Properties of Magnesium Chelatase in Greening Etioplasts: METAL ION SPECIFICITY AND EFFECT OF SUBSTRATE CONCENTRATIONS

Evidence was obtained by means of low temperature spectrofluorimetry and high pressure liquid chromatography for the exclusive chelation of Mg²⁺ into protoporphyrin IX by a plastid preparation extracted from greening cucumber cotyledons. Under the experimental conditions which were optimized for Mg²⁺ chelation, Zn²⁺ chelation was not detected. However, Zn²⁺ chelation was observed when ATP was omitted and ZnCl₂ was added to the incubation mixture. The observed Zn²⁺ chelation was partially nonenzymic. The requirements for the enzyme, Mg chelatase, are discussed in terms of the regulatory function of this enzyme.     

Characterization of the binding of deuteroporphyrin IX to the magnesium chelatase H subunit and spectroscopic properties of the complex

Magnesium protoporphyrin chelatase catalyzes the insertion of a Mg(2+) ion into protoporphyrin IX, which can be considered as the first committed step of (bacterio)chlorophyll synthesis. In the present work, the Mg chelatase H subunits from both Synechocystis and Rhodobacter sphaeroides were studied because of the differing requirements of these organisms for modified cyclic tetrapyrroles. Deuteroporphyrin was shown to be a substrate for Mg chelatase. Analytical HPLC gel filtration was used to show that an H-deuteroporphyrin complex can be reconstituted by incubating the magnesium chelatase H subunit with a molar excess of deuteroporphyrin and that these complexes are monomers. The binding process occurs in the absence of Mg(2+) or ATP or the I or D subunits of Mg chelatase. The emission from Trp residues in the H subunit is partly quenched when deuteroporphyrin is bound. Quantitative analysis of Trp fluorescence quenching led to determination of the K(d) values for deuteroporphyrin binding to BchH from Rb. sphaeroides and ChlH from Synechocystis, which are 1.22 +/- 0.42 microM and 0.53 +/- 0.12 microM for ChlH and BchH, respectively. In the case of ChlH, but not BchH, the K(d) increased 4-fold in the presence of MgATP(2-). Red shifts in absorbance and excitation peaks were observed in the B band of the bound porphyrin in comparison with deuteroporphyrin in solution, as well as reduced yield and red shifts of up to 8 nm in fluorescence emission. These alterations are consistent with a slightly deformed nonplanar conformation of the bound porphyrin. Mg deuteroporphyrin, the product of the Mg chelation reaction, was shown to form a complex with either ChlH or BchH; in each case the K(d) for Mg deuteroporphyrin is similar to that for deuteroporphyrin. The implications of the H-Mg protoporphyrin interaction for the next enzyme in the chlorophyll biosynthetic pathway, Mg protoporphyrin methyltransferase, are discussed.     

Zinc chelatase in human lymphocytes: detection of the enzymatic defect in erythropoietic protoporphyria

We describe a fluorometric assay for heme synthetase, the enzyme that is genetically deficient in erythropoietic protoporphyria. The method, which can readily detect activity in 1 microliter of packed human lymphocytes, is based on the formation of zinc protoheme from protoporphyrin IX. That zinc chelatase and ferrochelatase activities reside in the same enzyme was shown by the competitive action of ferrous ions and the inhibitory effects of N-methyl protoporphyrin (a specific inhibitor of heme synthetase) on zinc chelatase. The Km for zinc was 11 micrograms and that for protoporphyrin IX was 6 microM. The Ki fro ferrous ions was 14 microM. Zinc chelatase was reduced to 15.3% of the mean control activity in lymphocytes obtained from patients with protoporphyria, thus confirming the defect of heme biosynthesis in this disorder. The assay should prove to be useful for determining heme synthetase in tissues with low specific activity and to investigate further the enzymatic defect in protoporphyria.     

Inactivation of Mg chelatase during transition from anaerobic to aerobic growth in Rhodobacter capsulatus

The facultative photosynthetic bacterium Rhodobacter capsulatus can adapt from an anaerobic photosynthetic mode of growth to aerobic heterotrophic metabolism. As this adaptation occurs, the cells must rapidly halt bacteriochlorophyll synthesis to prevent phototoxic tetrapyrroles from accumulating, while still allowing heme synthesis to continue. A likely control point is Mg chelatase, the enzyme that diverts protoporphyrin IX from heme biosynthesis toward the bacteriochlorophyll biosynthetic pathway by inserting Mg(2+) to form Mg-protoporphyrin IX. Mg chelatase is composed of three subunits that are encoded by the bchI, bchD, and bchH genes in R. capsulatus. We report that BchH is the rate-limiting component of Mg chelatase activity in cell extracts. BchH binds protoporphyrin IX, and BchH that has been expressed and purified from Escherichia coli is red in color due to the bound protoporphyrin IX. Recombinant BchH is rapidly inactivated by light in the presence of O(2), and the inactivation results in the formation of a covalent adduct between the protein and the bound protoporphyrin IX. When photosynthetically growing R. capsulatus cells are transferred to aerobic conditions, Mg chelatase is rapidly inactivated, and BchH is the component that is most rapidly inactivated in vivo when cells are exposed to aerobic conditions. The light- and O(2)-stimulated inactivation of BchH could account for the rapid inactivation of Mg chelatase in vivo and provide a mechanism for inhibiting the synthesis of bacteriochlorophyll during adaptation of photosynthetically grown cells to aerobic conditions while still allowing heme synthesis to occur for aerobic respiration.     

Characterization of the Late Steps of Microbial Heme Synthesis: Conversion of Coproporphyrinogen to Protoporphyrin

Cell-free extracts of various cytochrome-containing, heterotrophic microorganisms were examined for ability to convert coproporphyrinogen to protoporphyrin. Extracts of Escherichia coli and Pseudomonas denitrificans readily accumulated large amounts of protoporphyrin when assayed under aerobic conditions. However, protoporphyrin did not accumulate under either aerobic or anaerobic conditions of assay or in the presence of various supplements in extracts of the aerobe Micrococcus lysodeikticus, the facultative anaerobe Staphylococcus aureus, or the anaerobe Vibrio succinogenes. Protoporphyrin also accumulated when extracts of E. coli and P. denitrificans were incubated aerobically with the early heme precursor, delta-amino levulinic acid (ALA). This protoporphyrin accumulation was markedly stimulated by the iron chelator, o-phenanthroline. Extracts of S. aureus and M. lysodeikticus accumulated coproporphyrin, but not protoporphyrin when incubated with ALA. The enzyme system in extracts of E. coli which converts coproporphyrinogen to protoporphyrin under aerobic conditions of assay was also partially characterized. This conversion was stimulated by the iron chelator, o-phenanthroline, the respiratory inhibitor, cyanide, and the reducing agent, thioglycolate. Dialysis of the extract did not diminish enzyme activity. Certain alternate electron acceptors and nitrite caused a marked inhibition of the conversion. These results indicate that this late step in heme synthesis, the conversion of coproporphyrinogen to protoporphyrin, can be readily demonstrated in extracts of some, but not all, cytochrome-containing bacteria and that the aerobic conversion in E. coli exhibits many characteristics similar to those demonstrated for the aerobic conversion previously studied in liver mitochondria.     

Magnesium-dependent ATPase activity and cooperativity of magnesium chelatase from Synechocystis sp. PCC6803

The first committed step in chlorophyll biosynthesis is catalyzed by magnesium chelatase, a complex enzyme with at least three substrates, cooperative Mg(2+) activation, and free energy coupling between ATP hydrolysis and metal-ion chelation. A detailed functional study of the behavior of the intact magnesium chelatase has been performed, including characterization of magnesium cooperativity and the stoichiometry of ATP consumption in relation to the magnesium porphyrin produced. It is demonstrated that, in vitro, this catalyzed reaction requires hydrolysis of approximately 15 MgATP(2-) and that the chelation partial reaction is energetically unfavorable, under our assay conditions, with a DeltaG degrees ' of 25-33 kJ mol(-1). Given the likely metabolite concentrations in vivo, this results in the chelatase reaction operating far from equilibrium. We have also determined the steady-state kinetic behavior of the intact enzyme and have compared the kinetic parameters obtained with those observed for the partial reactions of individual subunits. K(DIX) (where D(IX) represents deuteroporphyrin IX) is estimated to be 3.20 microm, and K(MgATP)(2-) is 0.45 mm. k(cat) for chelation is estimated to be 0.8 min(-1), suggesting that the ATP hydrolysis catalyzed by the isolated ChlI subunit is substantially slower in the intact chelatase. The magnesium-rich form of the chelatase is a more effective catalyst of the chelation reaction; magnesium activation of the chelatase increases V, as well as the specificity constant for the reaction of MgATP(2-) and D(IX), possibly as a result of a magnesium-triggered conformational change.     

Magnesium insertion by magnesium chelatase in the biosynthesis of zinc bacteriochlorophyll a in an aerobic acidophilic bacterium Acidiphilium rubrum

To elucidate the mechanism for formation of zinc-containing bacteriochlorophyll a in the photosynthetic bacterium Acidiphilium rubrum, we isolated homologs of magnesium chelatase subunits (bchI, -D, and -H). A. rubrum bchI and -H were encoded by single genes located on the clusters bchP-orf168-bchI-bchD-orf320-crtI and bchF-N-B-H-L as in Rhodobacter capsulatus, respectively. The deduced sequences of A. rubrum bchI, -D, and -H had overall identities of 59. 8, 40.5, and 50.7% to those from Rba. capsulatus, respectively. When these genes were introduced into bchI, bchD, and bchH mutants of Rba. capsulatus for functional complementation, all mutants were complemented with concomitant synthesis of bacteriochlorophyll a. Analyses of bacteriochlorophyll intermediates showed that A. rubrum cells accumulate magnesium protoporphyrin IX monomethyl ester without detectable accumulation of zinc protoporphyrin IX or its monomethyl ester. These results indicate that a single set of magnesium chelatase homologs in A. rubrum catalyzes the insertion of only Mg(2+) into protoporphyrin IX to yield magnesium protoporphyrin IX monomethyl ester. Consequently, it is most likely that zinc-containing bacteriochlorophyll a is formed by a substitution of Zn(2+) for Mg(2+) at a step in the bacteriochlorophyll biosynthesis after formation of magnesium protoporphyrin IX monomethyl ester.     

Effects of iron deficiency and chronic iron overloading on mitochondrial heme biosynthetic enzymes in rat liver

The effects of iron deficiency and iron overloading on the mitochondrial enzymes involved in heme synthesis were studied in rat livers. The in vitro activities of several of the enzymes in this pathway were differentially influenced by the in vivo iron status of the animals. delta-Aminolevulinic acid synthase was slightly increased in iron-overloaded animals, but remained normal in iron-deficient animals (0.58 +/- 0.09, 0.91 +/- 0.19 and 0.61 +/- 0.12 nmol delta-aminolevulinic acid/mg per h). Copro- and protoporphyrinogen oxidase activities were increased (20 and 60% above controls) in iron-deficient animals. In contrast, coproporphyrinogen oxidase was decreased by 20%, while protoporphyrinogen oxidase remained unchanged in iron-overloaded rats. These variations of activities were not due to changes in the affinity of these enzymes toward their substrates, as coporphyrinogen had the same Km in each case (0.62 +/- 0.05 M) as did protoporphyrinogen (0.22 +/- 0.035 M). Thus, the Km did not vary with the treatment received by the animals. Ferrochelatase activity was measured by both the pyridine hemochromogen method and by measurement of zinc protoporphyrin with endogenous zinc as substrate. In all cases, ferrochelatase was found to be able to synthesize zinc protoporphyrin with endogenous zinc as substrate. However, the apparent Km of zinc chelatase for protoporphyrin was significantly different in the three groups of animals with Km,appProto, app = 2.4 +/- 0.1 10(-7), 4 +/- 0.3 10(-7) and 9.10 +/- 0.05 10(-7) M in iron-overloaded, control and iron-deficient animals, respectively. When ferrochelatase activity was measured by pyridine hemochromogen, identical results were observed in iron-deficient and control animals but decreased by 45% in iron-overloaded animals. The mitochondrial heme content was also decreased by 40% in iron-overloaded rats but unchanged in either iron-deficient or control rats.     

Kinetic analyses of the magnesium chelatase provide insights into the mechanism, structure, and formation of the complex

The metabolic pathway known as (bacterio)chlorophyll biosynthesis is initiated by magnesium chelatase (BchI, BchD, BchH). This first step involves insertion of magnesium into protoporphyrin IX (proto), a process requiring ATP hydrolysis. Structural information shows that the BchI and BchD subunits form a double hexameric enzyme complex, whereas BchH binds proto and can be purified as BchH-proto. We utilized the Rhodobacter capsulatus magnesium chelatase subunits using continuous magnesium chelatase assays and treated the BchD subunit as the enzyme with both BchI and BchH-proto as substrates. Michaelis-Menten kinetics was observed with the BchI subunit, whereas the BchH subunit exhibited sigmoidal kinetics (Hill coefficient of 1.85). The BchI.BchD complex had intrinsic ATPase activity, and addition of BchH greatly increased ATPase activity. This was concentration-dependent and gave sigmoidal kinetics, indicating there is more than one binding site for the BchH subunit on the BchI.BchD complex. ATPase activity was approximately 40-fold higher than magnesium chelatase activity and continued despite cessation of magnesium chelation, implying one or more secondary roles for ATP hydrolysis and possibly an as yet unknown switch required to terminate ATPase activity. One of the secondary roles for BchH-stimulated ATP hydrolysis by a BchI.BchD complex is priming of BchH to facilitate correct binding of proto to BchH in a form capable of participating in magnesium chelation. This porphyrin binding is the rate-limiting step in catalysis. These data suggest that ATP hydrolysis by the BchI.BchD complex causes a series of conformational changes in BchH to effect substrate binding, magnesium chelation, and product release.     

Direct measurement of metal-ion chelation in the active site of the AAA+ ATPase magnesium chelatase

Magnesium chelatase catalyzes the first committed step in chlorophyll biosynthesis. This complex enzyme has at least three substrates and couples ATP hydrolysis to the insertion of Mg2+ into protoporphyrin IX. We directly observed metal-ion chelation fluorometrically, providing the first data describing the on-enzyme reaction. We describe the transient-state kinetics of magnesium chelatase with direct observation of the evolution of an enzyme-product complex EMgDIX. We demonstrate that MgATP2- binding occurs after the rate-determining step. As nucleotide hydrolysis is essential for the overall reaction this must also occur after the rate-determining step. This provides the first evidence for the synchronization of the ATPase and chelatase pathways and suggests a mechanism where nucleotide binding acts to clamp the chelatase in a product complex. Comparison of rate constants for the slow step in the reaction with further transient kinetics under conditions where multiple turnovers can occur reveals that an additional activation step is required to explain the behavior of magnesium chelatase. These data provide a new view of the sequence of events occurring in the reaction catalyzed by magnesium chelatase.     

Kinetic studies of human liver ferrochelatase. Role of endogenous metals

Ferrochelatase activity in human liver has been extensively characterized in the mitochondrial fraction by kinetic study of the enzyme in initial velocity conditions. We found that human liver mitochondrial membranes contain large amounts of endogenous metals that are substrates for the enzyme, leading to a lack of linearity of the activity as function of protein concentration. This lack of linearity is mainly due to a high zinc-chelatase activity with endogenous zinc. Under optimal experimental conditions, the maximum velocity for iron incorporation was 8.7 nmol of protoheme/h/mg of protein, and the maximum velocity for zinc incorporation was 4.3 nmol of zinc-protoporphyrin/h/mg of protein. The Michaelis constant for protoporphyrin IX was (i) dependent on the amount of protein when the overall chelatase reactions were measured but (ii) independent of the amount of protein when only zinc-chelatase activity was measured (Km = 0.5 microM). The Michaelis constants for iron and zinc were 0.35 and 0.08 microM, respectively, and the inhibitory constants for competitive incorporation of iron and zinc were KIFe/Zn = 0.12 microM and KIZn/Fe = 0.58 microM. The affinity of the enzyme for zinc lowers the actual determination of ferrochelatase activity with iron as substrate. Furthermore, when measuring ferrochelatase (e.g. in liver biopsy), endogenous zinc content in the biological sample must be taken into account.     

Nickel(II) chelatase variants directly evolved from murine ferrochelatase: porphyrin distortion and kinetic mechanism

The heme biosynthetic pathway culminates with the ferrochelatase-catalyzed ferrous iron chelation into protoporphyrin IX to form protoheme. The catalytic mechanism of ferrochelatase has been proposed to involve the stabilization of a nonplanar porphyrin to present the pyrrole nitrogens to the metal ion substrate. Previously, we hypothesized that the ferrochelatase-induced nonplanar distortions of the porphyrin substrate impose selectivity for the divalent metal ion incorporated into the porphyrin ring and facilitate the release of the metalated porphyrin through its reduced affinity for the enzyme. Using resonance Raman spectroscopy, the structural properties of porphyrins bound to the active site of directly evolved Ni(2+)-chelatase variants are now examined with regard to the mode and extent of porphyrin deformation and related to the catalytic properties of the enzymes. The Ni(2+)-chelatase variants (S143T, F323L, and S143T/F323L), which were directly evolved to exhibit an enhanced Ni(2+)-chelatase activity over that of the parent wild-type ferrochelatase, induced a weaker saddling deformation of the porphyrin substrate. Steady-state kinetic parameters of the evolved variants for Ni(2+)- and Fe(2+)-chelatase activities increased compared to those of wild-type ferrochelatase. In particular, the reduced porphyrin saddling deformation correlated with increased catalytic efficiency toward the metal ion substrate (Ni(2+) or Fe(2+)). The results lead us to propose that the decrease in the induced protoporphyrin IX saddling mode is associated with a less stringent metal ion preference by ferrochelatase and a slower porphyrin chelation step.     

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.     

ATPase activity of magnesium chelatase subunit I is required to maintain subunit D in vivo.

During biosynthesis of chlorophyll, Mg(2+) is inserted into protoporphyrin IX by magnesium chelatase. This enzyme consists of three different subunits of approximately 40, 70 and 140 kDa. Seven barley mutants deficient in the 40 kDa magnesium chelatase subunit were analysed and it was found that this subunit is essential for the maintenance of the 70 kDa subunit, but not the 140 kDa subunit. The 40 kDa subunit has been shown to belong to the family of proteins called "ATPases associated with various cellular activities", known to form ring-shaped oligomeric complexes working as molecular chaperones. Three of the seven barley mutants are semidominant mis-sense mutations leading to changes of conserved amino acid residues in the 40 kDa protein. Using the Rhodobacter capsulatus 40 and 70 kDa magnesium chelatase subunits we have analysed the effect of these mutations. Although having no ATPase activity, the deficient 40 kDa subunit could still associate with the 70 kDa protein. The binding was dependent on Mg(2+) and ATP or ADP. Our study demonstrates that the 40 kDa subunit functions as a chaperon that is essential for the survival of the 70 kDa subunit in vivo. We conclude that the ATPase activity of the 40 kDa subunit is essential for this function and that binding between the two subunits is not sufficient to maintain the 70 kDa subunit in the cell. The ATPase deficient 40 kDa proteins fail to participate in chelation in a step after the association of the 40 and 70 kDa subunits. This step presumably involves a conformational change of the complex in response to ATP hydrolysis.     

Lipoprotein Oxidation and Induction of Ferroxidase Activity in Stored Human Extracellular Fluids

It was observed that during the storage of human extracellular fluids at – 20°C the azide-inhibitable ferroxidase activity of caeruloplasmin declined, whilst a new azide-resistant ferroxidase activity (ARFA) developed. The literature suggested that storage-induced ARFA might be due to either a poorly defined enzymatic activity of a low density lipoprotein (LDL) or to lipid peroxides formed within the different lipoprotein fractions. To study this further, the major lipoprotein classes were separated from human serum by density gradient centrifugation. After storage of the lipoprotein fractions, it was found that the LDL fraction had the highest specific activity of ARFA and the highest content of lipid peroxidation products, as assessed by diene conjugates. The ARFA of LDL correlated with its content of diene conjugates and TBA reactive material, which initially suggested that the Fe(II) oxidising activity of peroxidised LDL arose from the reduction of peroxides by Fe(II) in the classical reaction between the metal ion and free radical reduction of lipid peroxides. However. steady state kinetic analysis indicated an enzymic role of LDL in Fe(II) oxidation, with lipid peroxides acting as a substrate for the enzyme. These results indicate that LDL may contain a peroxidase activity. catalysing the oxidation of Fe(II) by lipid peroxides, as well as a ferrous oxidase activity where O₂ is the oxidising substrate.     

CDP-diacylglycerol synthesis in rat liver mitochondria.

CDP-diacylglycerol for polyglycerophosphatide biogenesis can be synthesized within rat liver mitochondria. This membrane-associated enzyme was predominantly located in the inner mitochondrial membrane. GTP had a significant effect in activating the microsomal CDP-diacylglycerol synthase, especially if the microsomes were preincubated with GTP in the presence of phosphatidic acid. This stimulatory effect of GTP on the microsomal enzyme was not detected in the mitochondrial fractions. The enzymes could be solubilized from the membrane fractions using CHAPS, and the detergent-soluble activity partially restored by addition of phospholipids. Mitochondrial and microsomal CDP-diacylglycerol synthase activity could be completely separated by anion-exchange column chromatography. The mitochondrial and microsomal CDP-diacylglycerol synthases appear to be two distinct enzymes with different localization and regulatory characteristics.     

Inhibition of membrane erythrocyte (Ca2+ + Mg2+)-ATPase by hemin

Red blood cell lysis is a common symptom following severe or prolonged oxidative stress. Oxidative processes occur commonly in sickle cells, probably mediated through denatured hemoglobin and the accumulation of ferric hemes in the membranes. Calmodulin-stimulated (Ca2+ + Mg2+)-ATPase from sickle red cell membranes is partially inactivated (Leclerc et al. (1987) Biochim. Biophys. Acta 897, 33-40). In this study (Ca2+ + Mg2+)-ATPase activity from normal adult erythrocyte membranes was measured in the presence of hemin. We report a time- and concentration-dependent inhibition of the activity of the enzyme by hemin due to a decrease in the maximum velocity. Only a mild inhibitory effect was observed in the presence of iron-free protoporphyrin IX, indicating the catalytic influence of the iron. Experiments carried out with hemin (ferric iron) liganded with imidazole or with reduced protoheme (ferrous iron) liganded with carbon monoxide, demonstrated that the inhibition requires that hemin be capable of binding additional ligands. The inhibition was not influenced by the absence of oxygen but was prevented by addition of bovine serum albumin. Addition of butylated hydroxytoluene, a protective agent of lipid peroxidation, failed to prevent the inhibition of calmodulin-stimulated (Ca2+ + Mg2+)-ATPase. As dithiothreitol partially restores the enzyme activity, we postulated that hemin interacts with the thiol groups of the enzyme.     

Decreased and increased expression of the subunit CHL I diminishes Mg chelatase activity and reduces chlorophyll synthesis in transgenic tobacco plants

The chelation of Fe2+ and Mg2+ ions forms protoheme IX and Mg-protoporphyrin IX, respectively, and the latter is an intermediate in chlorophyll synthesis. Active magnesium protoporphyrin IX chelatase (Mg-chelatase) is an enzyme complex consisting of three different subunits. To investigate the function of the CHL I subunit of Mg-chelatase and the effects of modified Mg-chelatase activity on the tetrapyrrole biosynthetic pathway, we characterized N. tabacum transformants carrying gene constructs with the Chl I cDNA sequence in antisense and sense orientation under the control of the CaMV 35S promoter. Both elevated and diminished levels of Chl I mRNA and Chl I protein led to reduced Mg-chelatase activities, reflecting a perturbation of the assembly of the enzyme complex. The transformed plants did not accumulate the substrate of Mg-chelatase, protoporphyrin IX, but the leaves contained less chlorophyll and possessed increased chlorophyll a/b ratios, as well as a deficiency of light-harvesting chlorophyll binding proteins of photosystems I and II. The expression and activity of several tetrapyrrolic enzymes were reduced in parallel to lower the Mg-chelatase activity. Consistent with the lower chlorophyll contents, the rate-limiting synthesis of 5-aminolevulinate was also decreased in the transgenic lines analyzed. The consequence of reduced Mg-chelatase on early and late steps of chlorophyll synthesis, and on the organization of light harvesting complexes is discussed.     

Unraveling the substrate-metal binding site of ferrochelatase: an X-ray absorption spectroscopic study

Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, catalyzes the insertion of ferrous iron into the protoporphyrin IX ring. Ferrochelatases can be arbitrarily divided into two broad categories: those with and those without a [2Fe-2S] center. In this work we have used X-ray absorption spectroscopy to investigate the metal ion binding sites of murine and Saccharomyces cerevisiae (yeast) ferrochelatases, which are representatives of the former and latter categories, respectively. Co(2+) and Zn(2+) complexes of both enzymes were studied, but the Fe(2+) complex was only studied for yeast ferrochelatase because the [2Fe-2S] center of the murine enzyme interferes with the analysis. Co(2+) and Zn(2+) binding to site-directed mutants of the murine enzyme were also studied, in which the highly conserved and potentially metal-coordinating residues H207 and Y220 were substituted by residues that should not coordinate metal (i.e., H207N, H207A, and Y220F). Our experiments indicate four-coordinate zinc with Zn(N/O)(3)(S/Cl)(1) coordination for the yeast and Zn(N/O)(2)(S/Cl)(2) coordination for the wild-type murine enzyme. In contrast to zinc, a six-coordinate site for Co(2+) coordinated with oxygen or nitrogen was present in both the yeast and murine (wild-type and mutated) enzymes, with evidence of two histidine ligands in both. Like Co(2+), Fe(2+) bound to yeast ferrochelatase was coordinated by approximately six oxygen or nitrogen ligands, again with evidence of two histidine ligands. For the murine enzyme, mutation of both H207 and Y220 significantly changed the spectra, indicating a likely role for these residues in metal ion substrate binding. This is in marked disagreement with the conclusions from X-ray crystallographic studies of the human enzyme, and possible reasons for this are discussed.