The mitochondrial location of protoporphyrinogen oxidase

Deybach, Jean‐Charles; Silva, Vasco Da; Grandchamp, Bernard; Nordmann, Y

doi:10.1111/j.1432-1033.1985.tb08943.x

Cited by 66 publications

(37 citation statements)

References 15 publications

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“…mitochondrial membrane and requires molecular oxygen for catalysis to proceed [3][4][5]. The human, Bacillus subtilis, Myxococcus xanthus, Aquifex aeolicus, and mouse enzymes have previously been cloned, overexpressed in Escherichia coli, and purified to homogeneity [6][7][8][9][10].…”

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A continuous fluorimetric assay for protoporphyrinogen oxidase by monitoring porphyrin accumulation

Shepherd

Dailey

2005

Analytical Biochemistry

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A continuous spectro fluorimetric assay for protoporphyrinogen oxidase (PPO, EC 1.3.3.4) activity has been developed using a 96-well plate reader. Protoporphyrinogen IX, the tetrapyrrole substrate, is a colorless non fluorescent compound. The evolution of the fluorescent tetrapyrrole product, protoporphyrin IX, was detected using a fluorescence plate reader. The apparent K m (K app ) values for protoporphyrinogen IX were measured as 3.8± 0.3, 3.6± 0.5, and 1.0± 0.1 μM for the enzymes from human, Myxococcus xanthus, and Aquifex aeolicus, respectively. The K i for acifluorfen diphenylether herbicide,was measured as 0.53μM enzyme.Also, the specific activity of mouse liver mitochondrial PPO was measured as 0.043 nmol h -1 /mg mitochondria, demonstrating that this technique is useful for monitoring low-enzyme activities. This method can be used to accurately measure activities as low as 0.5 nM min -1 , representing a 50-fold increase in sensitivity over the currently used discontinuous assay. Furthermore, this continuous assay may be used to monitor up to 96 samples simultaneously. These obvious advantages over the discontinuous assay will be of importance for both the kinetic characterization of recombinant PPOs and the detection of low concentrations of this enzyme in biological samples.

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A continuous fluorimetric assay for protoporphyrinogen oxidase by monitoring porphyrin accumulation

Shepherd

Dailey

2005

Analytical Biochemistry

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“…VP manifests clinically with photosensitivity and acute attacks, which include various neuropsychiatric symptoms (4,5). PPOX is anchored to the inner membrane of mitochondria in eukaryotes (6) with its active site facing the cytosolic side of the membrane (7). The anchoring may involve amphipathic helical domains inserting PPOX into the inner mitochondrial membrane.…”

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Mitochondrial Targeting of Normal and Mutant Protoporphyrinogen Oxidase

Fraunberg

Nyrönen

Kauppinen

2003

Journal of Biological Chemistry

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We have investigated the signal sequence for mitochondrial transport of mutants (I12T, 78insC, IVS2-2a3c, 338G3 C, R152C, 470A3 C, and L401F) and the wild type protoporphyrinogen oxidase (PPOX), which is the penultimate enzyme in the heme biosynthesis. We constructed the corresponding green fluorescent protein fusion proteins and studied their intracellular localization in COS-1 cells. We showed that 28 amino acids in the amino terminus of PPOX contain an independently functioning signal for mitochondrial targeting. The experiments with amino-terminally truncated green fluorescent protein fusion proteins revealed that amino acids 25-477 of PPOX contained an additional mitochondrial targeting signal(s). We constructed a structural model for the interaction between the aminoterminal end of PPOX and the putative mitochondrial receptor protein Tom20. The model suggests that leucine and isoleucine residues Leu-8, Ile-12, and Leu-15 forming an ␣-helical hydrophobic motif, LXXXIXXL, were crucial for the recognition of the targeting signal. The validity of the model was tested using mutants L8Q, I12T, and L15Q disrupting the hydrophobic surface of the LXXXIXXL helix. The results from in vitro expression studies and molecular modeling were in accordance supporting the hypothesis that the recognition of the mitochondrial targeting signal is dependent on hydrophobic interactions between the targeting signal and the mitochondrial receptor.

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“…The terminal steps of the heme biosynthetic pathway in higher eukaryotes are catalyzed by enzymes that are either bound to or associated with the inner mitochondrial membrane (Jones & Jones, 1969;Elder & Evans, 1978;Grandchamp et al, 1978;Deybach et al, 1985;Harbin & Dailey, 1985; see Dailey, 1990). These enzymes, coproporphyrinogen oxidase (EC 1.3.3.3) (CPO), protoporphyrinogen oxidase (EC 1.3.3.4) (PPO), and ferrochelatase (EC 4.99.1.1) (FC), catalyze the oxidative decarboxylation The University of Georgia, Athens, Georgia 30602-2605.…”

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