Protoporphyrinogen oxidase (EC 1.3.3.4) is the penultimate enzyme of the heme biosynthetic pathway. This membrane-bound enzyme catalyzes the oxidative O-dependent aromatization of the colorless protoporphyrinogen IX to the highly conjugated protoporphyrin IX. The existence of an enzyme catalyzing this oxidative step has long been controversial, since porphyrinogens are rapidly oxidized to their corresponding porphyrins in the presence of air via a light-sensitive, autocatalytic reaction(1) . However, biochemical and genetic evidences now indicates the enzymatic nature of protoporphyrinogen oxidation in living cells. Protoporphyrinogen oxidase deficiency has been described in bacteria(2, 3, 4) , yeast(5) , and man, where a lack of protoporphyrinogen oxidase activity is responsible for the inherited disease porphyria variegata(6) . Studies on the structure and function of protoporphyrinogen oxidase were recently stimulated by the discovery that diphenyl ether-type herbicides are very potent inhibitors of the protoporphyrinogen oxidase activities of yeast, mammalian, and plant mitochondria and plant chloroplasts in vitro(7, 8) . The phytotoxicity of diphenyl ether-type herbicides is light-dependent and involves intracellular peroxidations promoted by protoporphyrin IX, the heme and chlorophyll precursor, leading to cell damage and lysis(9) , mimicking the symptoms of human patients with porphyria variegata. Protoporphyrin IX toxicity is due to the nonenzymatic oxidation of accumulated protoporphyrinogen that diffused away from its site of synthesis and is thus not further metabolized to metalloporphyrins by iron or magnesium chelatases. However, the mechanism of protoporphyrin accumulation in diphenyl ether-treated plants may also involve some nonspecific porphyrinogen-oxidizing enzymes, such as peroxidases(10, 11, 12) , that can oxidize protoporphyrinogen and also coproporphyrinogen or uroporphyrinogen to their corresponding porphyrins. These activities are insensitive to diphenyl ether inhibition, but are markedly inhibited by reducing agents such as dithiothreitol(11) . These nonspecific protoporphyrinogen-oxidizing activities may give rise to some discrepancies in the biochemical characterization of protoporphyrinogen oxidase in various organisms that are quite surprising, since the biochemical and structural properties of the enzymes of the heme biosynthesis pathway are remarkably well conserved through evolution (for a revue, see (13) ). In contrast, protoporphyrinogen oxidases purified from various sources appear to be rather different in terms of molecular mass, cofactor, and kinetic properties(14, 15, 16, 17) , electron acceptor(18, 19, 20, 21, 22) , and even subunit composition(20) . This biochemical diversity seems to be also found at the genetic level. The HemG gene of Escherichia coli, located at 87 min on the genetic map of E. coli K12(4) , has been cloned and sequenced(23) . This gene encodes a 181-amino acid protein (M 21,200) and complements a mutation affecting the oxidation of protoporphyrinogen and heme synthesis in E. coli. It restores the protoporphyrinogen oxidase activity to 7-25 times that of a nontransformed wild-type strain when introduced into the original mutant on a high copy number plasmid. However, a second genetic locus, HemK, has been recently described as being involved in protoporphyrinogen oxidation in E. coli(24) . This gene is part of a HemA-PrfA-HemK operon and encodes a 225-amino acid protein without homology to the HemG gene product, that may be a component of an alternative protoporphyrinogen oxidation pathway. The hemG mutant of E. coli was recently used to clone a human cDNA (25) and its murine homologue (26) that complement the mutation. These cDNAs encode a 477-amino acid protein with protoporphyrinogen oxidase activity and deduced sequences identical to that of peptides obtained from partially purified bovine protoporphyrinogen oxidase(26) . These mammalian protoporphyrinogen oxidases have substantial domains that are similar to the HemY gene product of Bacillus subtilis. HemY was isolated by complementation of a mutation affecting heme synthesis in vivo by altering either coproporphyrinogen oxidase, or protoporphyrinogen oxidase activities, or both(27) . This gene encodes a 472-amino acid extrinsic membrane-bound polypeptide that, when overexpressed in E. coli, is able to oxidize protoporphyrinogen IX to protoporphyrin IX in vitro and, more efficiently, coproporphyrinogen III to coproporphyrin III(28, 29) , unlike any eukaryotic protoporphyrinogen oxidase. The HemY gene product is not inhibited by diphenyl ether-type herbicides (29) and thus resembles more the nonspecific oxidases recently found in higher plants that may be involved in the rapid accumulation of protoporphyrin in diphenyl ether-treated plants.

As part of our efforts to understand the relationship between structure and function of eukaryotic protoporphyrinogen oxidase and eventually to elucidate the molecular basis of protoporphyrinogen oxidase and hence the mechanisms underlying the pathophysiology associated with the enzyme defect (human porphyria) or inhibition (herbicidal effects of diphenyl ethers), we recently purified protoporphyrinogen oxidase from the yeast Saccharomyces cerevisiae. This integral protein of the inner mitochondrial membrane is a 55-kDa cationic FAD-containing flavoprotein synthesized as a high molecular mass precursor (58 kDa) that is rapidly converted to the mature membrane-bound form in vivo(30) . Antibodies raised against purified yeast protoporphyrinogen oxidase were used to characterize the enzyme in a heme-deficient mutant lacking protoporphyrinogen oxidase activity (5) carrying a single nuclear mutation hem14-1(31) . This mutant strain was shown to synthesize normal amounts of an inactive protein that did not bind a tritiated inhibitor(32) . We therefore undertook the cloning of the yeast gene by functional complementation of the hem14-1 gene defect. Like any heme-deficient yeast strain, the mutant cannot grow aerobically on nonfermentative carbon sources (ethanol, glycerol, or lactate), but grows via fermentative metabolism, provided the growth medium contains Tween 80 and ergosterol as precursors of unsatured fatty acids and sterols, whose syntheses are dependent on specific hemoproteins. The present study describes the isolation and nucleotide sequence of the protoporphyrinogen oxidase gene from the yeast S. cerevisiae, the phenotypic effects of its disruption and further characterization of the protoporphyrinogen oxidase protein overexpressed in yeast cells. The mutant hem14-1 allele was also sequenced, and normal and mutated protoporphyrinogen oxidases were analyzed in a E. coli expression system.

EXPERIMENTAL PROCEDURES

Strains and Growth Conditions

Plasmid pools of partial Sau3A digests of yeast chromosomal DNA ligated at the BamHI site of the high copy number vector YEpEMBL23 (34) or partial HindIII digests of yeast chromosomal DNA ligated at the HindIII site of the low copy number vector pRS316 (35) were kindly provided by Dr. D. Thomas (Laboratoire d'Enzymologie, CNRS, Gif sur Yvette, France).

The E. coli strains used were (i) DH5 (from Life Technologies, Inc.) for cloning, maintenance, and propagation of plasmids; (ii) BL21(DE3) (from Novagen) for expression studies; and (iii) XLmutS (from Stratagene) for selection of altered plasmids in site-directed mutagenesis experiments. They were grown in LB medium containing 100 µg/ml ampicillin when necessary to maintain all of the Amp-based plasmids.

The pBluescript KS(+) phagemid vector (Stratagene) was the cloning vehicle for all HEM14 containing fragments used for sequencing reactions. The pT7-5 vector was used for expression studies of HEM14 in E. coli and as the template for in vitro site-directed mutagenesis of the cloned HEM14 gene.

Cloning and Sequencing Manipulations

The overlaps of the HEM14 clone with the PRP22 and ACS1 clones were recognized initially by sequencing the 5` and 3` ends of the 4.9-kb ()insert of plasmid pBS19-1 that complemented the hem14-1 mutation. The restriction site patterns of the cloned insert matched those published and prompted us to delete a 1.9-kb SalI-XhoI fragment yielding the plasmid pBS19-1XS9 that complemented the hem14-1 mutation. This plasmid was used for expression studies of the cloned protoporphyrinogen oxidase in yeast. The 3-kb XhoI-SmaI fragment of pBS19-1 was cloned at the XhoI-SmaI sites of the phagemid pBluescript(KS). This fragment was sequenced on subclones obtained by (i) internal deletions in the KSBS19-1XhSm plasmid taking into account unique restriction sites in the insert and adequate sites of the polylinker of pKS (NruI/SmaI, EcoR5/SmaI, EcoR1-Klenow/SmaI, XbaI/XbaI); and (ii) one-directional deletions generated by exonuclease III digestions (38) in plasmid KSBS19-1XhSm cut by SacII and SmaI using the Erase-a-Base kit (Promega, Madison, WI). The double-stranded plasmid DNA was sequenced by the chain termination method using the modified T7 DNA polymerase (39) from the Sequanase v. 2.0 sequencing kit (U. S. Biochemical Corp. and -S-dATP (1000Ci/mmol, Amersham Corp.). The sequencing primers used were universal(-40) and reverse primers available for pBluescript and various 17-mer oligonucleotides complementary to the HEM14 sequence already determined. The plasmid DNA (2-3 µg) was first denaturated in 0.4 N NaOH for 10 min at room temperature, precipitated with cold ethanol and annealed to the primer for 30 min at room temperature at a molar ratio of 1:1 when using pBluescript primers, and 1:20 when using synthetic HEM14-specific oligonucleotides. The oligonucleotides used as sequencing primers were synthesized on an Applied Biosystems DNA synthesizer and were used without further purification. To sequence the mutant allele hem14-1, the HEM14 locus was amplified by PCR from genomic DNA of the strains CC750-3D and W303-1A using the primers 5`-CGCGGATCCCTTTGTTGCTTCGGAGTCGC-3` (HindIII; (+) strand) and 5` CCCAAGCTTATAGTTCCAACCCATCATCG-3` (BamHI;(-) strand). The PCR products were purified from agarose gels (JetSorb, Genomed Inc.) digested by HindIII and BamHI and the repurified DNA (JetSorb, Genomed Inc.) was ligated into the dephosphorylated phagemid BlusScript KS+ linearized with HindIII and BamHI. The nucleotide sequence of the wild-type (control processed under the same conditions) and mutant alleles were obtained on DNAs pooled from seven independent clones. The nature of the mutations was confirmed on both strands. The DNA for PCR amplification of genomic sequences was prepared by the technique of Hoffman and Winston(40) .

HEM14 Gene Disruption

HEM14 Expression in E. coli and Site-directed Mutagenesis

The L422P mutation was introduced using the oligonucleotide 5`-AACCCAAACGCCCCCAACAAATAAACAAAAGTG-3`. The K424E mutation was introduced using the oligonucleotide 5`-CGCCCTCAACGAATATACAAAAGTGACTGCGATG-3`. Enrichment in mutated plasmid was done by eliminating the single ScaI restriction site in the ampicillin resistance gene of pT7-5. The plasmids were then transformed into the BL21(DE3) strain of E. coli. The T7 RNA polymerase gene was chemically induced by adding 0.2 mM isopropyl-1-thio--D-galactopyranoside to the cell cultures grown to an OD of 0.6-1 and incubation for 3 h at 25 °C. The cells were collected by centrifugation, resuspended in the lysis buffer (0.1 M potassium phosphate buffer, pH 7.2, containing 0.1 M KCl, 1% n-octyl glucoside, 1 mM EDTA, and 20 µg/ml PMSF), sonicated three times for 5 s each, and centrifuged. The protoporphyrinogen oxidase activity in the resulting cell-free extracts was measured. The homogenates were diluted twice in Laemmli sample buffer and processed for electrophoresis and immunodecoration as described below.

Purification of Yeast Protoporphyrinogen Oxidase

Miscellaneous

Protoporphyrinogen oxidase was assayed by measuring the rate of appearance of protoporphyrin fluorescence(46) . Enzyme assays were carried out at 30 °C. The incubation mixture was 0.1 M potassium phosphate buffer, pH 7.2, saturated with air, containing 2 µM protoporphyrinogen IX, 3 mM palmitic acid (in dimethyl sulfoxide, 0.5%, v/v, final concentration), 5 mM DTT, 1 mM EDTA, and 0.3 mg/ml (final concentration) Tween 80 to ensure a maximum fluorescence signal of protoporphyrin IX. Protoporphyrinogen was prepared by reducing protoporphyrin IX hydrochloride dissolved in KOH/EtOH (0.04 N, 20%) with 3% sodium amalgam(21) .

RESULTS

Identification and Deletion of the HEM14 Gene

The plasmid carrying the shortest insert (4.9 kb) was further analyzed. Subcloning and deletion analysis of the insert indicated that the complementing activity was restricted to a 2.5-kb fragment (Fig. 1). This fragment was sequenced (see ``Experimental Procedures'') and found to contain a 1.6-kb open reading frame encoding a 539-amino acid protein (Fig. 2). The phenotype of a cell lacking this gene was determined by removing a 439-bp NruI-EcoRV fragment containing the initiation codon and the 120 amino-terminal residues from the cloned insert and replacing it with a 820-bp fragment containing the TRP1 gene. A 2.7-kb linear BglI fragment, now containing the TRP1 substitution, was transformed into haploid trp1 cells. Tryptophan prototroph transformants were selected on a synthetic medium supplemented with Tween 80, ergosterol, and hemin. The transformants had a phenotype closely related to that of the original hem14-1 mutants. They were respiratory deficient (no growth on glycerol or ethanol as sole source of carbon), they accumulated porphyrins (Fig. 3) and had no detectable protoporphyrinogen oxidase activity in vitro. However, they were strictly auxotroph for Tween 80 and ergosterol (Te), while the original mutant could sustain some growth without Te. The cells disrupted for the HEM14 gene also lacked any immunodetectable protoporphyrinogen oxidase protein (Fig. 4). Similar phenotypes were obtained by deleting most of the open reading frame by replacing the 1493 bp NruI-EcoRI fragment of HEM14 (Fig. 2) by the 820-bp fragment containing the TRP1 gene.

Figure 1: Restriction map of the yeast genomic DNA fragment complementing the hem14-1 mutation and subcloning analysis. Upper maps, restriction sites from the cloned fragments; lower maps, restriction sites from the vectors. Protox, protoporphyrinogen oxidase.

Figure 2: Nucleotide sequence of the HEM14 gene and deduced amino acid sequence of protoporphyrinogen oxidase. Putative TATA box (&cjs0808;&cjs0808;) and transcription termination signals (-) are underlined. The NruI, EcoRV, and EcoRI recognition sequences are in boldface type. The stop codon of PRP22 and initiation codon of FAA2 are boxed.

Figure 3: Low temperature spectra of yeast cells (480-640 nm). All the strains were grown on YPG-Te. The cells were collected during the stationary phase of growth. A, S150-2B; B, CC750-3D; C, CC750-3D transformed by plasmid pBS19-1; D, S150-2Bhem14::TRP1; E, S150-2Bhem14::TRP1 transformed by plasmid pBS19XS9.

Figure 4: Immunocharacterization of yeast protoporphyrinogen oxidase after SDS-polyacrylamide gel electrophoresis of total protein extracts from yeast cells collected during the stationary phase of growth on YPG-Te. 1, S150-2B (10 µg of protein); 2, S150-2Bhem14::TRP1 (20 µg protein); 3, S150-2Bhem14::TRP1 transformed by plasmid pBS19XS9 (3 µg of protein). Panel A, Ponceau S staining of total proteins; Panel B, immunodetection of yeast protoporphyrinogen oxidase.

Mapping of the HEM14 Gene

Nucleotide Sequence of HEM14 and Features of the Deduced HEM14 Protein Sequence

Figure 5: Hydropathy plot of yeast protoporphyrinogen oxidase, according to Kyte and Doolittle, and distribution of acidic (A) and basic (B) amino acid residues in the protein.

Figure 6: Alignment (Clustal-w) of yeast (SCHEM14), B. subtilis (BSHEMY), human (HSPOX), and mouse (MMPOX) protoporphyrinogen oxidases. Residues identical in three or four sequences are boxed. Symbols over the yeast sequence are defining a putative ADP--binding domain according to Wierenga et al.(55) . (), basic or hydrophylic; (), small or hydrophobic; (), glycine; (), acid.

Optimization of the Overexpression of the HEM14 Gene Product

Figure 7: Protoporphyrinogen oxidase activity in cell-free extracts from cells harvested at various stages of aerobic growth on glucose, galactose or ethanol as source of carbon.

The physicochemical and kinetic properties of overexpressed protoporphyrinogen oxidase were further analyzed. The membrane bound overexpressed protoporphyrinogen oxidase was inhibited by diphenyl ether type herbicides, acifluorfen-methyl (methyl-5-[-2-chloro-4-(trifluoromethyl)-phenoxy-]-2-nitrobenzoic acid) and oxadiazon (5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol-2-one) at concentrations comparable that inhibit wild-type yeast; the IC were 2 and 30 nM, respectively (Fig. 8)(30) . Protoporphyrinogen oxidase was purified to homogeneity (200-fold, 60% recovery) from the mitochondrial membranes of the transformed cells. The specific activity of the enzyme was 43,600 nmol of protoporphyrinogen oxidizedhmg protein. The Michaelis constant for protoporphyrinogen was 0.02 µM. The enzyme was specific for protoporphyrinogen IX and did not oxidize coproporphyrinogen III to coproporphyrin III. The absorption spectrum of the enzyme as purified (Fig. 9) was typical of a flavoprotein with a flavin semiquinone. Preliminary electron spin resonance spectroscopy experiments did not detect the stable free radical, expected in such a redox state of the flavin (data not shown). The reactivity of purified protoporphyrinogen oxidase toward a photoaffinity probe (32) was identical to that of the wild-type enzyme. The protoporphyrinogen oxidase polypeptide appeared to be specifically labeled by diazo-[H]acifluorfen (Fig. 10, lane B). The labeling of the purified protein was completely blocked by 10 µM acifluorfen-methyl, an efficient inhibitor of protoporphyrinogen oxidase (Fig. 10, lane C). The NH-terminal peptide sequence of the protein was MLLPLTKLKPRAKVAVV. This sequence is identical to that deduced from the first ATG (Met) of the open reading frame of the HEM14 gene.

Figure 8: Inhibition of protoporphyrinogen oxidase activity from cells overproducing the enzyme, by the diphenyl ether-type herbicides acifluorfen-methyl () and oxadiazon ().

Figure 9: Absorption spectrum of purified overexpressed yeast protoporphyrinogen oxidase (230-650 nm). Insert, 5 magnification of the 350-650-nm region.

Figure 10: Photoaffinity labeling of purified overexpressed yeast protoporphyrinogen oxidase. Lane A, C-radiolabeled molecular mass markers; lane B, 10 ng of photolabeled enzyme; lane C, 10 ng of enzyme photolabeled in the presence of 10 µM acifluorfen-methyl as unlabeled competitor.

Sequencing of the hem14-1 Mutant Allele

Expression of Yeast Protoporphyrinogen Oxidase in E. coli and Site-directed Mutagenesis

DISCUSSION

We have isolated the HEM14 gene that encodes yeast protoporphyrinogen oxidase by functional complementation of a heme-deficient hem14-1 strain. It has been recently demonstrated that several oxidases are able to oxidize protoporphyrinogen to protoporphyrin in vitro(10, 11, 12) . Such oxidases may complement our mutant, depending on their intracellular location and concentration. It was therefore essential to establish nonambiguously that HEM14 is the structural gene of yeast protoporphyrinogen oxidase. This was done by genetic and biochemical studies. First, the hem14-1 mutation was complemented by the cloned gene on both single and multiple-copy plasmids. Second, yeast strains with a disrupted HEM14 open reading frame were constructed. These strains had the phenotypes of a protoporphyrinogen deficiency, producing a respiratory deficiency, accumulation of porphyrins and lack of immunodetectable protoporphyrinogen oxidase protein. Third, the HEM14 gene product (characterized by the NH-terminal protein sequence) was overexpressed in yeast. The product was recognized by anti-yeast protoporphyrinogen oxidase antibodies and was purified as authentic protoporphyrinogen oxidase. The protein was fully inhibited by a variety of diphenyl ether-type herbicides and labeled by photoaffinity using a highly specific probe. Fourth, two mutations in the hem14-1 allele were characterized, and the mutations were further analyzed by site-directed mutagenesis of the wild-type allele in an expression system for the HEM14 gene in E. coli.

The mapping of the HEM14 gene on chromosome V was demonstrated by both genetic linkage to URA3, a known marker of this chromosome and sequence specific in vitro amplification of the HEM14 locus on selected fragments of an ordered library of chromosome V(51) . The genes flanking HEM14 are PRP22 at the 5` end of HEM14 and FAA2 already mapped on chromosome V(50) , at the 3` end of HEM14. The approximate genetic distance between URA3 and HEM14 (17 cm, 16 tetrades analyzed) is compatible with the physical distance calculated from Isono's and Rile's maps (60 kb) and from the complete sequence of a set of ordered cosmids covering the entire chromosome V, as the contribution led by Dr. D. Bostein to the Yeast Genome Sequencing Project. Our results allow the ``putative 59.5-kDa open reading frame'' between PRP22 and FAA2 to be assigned to HEM14. HEM14 lies in a quite compact region of chromosome V, since the distance between the TAA stop codon of PRP22 and the ATG initiation codon of HEM14 is only 322 bp, and the distance between the TAA stop codon of HEM14 and the ATG initiation codon of FAA2 is only 321 bp. A single plasmid complementing the hem14 mutation was obtained by screening a yeast DNA library generated by partial HindIII digestion of genomic DNA. The insert was very long (>15 kb). This result is consistent with the fact that the PRP22-HEM14-FAA2 locus lies in a 20-kb HindIII fragment.

Several features of yeast protoporphyrinogen oxidase were revealed by the amino acid sequence of the protein deduced from the nucleotide sequence of the HEM14 gene which were not predicted by the biochemical characteristics of the enzyme. The predicted molecular mass of the HEM14 gene product is 59 kDa, close to the 58-kDa determined for the precursor form of the protein, with an isoelectric point of 9.3 comparable to that of the purified protein (pI >8.5). Yeast protoporphyrinogen oxidase was described as an integral protein of the inner mitochondrial membrane(30) . The predicted sequence of protoporphyrinogen oxidase should therefore contain those amino acid sequences that target the protein to the mitochondria and anchor it to the lipid bilayer. The first 13 amino acids of the open reading frame of the HEM14 gene contains all the information typical of a protein imported to the mitochondria, without acidic residues, but with high contents of Leu, Arg, Lys and Thr, and a propensity to form an amphiphilic helix. This sequence is immediately followed by a putative -dinucleotide binding fold, suggesting that the 13 amino-terminal residues may act as a cleaved presequence. We therefore determined the NH-terminal sequence of purified protoporphyrinogen oxidase. The terminal 17 residues are identical to the deduced NH-terminal sequence of the HEM14 gene product, and starts with the initiation methionine, as expected from the known specificity of the yeast methionine aminopeptidase that do not cleave the Met-Leu bond(56) . The lack of cleavage of the mitochondrial targeting sequence of the protein during the importation of the protein into the mitochondria is not usual for an inner membrane-bound protein, but a sequence as short as 11 residues has been characterized as the uncleaved targeting sequence of the mitochondrial isoform of the tRNA N-adenosine isopentyl transferase in yeast(57) . Although the sequence of protoporphyrinogen oxidase contains several hydrophobic regions, none of those is longer than 15 uncharged residues and they are therefore unlikely to form membrane spanning segments. However, shorter helical domains could be responsible for insertion of the protoporphyrinogen oxidase into the inner mitochondrial membrane, as described for prostaglandin H synthase-1, a monotopic membrane-bound protein of the endoplasmic reticulum(58) . Another possibility, compatible with a post-translational modification of the protein leading to the shift in electrophoretic mobility initially attributed to the proteolytic cleavage of a putative presequence previously described(30) , is that protoporphyrinogen oxidase is anchored to the inner mitochondrial membrane by a different mechanism, such as acylation. Preliminary experiments tend to support this hypothesis. ()

One of our goals is to obtain enough yeast protoporphyrinogen oxidase for structural studies on the protein. Commercially available yeast cells contain slightly more protoporphyrinogen oxidase activity and protein than laboratory grown cells. The industrial strains are usually grown on molasses and harvested in the late stationary phase of growth. This indicated that protoporphyrinogen oxidase seems to be fairly stable under such conditions, but the enzyme accounts for less than 0.001% of the membrane-bound proteins. We further optimized the production of the enzyme in our transformed laboratory strains by measuring protoporphyrinogen oxidase activity during the aerobic growth of yeast cells in media containing various carbon sources. Protoporphyrinogen oxidase activity was maximum during the stationary phase of growth of cells up to 8000 units (0.2 mg)/g (wet weight) of transformed cell in all cases. The activity during the exponential phase of growth was 2-3 times higher in cells grown on galactose or ethanol than in cells grown on glucose. Detailed analysis of the promotor of HEM14 will be needed to find out more about the mechanism and physiological relevance of HEM14 expression regulation in yeast. The overexpressed protein is associated with the membrane fraction. Both the membrane-bound and the purified enzyme are inhibited by diphenyl ether type herbicides and specifically photolabeled with an affinity probe, as is the enzyme from a wild-type strain.

The primary sequences of yeast protoporphyrinogen oxidase and related proteins (mammalian protoporphyrinogen oxidases and HemYp) are not easy to compare because of the overall low level of similarity of the proteins; the best conserved domain is the -ADP binding fold in the NH terminus of these proteins. If the targeting sequence of yeast protoporphyrinogen oxidase is short and not cleaved, the mammalian protoporphyrinogen oxidases are synthesized without such mitochondrial targeting sequences and therefore have a NH terminus starting with the -ADP binding fold as do monoamine oxidases, proteins associated with the outer membrane of mitochondria. As mentioned previously, this motif is found in the NH-terminal domain of several well characterized flavoproteins (monoamine oxidases, amino-acids oxidases, and so forth) and, in this area, of some proteins of unknown function. A search in the S. cerevisiae genome data base (at www.genome-stanford.edu) with ``protoporphyrinogen'' as a keyword identified the YHR009c open reading frame. Careful examination of this sequence showed that this gene product has a NH-terminal sequence very similar to that of HemYp (and therefore to the yeast protoporphyrinogen oxidase) but has no other domain similar to either HemYp or HEM14p. This unknown protein may, however, be a flavoprotein. The central question is to determine whether the conserved residues in the alignment of the yeast mammalian and bacterial proteins are involved in the substrate specificity of the enzymes and/or the catalytic activity. The B. subtilis enzyme has a poor substrate specificity, while the eukaryotic enzymes are more specific for protoporphyrinogen IX. However, the mammalian protoporphyrinogen oxidases are more closely related to HemYp than to the yeast enzyme, especially in their COOH-terminal domain. The two similar blocks in the mammalian and yeast enzymes are in poorly structured domains. These domains may, however, be important for the specificity of the enzyme.

The characterization of mutations affecting protoporphyrinogen oxidase activity should help determine the structure/function relationships. The short deletion affecting the HemYp function recently described (28) is located close to the end of the -ADP binding fold. The present paper, describes the first mutations affecting an eukaryotic protoporphyrinogen oxidase. The hem14-1 mutant allele carries two point mutations that introduce two changes in amino acids in a relatively COOH-terminal part of the protein, without significant similarity to the mammalian or bacterial protoporphyrinogen oxidases. The two mutations, L422P and K424E, are located in a domain close to a cysteine residue (Cys-435) in a Gly-Gly-Cys motif that may, by analogy to bovine monoamine oxidase(59) , be involved in the covalent binding of FAD to the enzyme. The two mutations are rather different in nature. A proline may drastically affect secondary structure in the protein, while the lysine to glutamic acid change may affect the catalytic properties of the enzyme. One model of how the enzyme acts involves the removal of hydrogen from protoporphyrinogen as at least one hydride involving the flavin, and one proton involving a basic residue in the protein. It is thus tempting to speculate that Lys-422 may be this basic residue. Alternatively, the lysine may help stabilize the flavin in an anionic semiquinone form necessary for activity, as in other oxidases (for a review, see (60) ). In a preliminary attempt to better characterize each mutation, we introduced the two mutations independently into the wild-type gene cloned in an expression system in E. coli. Both mutations led to the production (under standard conditions) of an inactive protein in E. coli. These mutated genes will now be introduced into the yeast strain lacking a functional HEM14 gene to detect residual activity through functional complementation or the involvement of some post-translational modification affecting the functioning of the mutated enzymes in vivo and in vitro.

The availability of transformed cells overexpressing protoporphyrinogen oxidase (yeast and E. coli) overcomes the basic barriers to investigating the structural properties of the enzyme and the topology of its active site, due to the small amounts of the protein in wild-type yeast strains. This approach will also help the characterization of the molecular changes involved in the mutation in the protoporphyrinogen oxidase-deficient yeast strain that provided the basis for site-directed mutagenesis of the yeast protoporphyrinogen oxidase gene. Our present results provide evidence that yeast protoporphyrinogen oxidase is an adequate model to elucidate the molecular basis of protoporphyrinogen oxidase function, and hence the mechanisms underlying the pathophysiology associated with the enzyme defect (human porphyria) and inhibition (herbicidal effects of diphenyl esters).

FOOTNOTES

*

This work was supported by grants from the Centre National de la Recherche Scientifique and Université Paris 7. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed. Tel.: 33-1-44 27 63 56; Fax: 33-1-44 27 57 16; jec{at}ccr.jussieu.fr.

()

The abbreviations used are: kb, kilobase pair(s); bp, base pair(s); PCR, polymerase chain reaction; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride.

()

S. Arnould and J.-M. Camadro, unpublished results.

ACKNOWLEDGEMENTS

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