maximally at pH 8.6 to 8.7. The effect of temperature on enzyme activity plotted according to Arrhenius gave a value of E of 9,100 calories per mol. Enzyme activity was inhibited in the presence of high salt concentrations and temperatures above 45 degrees. Oxygen was essential for protoporphyrinogen oxidase activity and an alternative elevtron acceptor has not yet been found. No requirement for a metal or other cofactor could be demonstrated. The presence of monothiol groups was indicated; however, it is not known whether the thiol groups are involved directly in the binding of substrate to the enzyme.     

Ferrochelatase activity and protoporphyrin IX utilization in Haemophilus influenzae.

Previous research showed that the heme-requiring human pathogen Haemophilus influenzae lacks the first six of the seven enzymes required for heme synthesis, starting with the precursor, 5-amino levulinic acid. In this study, I demonstrated either directly or by reasonable inference that all 57 strains of H. influenzae examined, including 2 unable to grow on protoporphyrin IX, possess ferrochelatase, which catalyzes heme formation by insertion of Fe2+ into the protoporphyrin IX nucleus and which is the last enzyme in the heme synthetic pathway. Further, I showed that this enzyme can also function in the reverse direction, releasing Fe2+ from heme.     

Role of Arabidopsis CHL27 protein for photosynthesis, chloroplast development and gene expression profiling

In Chl biosynthesis, aerobic Mg-protoporphyrin IX monomethyl ester (MPE) cyclase is a key enzyme involved in the synthesis of protochlorophyllide a, and its membrane-bound component is known to be encoded by homologs of CHL27 in photosynthetic bacteria, green algae and plants. Here, we report that the Arabidopsis chl27-t knock-down mutant exhibits retarded growth and chloroplast developmental defects that are caused by damage to PSII reaction centers. The mutant contains a T-DNA insertion within the CHL27 promoter that dramatically reduces the CHL27 mRNA level. chl27-t mutant plants grew slowly with a pale green appearance, suggesting that they are defective in Chl biosynthesis. Chl fluorescence analysis showed significantly low photosynthetic activity in chl27-t mutants, indicating damage in their PSII reaction centers. The chl27-t mutation also conferred severe defects in chloroplast development, including the unstacking of thylakoid membranes. Microarray analysis of the chl27-t mutant showed repression of numerous nuclear genes involved in photosynthesis, including those encoding components of light-harvesting complex I (LHCI) and LHCII, and PSI and PSII, which accounts for the defects in photosynthetic activity and chloroplast development. In addition, the microarray data also revealed the significant repression of genes such as PORA and AtFRO6 for Chl biosynthesis and iron acquisition, respectively, and, furthermore, implied that there is cross-talk in the Chl biosynthetic pathway among the PORA, AtFRO6 and CHL27 proteins.     

Fluorescence spectroscopy study of protoporphyrin IX metabolism level in cells.

Fluorescence spectroscopy was used to study the protoporphyrin IX (PpIX) metabolism level in chick embryos during the cell proliferation process. The emission spectra were measured for PpIX bound to albumin from nonincubated and incubated eggs. The relative characteristic emission intensity of PpIX was used to determine the level of PpIX metabolism as a function of the embryonic development time. This technique might be used to estimate tumor development time.     

Accumulation of protoporphyrin IX in light-sensitive mutants of Escherichia coli.

The accumulation of protoporphyrin IX (Proto IX) in light-sensitive mutants of Escherichia coli was detected by spectrofluorimetry. Fluorescence emission and excitation spectra were recorded from extracts of bacterial cells. Proto IX clearly accumulated in cells with mutations in the visA (hemH) gene but not in the wild-type strain CA274 or in visA mutants that had been rendered light-resistant by introduction of the wild-type visA⁺ gene. Accumulation of Proto IX was also not observed in cells with a mutation in the visB gene. These results confirm the hypothesis that the sensitivity of the visA mutants to light is caused by the abnormal accumulation of Proto IX, a substrate of ferrochelatase, as the result of a genetic defect in the gene for ferrochelatase.     

An Arabidopsis rhomboid protease has roles in the chloroplast and in flower development

Increasing numbers of cellular pathways are now recognized to be regulated via proteolytic processing events. The rhomboid family of serine proteases plays a pivotal role in a diverse range of pathways, activating and releasing proteins via regulated intramembrane proteolysis. The prototype rhomboid protease, rhomboid-1 in Drosophila, is the key activator of epidermal growth factor (EGF) receptor pathway signalling in the fly and thus affects multiple aspects of development. The role of the rhomboid family in plants is explored and another developmental phenotype, this time in a mutant of an Arabidopsis chloroplast-localized rhomboid, is reported here. It is confirmed by GFP-protein fusion that this protease is located in the envelope of chloroplasts and of chlorophyll-free plastids elsewhere in the plant. Mutant plants lacking this organellar rhomboid demonstrate reduced fertility, as documented previously with KOM-the one other Arabidopsis rhomboid mutant that has been reported in the literature-along with aberrant floral morphology.     

Dual‐wavelength excitation for fluorescence‐based quantification of zinc protoporphyrin IX and protoporphyrin IX in whole blood

Quantification of erythrocyte zinc protoporphyrin IX (ZnPP) and protoporphyrin IX (PPIX), individually or jointly, is useful for the diagnostic evaluation of iron deficiency, iron‐restricted erythropoiesis, lead exposure, and porphyrias. A method for simultaneous quantification of ZnPP and PPIX in unwashed blood samples is described, using dual‐wavelength excitation to effectively eliminate background fluorescence from other blood constituents. In blood samples from 35 subjects, the results of the dual‐wavelength excitation method and a reference high performance liquid chromatography (HPLC) assay were closely correlated both for ZnPP (r_s = 0.943, p < 0.0001; range 37–689 μmol ZnPP/mol heme, 84–1238 nmol/L) and for PPIX (r_s = 0.959, p < 0.0001; range 42–4212 μmol PPIX/mol heme, 93–5394 nmol/L). In addition, for ZnPP, the proposed method is compared with conventional single‐wavelength excitation and with commercial front‐face fluorimetry of washed erythrocytes and whole blood. We hypothesize that dual‐wavelength excitation fluorimetry will provide a new approach to the suppression of background fluorescence in blood and tissue measurements of ZnPP and PPIX. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)