Judith M. Jacobs

Porphyrin Accumulation and Export by Isolated Barley (Hordeum vulgare) Plastids (Effect of Diphenyl Ether Herbicides)

We have investigated the formation of porphyrin intermediates by isolated barley (Hordeum vulgare) plastids incubated for 40 min with the porphyrin precursor 5-aminolevulinate and in the presence and absence of a diphenylether herbicide that blocks protoporphyrinogen oxidase, the enzyme in chlorophyll and heme synthesis that oxidizes protoporphyrinogen IX to protoporphyrin IX. In the absence of herbicide, about 50% of the protoporphyrin IX formed was found in the extraplastidic medium, which was separated from intact plastids by centrifugation at the end of the incubation period. In contrast, uroporphyrinogen, an earlier intermediate, and magnesium protoporphyrin IX, a later intermediate, were located mainly within the plastid. When the incubation was carried out in the presence of a herbicide that inhibits protoporphyrinogen oxidase, protoporphyrin IX formation by the plastids was completely abolished, but large amounts of protoporphyrinogen accumulated in the extraplastidic medium. To detect extraplastidic protoporphyrinogen, it was necessary to first oxidize it to protoporphyrin IX with the use of a herbicide-resistant protoporphyrinogen oxidase enzyme present in Escherichia coli membranes. Protoporphyrinogen is not detected by some commonly used methods for porphyrin analysis unless it is first oxidized to protoporphyrin IX. Protoporphyrin IX and protoporphyrinogen found outside the plastid did not arise from plastid lysis, because the percentage of plastid lysis, measured with a stromal marker enzyme, was far less than the percentage of these porphyrins in the extraplastidic fraction. These findings suggest that of the tetrapyrrolic intermediates synthesized by the plastids, protoporphyrinogen and protoporphyrin IX, are the most likely to be exported from the plastid to the cytoplasm. These results help explain the extraplastidic accumulation of protoporphyrin IX in plants treated with photobleaching herbicides. In addition, these findings suggest that plastids may export protoporphyrinogen or protoporphyrin IX for mitochondrial heme synthesis.

Nitric oxide hemoglobin in mice and rats in endotoxic shock

Mice given ip bacterial endotoxin (LPS) at 10 mg/kg showed a statistically significant decrease in plasma glucose and an increase in hematocrit at 2 h after injection. Glucose was still decreased at 4 h, but the hematocrit had returned to control values. Nitrosylated hemoglobin (HbNO) was detected at 3, but not at 2 h. By 4 h it had increased 5-fold. When N-monomethylarginine (NMMA) at 100 mg/kg, ip was given 2 h after LPS in mice, the HbNO concentration at 4 h was significantly reduced, but the hypoglycemia was worsened because NMMA itself produced a significant hypoglycemia. Rats given iv LPS, 20 mg/kg, showed a fleeting, transient rise in mean arterial pressure (MAP) lasting only a few min. Thereafter, the MAP tended to drift slowly downward over 4 h, but when the MAP at 30 min intervals was compared to the pre-LPS MAP, there were no significant differences. Plasma glucose in unanesthetized rats was significantly elevated at 1 h, back to control at 2 h, and significantly decreased at 3 h. HbNO was detected as early as 1 h after injection. By 2 h the HbNO concentrations exceeded the highest levels found in mice, and they were still increasing as late as 5 h after injection. Unanesthetized rats showed toxic signs and 3/12 rats died within 4 hours of LPS administration. These results are consistent with a model for endotoxic shock in which LPS stimulates an inducible pathway for NO synthesis.

Measurement of Heme Concentration

Heme (iron protoporphyrin IX) is a prosthetic group for a number of hemoproteins in different tissues (e.g., hemoglobin, myoglobin, cytochrome P‐450s, mitochondrial cytochromes, catalases, and peroxidases). Mutations in the biosynthetic pathway can affect the synthesis and/or degradation of heme. Several assays are provided in this unit for quantifying heme: a spectophotometric assay based on the characteristic absorption spectrum of oxidized and reduced form of the hemochrome formed by replacing the nitrogen ligands with pyridine; a fluorescence assay based on removal of the iron by a heated, strong oxalic acid solution to produce fluorescent protoporphyrin; a reversed‐phase HPLC assay to measure heme and intermediates in the synthetic pathway; and a radiometric assay to measure newly synthesized heme in tissue culture cells.

Effects of the photobleaching herbicide, acifluorfen-methyl, on protoporphyrinogen oxidation in barley organelles, soybean root mitochondria, soybean root nodules, and bacteria

The photobleaching herbicide, acifluorfen-methyl (AFM), has been reported to be an inhibitor of the heme and chlorophyll biosynthetic enzyme protoporphyrinogen oxidase (Protox) in several plant species. However, AFM had no effect on the levels of Protox activity measured in a mitochondrial fraction from soybean roots. In contrast, AFM inhibited Protox activity in etioplasts from barley leaves and in mitochondria from barley roots, but the extent of inhibition varied depending upon the assay conditions and was maximal only in the presence of 5 mM dithiothreitol (DTT). AFM inhibition was enhanced by preincubation of barley organelle extract in the presence of DTT. Preincubation of barley extract with DTT and AFM together (but not with AFM alone) caused extensive enzyme inhibition which was not reversible by dialysis. These findings have implications for the mechanism of AFM action and for the differential effect of these herbicides on crop and weed species. AFM had no effect on the Protox activity of membranes from free-living bacterial cell of Bradyrhizobium japonicum or Escherichia coli, or on the high levels of Protox activity associated with the plant-derived membrane surrounding the symbiotic bacteria within the soybean root nodule.

Fumarate as alternate electron acceptor for the late steps of anaerobic heme synthesis in Escherichia coli

Summary The anaerobic formation of protoheme from added excess delta amino levulinic acid was markedly reduced if fumarate was omitted from an anaerobic incubation mixture containing resting suspensions of E. coli grown anaerobically on a medium containing fumarate. However, the formation of coproporphyrinogen did not require fumarate, suggesting a role for fumarate at some step between coproporphyrinogen and heme in the anaerobic heme biosynthetic pathway. The appearance of fluorescence during the anaerobic incubation of these cell suspensions was also dependent upon the presence of fumarate, suggesting that fumarate could anaerobically oxidize protoporphyrinogen to protoporphyrin. This was confirmed by demonstrating that fumarate could serve as an alternate electron acceptor to replace oxygen in the oxidation of chemically reduced protoporphyrinogen under anaerobic conditions inextracts of these cells. This was the first demonstration of the enzymatic oxidation of protoporphyrinogen to protoporphyrin by a physiological electron acceptor other than oxygen in any type of cell.

Protoporphyrinogen oxidation, an enzymatic step in heme and chlorophyll synthesis: Partial characterization of the reaction in plant organelles and comparison with mammalian and bacterial systems☆

High rates of oxidation of protoporphyrinogen to protoporphyrin were demonstrable in etioplasts, chloroplasts, and mitochondria from young barley shoots. Much lower rates were observed in chloroplasts from older barley or mature spinach, in mitochondria from potatoes or rat liver, and in membranes from the bacteria Escherichia coli and Rhodopseudomonas spheroides. The presence of high activity in cells capable of rapid synthesis of large amounts of chlorophyll suggests a role for this activity in chlorophyll synthesis. Characteristics of the plant protoporphyrinogen-oxidizing activity were compared to the activity in rat liver mitochondria. The activity in spinach chloroplasts exhibited a pH optimum of 7, which was lower than that of the mammalian enzyme. The plant activity was more sensitive to inhibition by glutathione or excess detergent, and was more readily inactivated at room temperature. The plant activity exhibited less specificity toward porphyrinogen substrates, oxidizing mesoporphyrinogen as rapidly as protoporphyrinogen. The mammalian enzyme oxidized mesoporphyrinogen slowly, and neither system oxidized coproporphyrinogen or uroporphyrinogen. Both the plant and the mammalian activity were bound to organelle membranes, but could be extracted with detergents. In contrast, activity from membranes of the bacteria E. coli and R. spheroides was inactivated by detergent treatment. The plant extracts could be fractionated with ammonium sulfate and retained activity after dialysis or Sephadex G-25 treatment, suggesting no readily dissociable cofactor. The activity extracted from spinach chloroplasts was mostly inactivated by trypsin digestion, which was additional evidence for the protein nature of the plant activity.

Protoporphyrinogen oxidation in Rhodopseudomonas spheroides, a step in heme and bacteriochlorophyll synthesis.

Abstract Protoporphyrinogen oxidizing activity in photosynthetically grown, bacteriochlorophyll-rich extracts of Rhodopseudomonas spheroides was heat labile, destroyed by trypsin digestion, associated with membranes, and proportional to extract concentration. Substrate specificity was indicated by the inability to oxidize other porphyrinogens. These properties are consistent with an enzymatic reaction. Activity was markedly inhibited by respiratory inhibitors such as cyanide, azide, and hydroxylamine, by reducing agents such as glutathione and 2-mercaptoethanol, and by respiratory substrates such as succinate and NADH. These agents as well as protoporphyrinogen also caused reduction of cytochromes during the assay. Extraction of quinones from membranes with pentane also inhibited protoporphyrinogen oxidation. These results clearly indicate that this oxidation in R. spheroides is closely linked with components of the respiratory electron transport chain, suggesting possibilities for regulation by light and oxygen. Cyanide did not cause inhibition of Protoporphyrinogen oxidation by rat liver mitochondria, suggesting different mechanisms for the bacterial and mitochondrial systems. In addition, an enzyme able to link protoporphyrinogen oxidation directly to oxygen was solubilized with Triton from rat liver mitochondria, but not from R. spheroides membranes. Membranes from R. spheroides grown aerobically, when bacteriochlorophyll synthesis does not occur, exhibited about half the protoporphyrinogen oxidizing activity as membranes from photosynthetically grown cells. This activity was also markedly inhibited by cyanide and could not be solubilized with Triton, but was not inhibited by glutathione.

Microbial oxidation of protoporphyrinogen, an intermediate in heme and chlorophyll biosynthesis

Abstract The oxidation of protoporphyrinogen to protoporphyrin, a late step in heme and chlorophyll synthesis, is catalyzed aerobically by a particulate fraction of Escherichia coli at a rate significantly higher than the rate of autooxidation. This activity is heat labile and is markedly inhibited by addition of respiratory substrates such as NADH. NADH is oxidized at a rate 100-fold higher than protoporphyrinogen. Particles from a cytochrome-less mutant of E. coli were markedly deficient in protoporphyrinogen oxidizing activity. Particles from a quinone-deficient mutant were also deficient. These findings suggest a possible role for the electron transport system in aerobic protoporphyrinogen oxidation. This activity was also examined in a variety of other bacteria. Particles from Streptococcus faecalis , which does not synthesize heme, were unable to oxidize protoporphyrinogen, confirming the specificity of this activity. Particles from aerobically grown Staphylococcus aureus exhibited protoporphyrinogen oxidizing activity, but particles from anaerobically grown cells had no activity above that of the nonenzymatic control. This indicates the repressible nature of this activity, and may also explain why Staphylococci synthesize cytochromes during aerobic, but not during anaerobic growth. Particles from photosynthetically grown Rhodopseudomonas spheroides , which contain both chlorophyll and heme, oxidized protoporphyrinogen at a rate no higher than the nonenzymic control. However, particles from cells grown aerobically, when bacteriochlorophyll synthesis is markedly repressed, readily exhibited protoporphyrinogen oxidizing activity. These initial findings suggest that this activity is detectable in cells primarily synthesizing heme, but not in cells primarily synthesizing bacteriochlorophyll, and could have implications both for the mechanism and regulation of the heme and bacteriochlorophyll pathways.

Characteristics of purified protoporphyrinogen oxidase from barley.

The membrane bound enzyme oxidizing protoporphyrinogen to protoporphyrin, a step in heme and chlorophyll synthesis, was purified to a single prominent polypeptide band on SDS/PAGE from barley mitochondrial fractions. It contained a variety of lipids including 0.66 mg of phosphatidyl ethanolamine and 0.46 mg of free fatty acid per mg of protein. Iron, but no flavins or cytochromes, was detected. In the presence of glutathione, enzymatic oxidation was inhibited by the iron chelator o-phenanthroline but was stimulated by iron EDTA. The purified enzyme was inhibited by reductants such as glutathione, ascorbate, NADH and NADPH. These findings are compatible with some direct or indirect involvement of lipids and iron in this oxidation in plants.

Protoporphyrinogen oxidation in chloroplasts and plant mitochondria, a step in heme and chlorophyll synthesis☆

Abstract The oxidation of protoporphyrinogen to protoporphyrin was demonstrated in greening plastids and mitochondria from greening barley shoots. The plastids, purified by sucrose gradient centrifugation, were essentially free of a mitochondrial marker enzyme. The plastid activity was destroyed by mild heating and was proportional to plastid concentration suggesting, an enzymatic reaction. Uroporphyrinogen I was not oxidized at an appreciable rate. Activity was also demonstrated in etioplasts and mitochondria from dark-grown barley, and in chloroplasts from commercial spinach leaves. The chelating agent 1,10-phenanthroline partially decreased activity in plant organelles, but cyanide did not. The plastid activity, like the activity in liver mitochondria, was readily demonstrable at pH 8.4 in the presence of glutathione as reducing agent. However, the plastid activity was markedly enhanced by assay at pH 7.0 and the absence of reducing agents. These properties distinguish the activity in plants from that previously described in mammalian mitochondria and photosynthetic bacteria.