Identification of porphyrin present in apo-cytochrome c oxidase of copper-deficient yeast cells

Heme a was not detected either in mitochondria isolated from copper-deficient yeast or in the intact cells. Nevertheless, the intracellular concentration of free porphyrins indicated that the pathway of porphyrin and heme synthesis was not impaired in copper-deficient cells. The immunoprecipitated apo-oxidase from copper-deficient cells revealed an absorption spectrum with maxima at 645, 592, 559, 519 and 423 nm, similar to that of purified porphyrin a. When solubilized mitochondria from [³H]leucine and δ-amino[¹⁴C]levulinic acid-labeled copper-deficient yeast cells were incubated with rabbit antiserum against cytochrome c oxidase, a precipitate was obtained. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of this immunoprecipitate showed [³H]leucine associated with six bands and δ-amino[¹⁴C]levulinic acid resolved in a single band. HCl fractionation of copper-deficient mitochondria labeled with δ-amino[¹⁴C]levulinic acid showed a high specific radioactivity in the fraction extracted by 20% HCl, a solvent which extracts porphyrin a. Thinlayer chromatography of the radioactivity found in 20% HCl showed an R_F value identical to that of purified porphyrin a. When δ-amino[³H]levulinic acid-labeled, copper-deficient yeast cells are grown in copper-supplemented medium, the porphyrin a accumulated in copper-deficient cells wa converted into heme a, and this conversion was prevented by cycloheximidine.These observations suggest that porphyrin a is present in the apo-oxidase of copper-deficient cells, but that the conversion to heme a does not occur. This conversion reaction appears to be a point in the biosynthetic pathway of cytochrome c oxidase which is blocked by copper deficieny.     

Reaction of hemoglobin with HOCl: mechanism of heme destruction and free iron release

Hypochlorous acid (HOCl) is generated by myeloperoxidase using chloride and hydrogen peroxide as substrates. HOCl and its conjugate base (OCl⁻) bind to the heme moiety of hemoglobin (Hb) and generate a transient ferric species whose formation and decay kinetics indicate it can participate in protein aggregation and heme destruction along with subsequent free iron release. The oxidation of the Hb heme moiety by OCl⁻ was accompanied by marked heme destruction as judged by the decrease in and subsequent flattening of the Soret absorbance peak at 405 nm. HOCl-mediated Hb heme depletion was confirmed by HPLC analysis and in-gel heme staining. Exposure of Hb to increasing concentrations of HOCl produced a number of porphyrin degradation products resulting from oxidative cleavage of one or more of the carbon-methene bridges of the tetrapyrrole ring, as identified by their characteristic HPLC fluorescence and LC-MS. A nonreducing denaturing SDS-PAGE showed several degrees of protein aggregation. Similarly, porphyrin degradation products were identified after exposure of red blood cells to increasing concentrations of HOCl, indicating biological relevance of this finding. This work provides a direct link between Hb heme destruction and subsequent free iron accumulation, as occurs under inflammatory conditions where HOCl is formed in substantial amounts.     

Recovery of erythropoietin from anemic sheep plasma

A method is described for the large-scale preparation of erythropoietin from anemic sheep plasma. DEAE-cellulose and carboxymethylcellulose column chromatography was used to prepare Step II erythropoietin. A total of 168 sheep yielded 499 liters of plasma from which 323,000 IU of Step II erythropoietin was obtained.     

Porphyrinogenic effects in chick embryo liver cell culture of chloramphenicol analogues that are mechanism-based inactivators of cytochrome P-450

Structural analogues of chloramphenicol (CAP) cause mechanism-based inactivation of rat liver cytochrome P-450 (P450) either via protein acylation or destruction of the heme prosthetic group. The goal of the present work was to determine whether CAP analogues that cause loss of the P450 heme moiety also cause porphyrin accumulation in chick embryo liver cell culture. The porphyrin profiles produced by exposure of cells to CAP analogues (160 microM) were determined by high-performance liquid chromatography with fluorescence detection. Of three CAP analogues that do not cause loss of the heme moiety of rat liver P450IIB1, two dichloroacetamides were not porphyrinogenic. The third compound, a chlorofluoroacetamide, caused porphyrin accumulation. This result may be due to the presence of P450 isozymes in chick embryo hepatocytes, distinct from rat liver P450IIB1, that are susceptible to destruction by this analogue. Of four CAP analogues that inactivate rat liver P450IIB1 with concomitant heme loss, a dichloroacetamide and two chlorofluoroacetamides caused porphyrin accumulation. The remaining compound, a monochloroacetamide, was not porphyrinogenic, perhaps because the P450 apoprotein cannot be reconstituted with fresh heme drawn from the regulatory "free heme pool" following inactivation by this analogue. Alternatively, there may be no P450 isozyme in chick embryo liver cell culture that is susceptible to inactivation by this compound.     

Proteasome inhibition and oxidative reactions disrupt cellular homeostasis during heme stress

Dual control of cellular heme levels by extracellular scavenger proteins and degradation by heme oxygenases is essential in diseases associated with increased heme release. During severe hemolysis or rhabdomyolysis, uncontrolled heme exposure can cause acute kidney injury and endothelial cell damage. The toxicity of heme was primarily attributed to its pro-oxidant effects; however additional mechanisms of heme toxicity have not been studied systematically. In addition to redox reactivity, heme may adversely alter cellular functions by binding to essential proteins and impairing their function. We studied inducible heme oxygenase (Hmox1)-deficient mouse embryo fibroblast cell lines as a model to systematically explore adaptive and disruptive responses that were triggered by intracellular heme levels exceeding the homeostatic range. We extensively characterized the proteome phenotype of the cellular heme stress responses by quantitative mass spectrometry of stable isotope-labeled cells that covered more than 2000 individual proteins. The most significant signals specific to heme toxicity were consistent with oxidative stress and impaired protein degradation by the proteasome. This ultimately led to an activation of the response to unfolded proteins. These observations were explained mechanistically by demonstrating binding of heme to the proteasome that was linked to impaired proteasome function. Oxidative heme reactions and proteasome inhibition could be differentiated as synergistic activities of the porphyrin. Based on the present data a novel model of cellular heme toxicity is proposed, whereby proteasome inhibition by heme sustains a cycle of oxidative stress, protein modification, accumulation of damaged proteins and cell death.Free heme can accumulate in hemolytic conditions during rhabdomyolysis and locally in wounded or inflamed tissues.¹ The concentration of free heme in the extracellular space and within cells must be controlled within a narrow homeostatic range to avoid cytotoxicity and tissue damage caused by heme stress.²Extracellular release from hemoproteins, cellular uptake, and intracellular metabolism determine the cumulative exposure of cells and tissues to heme.¹ The hemoglobin (Hb) and heme scavenger proteins haptoglobin and hemopexin restrict the accumulation of free heme within the extracellular space and prevent uncontrolled translocation into susceptible cells.^3,4 Within cells, heme is continuously degraded by heme oxygenases (Hmox).^{5, 6, 7, 8} The heme oxygenase system includes the constitutively expressed Hmox2 and inducible Hmox1 that is induced by acute increases in cellular heme such as during exogenous heme exposure.⁹ Cellular heme toxicity can result if excessive extracellular release exceeds the metabolic heme degradation capacity or if Hmox activity is inadequately low, such as that observed in rare conditions associated with loss-of-function mutations in the Hmox1 gene.¹⁰Several mechanisms of heme-triggered cell damage have been explored previously, with a focus on oxidative processes that can be catalyzed by free heme as well as on the activation of innate immunity receptors by the porphyrin.^{3,11, 12, 13, 14, 15, 16, 17} However, there is limited understanding of the ‘metabolic'' disruption that occurs in cells when intracellular free heme exceeds homeostatic levels and causes toxicity. To identify novel mechanisms of heme-triggered cell damage, we systematically explored heme-driven deviations of the cellular proteome phenotype and their underlying molecular mechanisms.The primary signals that consistently appeared throughout our studies suggested that secondary to oxidative processes, the dysfunction of cellular protein homeostasis was the most important component of heme toxicity. These effects could be traced mechanistically to an inhibitory function of the porphyrin in the principal cellular protein degradation machinery: the proteasome.     

Early labeled heme synthesis in normal rats and rats with iron deficiency anemia

Heme synthesis from [2-¹⁴C]glycine was studied in liver and red blood cells. In normal rats liver contained two early [¹⁴C] heme peaks maximal at 1 and 4.5 h, followed by a long plateau of heme labeling. These phases were present in both microsomes and mitochondria. Cycloheximide suppressed formation of the first but not the second heme component. All phases of hepatic heme labelling were reduced in iron-deficient rats, with better preservation of the microsomal fraction. In iron-deficient rats responding to iron therapy, the first peak merged with an enlarged and premature second component; the increase was most marked in mitochondria. Thus, labeled heme metabolism was less perturbed in microsomes than mitochondria in both of these conditions. Peripheral blood also contained a [¹⁴C]heme peak at 1 h in all experimental groups. This was highest with the increased eythroid response observed in irontreated rats. The first heme peak, present in both hepatic and erythroid cells, may represent a pool of free or unassigned heme. The later heme component may reflect formation of hemoproteins, which could be related directly or indirectly to the initial, rapid turnover heme component.     

Hemoglobin switching in sheep: commitment of erythroid stem cells to expression of the betaC-globin gene and accumulation of betaC-globin mRNA

The switch from HbA (α₂β₂^A) to HbC (α₂β₂^C) synthesis was induced by injection of erythropoietin into a lamb homozygous for HbA. Serial samples of bone marrow were analyzed to detect the initial commitment of erythroid stem cells (CFU-E) to form colonies which made HbC in vitro, and to detect the initial accumulation of β^C-globin mRNA and the onset of HbC synthesis in erythroblasts in vivo. CFU-E-derived erythroid colonies were formed in plasma clot culture at a low erythropoietin concentration, and the relative amounts of β^A- and β^C-globin synthesized were determined after a 24 hr pulse of ³H-leucine, added after 84 hr in culture. RNA was extracted from nuclei and cytoplasm of “early” and “late” populations of bone marrow erythroblasts which had been fractionated by Ficoll-Hypaque density centrifugation. The concentration of β^A- and β^C-globin mRNA was determined by annealing to purified synthetic DNAs (cDNAs) complementary to β^A and β^C mRNA. No β^C-globin was synthesized in erythroblasts or in CFU-E-derived erythroid colonies prior to the injection of erythropoietin. An increase in the concentration of CFU-E in the bone marrow and the appearance of β^C-globin synthesis in CFU-E-derived colonies were detected 12 hr after the erythropoietin injection. In contrast, β^C mRNA was not detected in either “early” or “late” erythroid cells until 36 hr later. The first measurable β^C-globin mRNA was accompanied by the appearance of β^C-globin synthesis in bone marrow erythroblasts. Our results suggest that the accumulation of β^C-globin mRNA is a relatively late event following induction of HbA to HbC switching by erythropoietin. The expansion of the compartment of erythroid stem cells and the commitment of CFU-E to β^C-globin synthesis appear to precede the detectable accumulation of β^C mRNA by 24–36 hr.     

The effects of selected monopyrroles on various aspects of heme biosynthesis and degradation in the rat

The effects of four monopyrroles on porphyrin biosynthesis and excretion in the rat were studied. All four compounds investigated significantly increased total urinary porphyrin excretion and hepatic porphyrin levels while the effects on fecal excretion were equivocal. Peak porphyrin production elicited by treatment with ethyl 3-acetyl-2,4-dimethylpyrrole-5-carboxylate was found to be dose dependent, as was the time of maximum excretion. The effects of 3-ethyl-5-hydroxy-4,5-dimethyl-Δ³-pyrrolin-2-one, a compound excreted in abnormally high levels in the urine of patients with hepatic porphyria, were studied in greater depth. It was found that this compound caused an increase in the activity of δ-aminolevulinic acid synthase, in vivo, which was associated with a depression of microsomal levels of heme and cytochrome P-450. This depression of heme levels could not be related to increased catabolism or nonenzymic breakdown. It is suggested that the primary effect of this and the other compounds on porphyrin metabolism is a reduction in heme formation by a mechanism at present unclear.     

Inhibition of heme synthesis induces apoptosis in human erythroid progenitor cells

Heme synthesis by erythroid progenitor cells is maintained by erythropoietin (EP), insulin-like growth factor-I (IGF-I), and stem cell factor (SCF), and without these growth factors apoptosis (programmed cell death) occurs. To clarify the possible interaction between heme synthesis and programmed cell death of human erythroid progenitor cells, the effect of specific inhibition of heme synthesis on apoptosis of highly purified human erythroid colony forming cells (ECFC) was studied. When the amount of uncleaved DNA was determined as a measure of apoptosis, the heme synthesis inhibitors, succinylacetone (SA) (0.1 mmol/L) or isonicotinic acid hydrazide (INH) (10 mmol/L), significantly decreased the amount of uncleaved DNA (P < 0.01) in the presence of erythropoietin (EP). Addition of recombinant heavy-chain ferritin (rHF) (10 nmol/L), or deprivation of transferrin from the culture medium, which decreased heme synthesis, also reduced the amount of uncleaved DNA (P < 0.01). The production of apoptosis by diverse inhibitors of heme synthesis was in each case reversed by the addition of hemin (0.1 mmol/L) and did not occur with HL-60 cells. When the colony-forming capacity of ECFC was determined by plasma clot assay, SA, INH, or rHF reduced the number of CFU-E (P < 0.01), and the effect of SA was reversed by hemin. The addition of SA did not alter the c-myc response of ECFC to EP. These data indicate that inhibition of heme synthesis induces apoptosis of human erythroid progenitor cells, in a manner independent of an early c-myc response, and suggest that the presence of apoptosis in ineffective erythropoiesis may be secondary to impaired heme synthesis. © 1995 Wiley-Liss, Inc.     

Growth Cycle-Dependent Overproduction and Accumulation of Protoporphyrin IX in Tetrahymena: Effect of Heavy Metals1

Cells of the ciliate Tetrahymena pyriformis GL overproduce and accumulate massive quantities of the heme intermediate, protoporphyrin IX. Protoporphyrin is localized intracellularly in discrete membranous compartments. The amount of porphyrin stored in the cell changes dramatically as cells progress through the growth cycle. Porphyrin overproduction is stimulated by δ-aminolevulinic acid, but only during the mid-stationary phase. Overproduction of protoporphyrin IX apparently results from an increase, late in the growth cycle, of activities subsequent to δ-aminolevulinic acid synthetase. Feedback inhibition in the pathway by accumulated protoporphyrin IX does not occur. The presence of Co²⁺ completely inhibits accumulation of protoporphyrin IX in a manner reversed by δ-aminolevulinic acid. Sn⁴⁺ stimulates protoporphyrin IX accumulation in the culture.     

The role of coproporphyrinogen III oxidase and ferrochelatase genes in heme biosynthesis and regulation in Aspergillus niger

Heme is a suggested limiting factor in peroxidase production by Aspergillus spp., which are well-known suitable hosts for heterologous protein production. In this study, the role of genes coding for coproporphyrinogen III oxidase (hemF) and ferrochelatase (hemH) was analyzed by means of deletion and overexpression to obtain more insight in fungal heme biosynthesis and regulation. These enzymes represent steps in the heme biosynthetic pathway downstream of the siroheme branch and are suggested to play a role in regulation of the pathway. Based on genome mining, both enzymes deviate in cellular localization and protein domain structure from their Saccharomyces cerevisiae counterparts. The lethal phenotype of deletion of hemF or hemH could be remediated by heme supplementation confirming that Aspergillus niger is capable of hemin uptake. Nevertheless, both gene deletion mutants showed an extremely impaired growth even with hemin supplementation which could be slightly improved by media modifications and the use of hemoglobin as heme source. The hyphae of the mutant strains displayed pinkish coloration and red autofluorescence under UV indicative of cellular porphyrin accumulation. HPLC analysis confirmed accumulation of specific porphyrins, thereby confirming the function of the two proteins in heme biosynthesis. Overexpression of hemH, but not hemF or the aminolevulinic acid synthase encoding hemA, modestly increased the cellular heme content, which was apparently insufficient to increase activity of endogenous peroxidase and cytochrome P450 enzyme activities. Overexpression of all three genes increased the cellular accumulation of porphyrin intermediates suggesting regulatory mechanisms operating in the final steps of the fungal heme biosynthesis pathway.     

Porphyrogenic effects and induction of heme oxygenase in vivo by δ-aminolevulinic acid

The effects of single large doses of the porphyrin-heme precursor ?d-aminolevulinic acid on tissue porphyrins and on δ-aminolevulinate synthase and heme oxygenase, the rate-living enzymes of liver heme synthesis and degradation respectively, were studied in the chick embryo in ovo, in the mouse and in the rat. δ-Aminolevulinic acid treatment produced a distinctive pattern characterized by extensive tissue porphyrin accumulation and alterations in these rate-limiting enzymes in the liver. Repression of basal or allylisopropylacetamide-induced liver δ-aminolevulinate synthase was observed and, in the mouse and the rat, induction of liver heme oxygenase after δ-aminolevulinic acid treatment, in a manner similar to the known effects of hemin on these enzymes. In the chick embryo liver in ovo heme oxygenase was substantially higher than in rat and mouse liver, and was not significantly induced by δ-aminolevulinic acid or other compounds, including hemin, CS₂ and CoCl₂. Levulinic acid, an analogue of δ-aminolevulinic acid, did not induce heme oxygenase in mouse liver. δ-Aminolevunilic acid treatment did not impair ferrochelatase activity but was associated with slight and variable decreases in liver cytochrome P-450. Treatment of chick embryos with a small ‘priming’ dose of 1,4-dihydro-3,5-dicarbethoxycollidine, which impairs liver ferrochelatase activity, accentuated porphyrin accumulation after δ-aminolevulinic acid in the liver. These observations indicate that exogenous δ-aminolevulinic acid is metabolized to porphyrins in a number of tissues and, at least in the liver, to a physiologically significant amount of heme, thereby producing an increase in the size of one or more of the heme pools that regulate both heme systhesis and degradation. It is also possible than when δ-aminolevulinic acid is markedly overproduced in vivo it may be transported to many tissues and re-enter the heme pathway and alter porphyrin-heme metabolism in cells and tissues other than those in which its overproduction primarily occurs.     

Porphyrin accumulation in mitochondria is mediated by 2-oxoglutarate carrier

Heme (Fe-protoporphyrin IX), an endogenous porphyrin derivative, is an essential molecule in living aerobic organisms and plays a role in a variety of physiological processes such as oxygen transport, respiration, and signal transduction. For the biosynthesis of heme or the mitochondrial heme proteins, heme or its biosynthetic precursor porphyrin must be transported into mitochondria from cytosol. The mechanism of porphyrin accumulation in the mitochondrial inner membrane is unclear. In the present study, we analyzed the mechanism of mitochondrial translocation of porphyrin derivatives. We showed that palladium meso-tetra(4-carboxyphenyl)porphyrin (PdTCPP), a phosphorescent porphyrin derivative, accumulated in the mitochondria of several cell lines. Using affinity latex beads, we showed that 2-oxoglutarate carrier (OGC), the mitochondrial transporter of 2-oxoglutarate, bound to PdTCPP, and in vitro PdTCPP inhibited 2-oxoglutarate uptake into mitochondria in a competitive manner (Ki = 15 microM). Interestingly, all types of porphyrin derivatives examined in this study competitively inhibited 2-oxoglutarate uptake into mitochondria, including protoporphyrin IX, coproporphyrin III, and hemin. Furthermore, mitochondrial accumulation of porphyrins was inhibited by 2-oxoglutarate or OGC inhibitor. These results suggested that porphyrin accumulation in mitochondria is mediated by OGC and that porphyrins are able to competitively inhibit 2-oxoglutarate uptake into mitochondria. This is the first report of a putative mechanism for accumulation of porphyrins in the mitochondrial inner membrane.     

Inhibition of the heme-induced hemolysis of red blood cells by the chlorite-based drug WF10

Excessive release of hemoglobin from red blood cells markedly disturbs the health status of patients due to cytotoxic effects of free hemoglobin and heme. The latter component is able to initiate novel hemolytic events in unperturbed red blood cells. We modeled this process by incubation of ferric protoporphyrin IX with freshly isolated red blood cells from healthy volunteers. The heme-induced hemolysis was inhibited in a concentration-dependent manner by the chlorite-based drug WF10, whereby the hemolysis degree was totally abolished at a molar ratio of 1:2 between chlorite and heme. Upon incubation of heme with WF10, the ultraviolet-visible spectrum changed, whereas the release of iron from heme and the appearance of fluorescent breakdown products of the porphyrin ring were negligible at this ratio, but increased with increasing excess of chlorite over heme. Thus, inhibition of hemolysis by WF10 takes already place at those chlorite concentrations, where no degradation of the porphyrin ring occurs. As WF10 is applied in form of an intravenous infusion to patients with severe inflammatory states, these data support the hypothesis that the beneficial WF10 effects are closely associated with inactivation of free heme.     

LIPOPROTEIN INHIBITOR OF BQNE MARROW CELLS IN TUMOR-BEARING RATS

The role of a plasma inhibitor of erythropoiesis is evaluated in rats with Walker-256 carcinoma (W-256). Plasma from tumor-bearing rats was treated by gel filtration chromatography (Sephadex G-150) and fractions were combined into four pools on the basis of mol. wt. Inhibitory activity was assayed by adding an aliquot of the plasma fractions to normal rat marrow cells which were cultured for 24 hr with and without erythropoietin. ⁵⁹Fe-heme synthesis, [³H]thymidine DNA synthesis, and ¹⁴C-leucine protein synthesis were studied. The results indicated that cultures containing the high mol. wt. pool (>400,000 daltons) had significantly decreased heme, DNA and protein synthesis. This inhibitor also diminished the response to erythropoietin in polycythemic mice. The lower mol. wt. pool stimulated heme synthesis in vitro. To identify the inhibitor further, plasma lipo-protein classes were isolated by density gradient ultracentrifugation. The very low density lipoprotein (VLDL) and chylomicron fractions markedly inhibited DNA, protein and heme synthesis. Low density and high density lipoprotein fractions were inactive. A lipoprotein inhibitor of erythropoiesis was also identified in cancerous ascitic fluid, and to a lesser degree, in normal rat plasma. We suggest that this VLDL inhibitor of marrow erythropoiesis is a contributing factor in the anaemia of cancer.     

5-Aminolevulinic acid stimulation of porphyrin and hemoglobin synthesis by uninduced Friend erythroleukemic cells.

Porphyrin synthesis and iron accumulation was stimulated by exogenous 5-aminolevulinic acid (ALA) in uninduced Friend erythroleukemic cells (FELC). Uroporphyrin and protoporphyrin were the major intermediated precursors produced. All porphyrin types were conjugated to protein insoluble cellular components and could be extracted only by methanol sulfuric acid esterification. Heme content of the uninduced FELC was increased 6-fold in the presence of 5 x 10(-4) M ALA. As a consequence, the synthesis of the minor murine hemoglobin component was preferentially induced, an effect similar to that expressed by exogenous hemin. Addition of exogenous ALA to 0.5% DMSO-induced cells increased total hemoglobin synthesis with a higher efficiency of the minor hemoglobin. The endogenous synthesis of porphyrin from exogenous ALA was markedly reduced by hemin. Uroporphyrin, coproporphyrin, protoporphyrin and heme were equally repressed, indicating an inhibitory effect of hemin on ALA dehydrase and urosynthetase activities. In addition, hemin repressed [3H]leucine incorporation into protein by uninduced cells. Incubation of uninduced cells in culture medium without serum in the presence of hemin blocked their protein synthesis activity, whereas addition of serum exerted a protective effect on living FELC.     

Effect of Oxygen on Heme and Porphyrin Accumulation from δ-Aminolevulinic Acid by Suspensions of Anaerobically Grown Staphylococcus epidermidis

The effect of various conditions on the accumulation of porphyrins and heme by resting suspensions of anaerobically grown cells of Staphylococcus epidermidis was examined. Anaerobically grown cells contain 10 to 15% of the amount of protoheme found in cells grown aerobically. Resting suspensions of anaerobically grown cells, when incubated aerobically in buffer with delta-aminolevulinic acid and glucose for 60 min, exhibited a fourfold increase in protoheme content. At high levels of delta-aminolevulinic acid, there was also a significant accumulation of porphyrins with the solubility and chromatographic properties of coproporphyrin and uroporphyrin. Protoporphyrin was not accumulated. When oxygen was excluded from the incubation mixture, accumulation of protoheme was prevented, but accumulation of coproporphyrin and total porphyrin was enhanced. Nitrate served as an electon acceptor as indicated by its reduction to nitrite; however, nitrate did not substitute for oxygen in causing the accumulation of protoheme. These results suggested that oxygen is required for one of the late steps of heme synthesis in S. epidermidis, possibly for the conversion of coproporphyrinogen to protoporphyrin. The inability of nitrate to substitute for oxygen suggests a role for molecular oxygen as a substrate rather than as an electron acceptor for heme synthesis.     

Transport of Intact Porphyrin by HpuAB, the Hemoglobin-Haptoglobin Utilization System of Neisseria meningitidis

The meningococcal hemA gene was cloned and used to construct a porphyrin biosynthesis mutant. An analysis of the hemA mutant indicated that meningococci can transport intact porphyrin from heme (Hm), hemoglobin (Hb), and Hb-haptoglobin (Hp). By constructing a HemA⁻ HpuAB⁻ double mutant, we demonstrated that HpuAB is required for the transport of porphyrin from Hb and Hb-Hp.     

Air-stable,heme-like water-soluble iron(II) porphyrin: in situ preparation and characterization

Preparation of the water-soluble, kinetically labile, high-spin iron(II) tetrakis(4-sulfonatophenyl)porphyrin, Fe(II)TPPS⁴⁻, has been realized in neutral or weakly acidic solutions containing acetate buffer. The buffer played a double role in these systems: it was used for both adjusting pH and, via formation of an acetato complex, trapping trace amounts of iron(III) ions, which would convert the iron(II) porphyrins to the corresponding iron(III) species. Fe(II)TPPS⁴⁻ proved to be stable in these solutions even after saturation with air or oxygen. In the absence of acetate ions, however, iron(II) ions play a catalytic role in the formation of iron(III) porphyrins. While the kinetically inert iron(III) porphyrin, Fe(III)TPPS³⁻, is a regular one with no emission and photoredox properties, the corresponding iron(II) porphyrin displays photoinduced features which are typical of sitting-atop complexes (redshifted Soret absorption and blueshifted emission and Q absorption bands, photoinduced porphyrin ligand-to-metal charge transfer, LMCT, reaction). In the photolysis of Fe(II)TPPS⁴⁻ the LMCT process is followed by detachment of the reduced metal center and an irreversible ring-opening of the porphyrin ligand, resulting in the degradation of the complex. Possible oxygen-binding ability of Fe(II)TPPS⁴⁻ (as a heme model) has been studied as well. Density functional theory calculations revealed that in solutions with high acetate concentration there is very little chance for iron(II) porpyrin to bind and release O₂, deviating from heme in a hydrophobic microenvironment in hemoglobin. In the presence of an iron(III)-trapping additive that is much less strongly coordinated to the iron(II) center than the acetate ion, Fe(II)TPPS⁴⁻ may function as a heme model.     

Inhibition of heme biosynthesis in erythroid precursors (BFU-e) isolated from human peripheral blood: effects on globin synthesis

We studied the relationship between heme accumulation and globin synthesis in human erythroid precursors which were stimulated by 2 I.U. of erythropoietin in semi-solid cultures (1% methyl-cellulose, 20% fetal calf serum) and treated with 6-9 micrograms/ml of desferrioxamina (DF), a potent inhibitor of heme synthesis (6). Heme accumulation was detected by specific reaction with benzidine (4), globin synthesis by CM-cellulose column chromatography. Our results demonstrate that globin gene expression occurs in DF-treated erythroid cells which do not accumulate heme molecules. As heme does affect translation and stability of globin mRNA (10) our system might be suitable for studies focused on pathological alterations of erythropoiesis associated with the presence of unstable globin mRNAs and/or unstable globins.