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Query: CAS:7782-44-7 (oxygen)
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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.
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PMID:The enzymic conversion of protoporphyrinogen IX to protoporphyrin IX in mammalian mitochondria. 0 61

Protoporphyrinogen oxidase has been solubilized from plasma membranes of Desulfovibrio gigas. The enzyme was purified to apparent homogeneity with single silver-stained protein bands on isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels. This protoporphyrinogen oxidase has a molecular weight (Mr) of 148,000 and is composed of three dissimilar subunits of Mrs 12,000, 18,500, and 57,000, which are held together by sulfhydryl bonds. Unlike other protoporphyrinogen oxidases, which use molecular oxygen as an electron acceptor, this enzyme does not couple to oxygen. The protoporphyrinogen oxidase donates electrons to 2,6-dichlorophenol-indophenol but not to NAD+, NADP+, flavin adenine dinucleotide, or flavin mononucleotide. The natural physiological electron acceptor of the protoporphyrinogen oxidase from D. gigas is unknown. By using 2,6-dichlorophenol-indophenol as the electron acceptor, the Km and Vmax values for oxidation of protoporphyrinogen were determined to be 21 microM and 8.38 nmol/min per 70 micrograms of protein, respectively. The catalytic rate constant, Kcat, was calculated to be 17.7 mol of protoporphyrin formed per mole of enzyme per min of incubation, and the Kcat/Km was 0.84. Energies of activation were calculated from Arrhenius plots with 7,429 cal (ca. 31,080 J)/mol per degree below 10 degrees C and 1,455 cal (ca. 6,088, J)/mol per degree above 10 degrees C. Optimum enzyme activity was at 23 degrees C, and inhibition was observed with both N-ethylmaleimide and iodoacetamide.
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PMID:Purification and properties of protoporphyrinogen oxidase from an anaerobic bacterium, Desulfovibrio gigas. 366 28

Protoporphyrinogen oxidase, the molecular target of diphenylether-type herbicides, was purified to homogeneity from yeast mitochondrial membranes and found to be a 55-kDa polypeptide with a pI of 8.5 and a specific activity of 40,000 nmol of protoporphyrin/h/mg of protein at 30 degrees C. The Michaelis constant (Km) for protoporphyrinogen IX was 0.1 microM. Due to the high affinity of the enzyme toward oxygen, the Km for oxygen could only be approximated to 0.5-1.5 microM. The purified enzyme contained a flavin as cofactor. Studies with rabbit antibodies to yeast protoporphyrinogen oxidase showed that the enzyme is synthesized as a high molecular weight precursor (58 kDa) that is rapidly converted in vivo to the mature (55 kDa) membrane-bound form. Protoporphyrinogen oxidase activity was found only in purified yeast mitochondrial inner membrane (not in the outer membrane). Acifluorfen-methyl, a potent diphenylether-type herbicide, competitively inhibited the purified enzyme (Ki = 10 nM). The mixed inhibition by acifluorfen-methyl previously reported for the membrane-bound protoporphyrinogen oxidase (Camadro, J.M., Matringe, M., Scalla, R., and Labbe, P. (1991) Biochem. J. 277, 17-21) was shown to be related to partial proteolysis of the enzyme.
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PMID:Purification and properties of protoporphyrinogen oxidase from the yeast Saccharomyces cerevisiae. Mitochondrial location and evidence for a precursor form of the protein. 779 2

In the conversion of uroporphyrinogen III into protoporphyrin IX and thence into chlorophylls, all eight carboxylic side chains, as well as the four meso positions, are modified, and four enzymes are involved. In the uroporphyrinogen decarboxylase-catalysed reaction all four acetate side chains are converted into methyl groups by the same mechanism, to produce coproporphyrinogen III. Both methylene hydrogen atoms remain undisturbed and the reaction occurs with the retention of stereochemistry. Several questions regarding the enzymology of the decarboxylase are posed. Do all the decarboxylations occur at the same active site and, if so, are the four acetate chains handled in a particular sequence? Is the decarboxylation reaction aided by the transient formation of an electron-withdrawing functionality in the pyrrole ring? Coproporphyrinogen oxidase converts the two propionate side chains of rings A and B into vinyl groups, with an overall anti-periplanar removal of the carboxyl group and the Hsi from the neighbouring position. Evidence is examined to evaluate whether a hydroxylated compound acts as an intermediate in the oxidative decarboxylation reaction. Protoporphyrinogen oxidase then converts the methylene-interrupted macrocycle of protoporphyrinogen IX into a conjugated system. The conversion has been suggested to involve three consecutive dehydrogenation reactions followed by an isomerization step. The face of the macrocycle from which the three meso hydrogen atoms are removed in the dehydrogenation reaction is thought to be opposite to that from which the fourth meso hydrogen is lost during the prototropic rearrangement. In an investigation of the in vivo mechanism for the esterification of the ring D propionic acid group with a C20 isoprenyl group 5-aminolaevulinic acid was labelled with 13C and 18O at C-1 and incorporated into bacteriochlorophyll a. The 18O-induced shift of the 13C resonance in the NMR spectrum showed that both oxygen atoms of the carboxyl group are retained in the ester bond. This and other results suggest that the reaction occurs by the nucleophilic attack of the ring D carboxylate anion on the activated form of an isoprenyl alcohol.
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PMID:The modification of acetate and propionate side chains during the biosynthesis of haem and chlorophylls: mechanistic and stereochemical studies. 784 50

Protoporphyrinogen oxidase (EC 1.3.3.4) catalyzes the six electron oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from the bacterium Myxococcus xanthus has been cloned, expressed, purified, and characterized. The protein has been expressed in Escherichia coli using a Tac promoter-driven expression plasmid and purified to apparent homogeneity in a rapid procedure that yields approximately 10 mg of purified protein per liter of culture. Based upon the deduced amino acid sequence the molecular weight of a single subunit is 49,387. Gel permeation chromatography in the presence of 0.2% n-octyl-beta-D-glucopyranoside yields a molecular weight of approximately 100,000 while SDS gel electrophoresis shows a single band at 50,000. The native enzyme is, thus, a homodimer. The purified protein contains a non-covalently bound FAD but no detectable redox active metal. The M. xanthus enzyme utilizes protoporphyrinogen IX, but not coproporphyrinogen III, as substrate and produces 3 mol of H2O2/mol of protoporphyrin. The apparent Km and kcat for protoporphyrinogen in assays under atmospheric concentrations of oxygen are 1.6 microM and 5.2 min-1, respectively. The diphenyl ether herbicide acifluorfen at 1 microM strongly inhibits the enzyme's activity.
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PMID:Protoporphyrinogen oxidase of Myxococcus xanthus. Expression, purification, and characterization of the cloned enzyme. 862 4

Protoporphyrinogen oxidase, which catalyzes the oxygen-dependent aromatization of protoporphyrinogen IX to protoporphyrin IX, is the molecular target of diphenyl ether type herbicides. The structural gene for the yeast protoporphyrinogen oxidase, HEM14, was isolated by functional complementation of a hem14-1 protoporphyrinogen oxidase-deficient yeast mutant, using a novel one-step colored screening procedure to identify heme-synthesizing cells. The hem14-1 mutation was genetically linked to URA3, a marker on chromosome V, and HEM14 was physically mapped on the right arm of this chromosome, between PRP22 and FAA2. Disruption of the HEM14 gene leads to protoporphyrinogen oxidase deficiency in vivo (heme deficiency and accumulation of heme precursors), and in vitro (lack of immunodetectable protein or enzyme activity). The HEM14 gene encodes a 539-amino acid protein (59,665 Da; pI 9.3) containing an ADP- beta alpha beta-binding fold similar to those of several other flavoproteins. Yeast protoporphyrinogen oxidase was somewhat similar to the HemY gene product of Bacillus subtilis and to the human and mouse protoporphyrinogen oxidases. Studies on protoporphyrinogen oxidase overexpressed in yeast and purified as wild-type enzyme showed that (i) the NH2-terminal mitochondrial targeting sequence of protoporphyrinogen oxidase is not cleaved during importation; (ii) the enzyme, as purified, had a typical flavin semiquinone absorption spectrum; and (iii) the enzyme was strongly inhibited by diphenyl ether-type herbicides and readily photolabeled by a diazoketone derivative of tritiated acifluorfen. The mutant allele hem14-1 contains two mutations, L422P and K424E, responsible for the inactive enzyme. Both mutations introduced independently in the wild-type HEM14 gene completely inactivated the protein when analyzed in an Escherichia coli expression system.
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PMID:Cloning and characterization of the yeast HEM14 gene coding for protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides. 862 63

Protoporphyrinogen oxidase (E.C.1.3.3.4) catalyzes the oxygen-dependent oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from human placenta has been cloned, sequenced, expressed in Escherichia coli, purified to homogeneity, and characterized. Northern blot analysis of eight different human tissues show evidence for only a single transcript in all tissue types and the size of this transcript is approximately 1.8 kb. The human cDNA has been inserted into an expression vector for E. coli and the protein produced at high levels in these cells. The protein is found in both membrane and cytoplasmic fractions. The enzyme was purified to homogeneity in the presence of detergents using a metal chelate affinity column. The purified protein is a homodimer composed of subunits of molecular weight of 51,000. The enzyme contains one noncovalently bound FAD per dimer, has a monomer extinction coefficient of 48,000 at 270 nm and contains no detectable redox active metals. The apparent K(m) and Kcat for protoporphyrinogen IX are 1.7 microM and 10.5 min-1, respectively. The enzyme does not use coproporphyrinogen III as a substrate and is inhibited by micromolar concentrations of the herbicide acifluorfen. Protein database searches reveal significant homology between protoporphyrinogen oxidase and monoamine oxidase.
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PMID:Human protoporphyrinogen oxidase: expression, purification, and characterization of the cloned enzyme. 877 Dec 1

Protoporphyrinogen oxidase, the last enzyme of the common branch of the heme and chlorophyll pathways in plants, is the molecular target of diphenyl ether-type herbicides. These compounds inhibit the enzyme competitively with respect to the tetrapyrrole substrate, protoporphyrinogen IX. We used the flavinic nature of protoporphyrinogen oxidase to investigate the reactivity of the enzyme toward the 2,2'-diphenyleneiodonium cation, a known inhibitor of several flavoproteins. Diphenyleneiodonium inhibited the membrane-bound yeast protoporphyrinogen oxidase competitively with molecular oxygen. The typical slow-binding kinetics suggested that the enzyme with a reduced flavin rapidly combined with the inhibitor to form an initial complex which then slowly isomerized to a modified enzyme-inhibitor complex (Ki = 6.75 x 10(-8) M, Ki* = 4.1 x 10(-9) M). This inhibition was strongly pH-dependent and was maximal at pH 8. Substituted diphenyleneiodoniums were synthesized and shown to be even better inhibitors than 2,2'-diphenyleneiodonium: Ki = 4.4 x 10(-8) M and Ki* = 1.3 x 10(-9) M for 4-methyl-2,2'-diphenyleneiodonium, Ki = 2.2 x 10(-8) M and Ki * = 1.1 x 10(-9) M for 6-methyl-2,2'-diphenyleneiodonium, and Ki = 6.4 x 10(-9) M and Ki* = 1.2 x 10(-1)2 M for 4-nitro-2,2'-diphenyleneiodonium. The 4-nitro-2,2'-diphenyleneiodonium was a quasi irreversible inhibitor (k5/k6 > 5000). Diphenyleneiodoniums are a new class of protoporphyrinogen oxidase inhibitors that act via a mechanism very different from that of diphenyl ether-type herbicides and appear to be promising tools for studies on the structure-function relationships of this agronomically important enzyme.
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PMID:Kinetics of protoporphyrinogen oxidase inhibition by diphenyleneiodonium derivatives. 925 15

Protoporphyrinogen oxidase catalyzes the oxygen-dependent aromatization of protoporphyrinogen IX to protoporphyrin IX and is the molecular target of diphenyl ether-type herbicides. Structural features of yeast protoporphyrinogen oxidase were assessed by circular dichroism studies on the enzyme purified from E. coli cells engineered to overproduce the protein. Coexpression of the bacterial gene ArgU that encodes tRNAAGA,AGG and a low induction temperature for protein synthesis were critical for producing protoporphyrinogen oxidase as a native, active, membrane-bound flavoprotein. The secondary structure of the protoporphyrinogen oxidase was 40.0 +/- 1. 5% alpha helix, 23.5 +/- 2.5% beta sheet, 18.0 +/- 2.0% beta turn, and 18.5 +/- 2.5% random-coil. Purified protoporphyrinogen oxidase appeared to be a monomeric protein that was relatively heat-labile (Tm of 44 +/- 0.5 degreesC). Acifluorfen, a potent inhibitor that competes with the tetrapyrrole substrate, and to a lower extent FAD, the cofactor of the enzyme, protected the protein from thermal denaturation, raising the Tm to 50.5 +/- 0.5 degreesC (acifluorfen) and 46.5 +/- 0.5 degreesC (FAD). However, diphenyleneiodonium, a slow tight-binding inhibitor that competes with dioxygen, did not protect the enzyme from heat denaturation. Acifluorfen binding to the protein increased the activation energy for the denaturation from 15 to 80 kJ.mol-1. The unfolding of the protein was a two-step process, with an initial fast reversible unfolding of the native protein followed by slow aggregation of the unfolded monomers. Functional analysis indicated that heat denaturation caused a loss of enzyme activity and of the specific binding of radiolabeled inhibitor. Both processes occurred in a biphasic manner, with a transition temperature of 45 degreesC.
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PMID:Stability of recombinant yeast protoporphyrinogen oxidase: effects of diphenyl ether-type herbicides and diphenyleneiodonium. 973 59

Protoporphyrinogen oxidase (PPO) catalyzes the penultimate reaction in heme biosynthesis. The 'oxygen dependent' form of this enzyme can utilize three molecules of oxygen as electron acceptors in the reaction. In the current study, the ability of cytochrome c to serve as an electron acceptor for PPO was examined. Cytochrome c was found to enhance the catalytic rate of Drosophila melanogaster PPO under reduced oxygen conditions, and cytochrome c became reduced during PPO catalysis. Further kinetic analysis under anaerobic conditions revealed that hydrogen peroxide, a byproduct of the PPO reaction, is required for this rate enhancement to occur. This suggests that the generation of free radicals via the peroxidase activity of cytochrome c plays a part in this rate enhancement, rather than cytochrome c acting as an electron acceptor for the PPO reaction. Given the abundance of cytochrome c in the intermembrane space of mitochondria, the cellular location of PPO, this process may potentially impact on the synthesis of heme in vivo particularly in conditions of low oxygen or hypoxia.
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PMID:Peroxidase activity of cytochrome C facilitates the protoporphyrinogen oxidase reaction. 1926 95


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