the Yeast Saccharomyces cerevisiae

Vol. 269, No. 51, Issue of December 23, pp. 32085-32091, 1994 Printed in U.S.A.

THEJOURNAL OF BIOL~CICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Purification and Propertiesof Protoporphyrinogen Oxidasefrom the Yeast Saccharomyces cerevisiae MITOCHONDRIAL LOCATIONAND EVIDENCE FOR A PRECURSOR FORM OF THE PROTEIN* (Received for publication, May 18, 1994, and in revised form, September 22, 1994)

Jean-Michel CamadroS, Franqoise Thome, Nicole Brouillet, and Pierre Labbe From the Laboratoire de Biochimie des Porphyrines, Ddpartement de Microbiologie, Znstitut Jacques Monod, 2 place Jussieu, F-75251 Paris Cedex 05, France

Protoporphyrinogen oxidase, the molecular target of to maintaining anaerobic conditions during growth, there is diphenylether-type herbicides, was purified to homoge- enough oxygen present for protoporphyrinogen oxidase to function if the affinity of the enzyme for oxygen is extremely high. neity from yeast mitochondrial membranes and found to be a 55-kDa polypeptide with a PIof 8.5 and a specific The second particularity of the yeast S. cerevisiae is that the activity of 40,000 nmol of protoporphyriddmg of pro- terminal enzymes of the heme biosynthesis pathway are not tein at 30 "C. The Michaelis constant (K,)for protopor- distributed in the same way as they are in mammalian cells. phyrinogen M was 0.1p ~Due . to the high affinity of the Coproporphyrinogen oxidase, the enzyme preceding protoporenzyme toward oxygen,the K,,, for oxygen could only bephyrinogen oxidase in the heme biosynthesis pathway, lies in . purified enzyme con- the cytosol of yeast cells (5) rather than in the intermembrane approximated to 0.5-1.5 p ~The tained a flavinas cofactor. Studies with rabbit antibod- space of mitochondria as in higher eukaryotes (6, 7). Yeast coproporphyrinogenoxidasealso requires molecular oxygen ies to yeast protoporphyrinogen oxidase showed that the enzyme is synthesized as a high molecular weight and is present in large amounts inanaerobically grown yeast precursor (58 kDa) that is rapidly converted in vivo to cells, since its synthesisis negatively regulated by oxygen (and the mature (55 kDa) membrane-bound form. Protopor- heme) (8, 9). This raises the questionof whether, in addition to phyrinogen oxidaseactivity was found onlyin purified the problem of oxygen availability for both oxidases, protoporyeast mitochondrial inner membrane (not in the outer phyrinogendelivery to protoporphyrinogenoxidase mayin membrane). Acifluorfen-methyl, a potent diphenylethersome way regulate heme synthesis if that enzyme, like the type herbicide, competitively inhibited the purified en- mammalian enzyme, is an inner mitochondrial membrane= 10 m). The mixed inhibition by acifluorfen- bound enzyme. zyme (Ki methyl previously reported for the membrane-bound Poulson and Polglase (2) were the first to partially purify J. M., Matringe, protoporphyrinogen oxidase from yeast cells. The enzyme was protoporphyrinogen oxidase (Camadro, M., Scalla, R., and Labbe, P. (1991) Biochern. J. 277,17-21) described as a 180 f 18-kDa protein whose activity had a opwas shown to be related to partial proteolysis of the timum pHof 7.5 and was inhibitedby sulfhydryl reagents. The enzyme. measured K,,, for protoporphyrinogenoxidase was 4.8 (2). Protoporphyrinogenoxidase has beenpurifiedfrom other sources and appears to be rather heterogenous in terms of Protoporphyrinogen oxidase (EC 1.3.3.4) is the penultimate molecular and kinetic properties. The beef liver enzyme (57 enzyme of the hemebiosynthetic pathway. It catalyzes theoxi- kDa) was reported having peptides and antigenic determinants dative 0,-dependent aromatization of the colorless protopor- in common with ferrochelatase, the last enzyme of the heme phyrinogen IX to highly conjugated protoporphyrin M (Fig. 1). biosynthesis pathway, which is an inner mitochondrial memThe existence of an enzyme catalyzing this oxidative step has brane-bound enzyme in mammals(10). The beef liver protoporlong been controversial, since porphyrinogensoxidize rapidly to phyrinogen oxidase was also shown to be a flavoprotein contheir corresponding porphyrins in the presence of air via a taining FAD (11).The mouse liver protoporphyrinogen oxidase light-sensitive autocatalytic reaction (1) was described as a 65-kDa polypeptide devoid of any cofactor The function of the yeastSaccharomyces cerevisiaeprotopor- (121, but recentwork indicates that the mouse enzymecontains phyrinogen oxidase in vivo raises two main questions. First, FMN (13).The enzyme partially purified from barley (35 kDa) molecular oxygen was described as the only electron and pro- was found to be identical in mitochondria and etioplasts (14). tons acceptor of the enzyme (21, but anaerobically grown yeast The only bacterial protoporphyrinogen oxidase purifiedso far is cells synthesize heme and hemoproteins (cytochromes b5 and that of Desulfovibrio gigas, which appeared to be a multimeric P450) (31, and protoporphyrinogen oxidase activity was meas- polypeptide (M, = 148,000, dissociating into three peptides of uredinthe promitochondrial membranes of anaerobically M , = 57,000, 18,500, and 12,000 under reducing conditions) grown cells (4). Therefore, there is either an unknown electron (15). The catalytic properties of the differentenzymes display a acceptor for protoporphyrinogen oxidase activity in Saccharo- similar diversity with different optimum pHs, maximum vemyces cereuisiae, a facultative aerobe, or despite the care taken locities,Michaelis constants for protoporphyrinogen IX, and substrate specificities (11, 12, 14, 16, 17). It has been recently demonstrated that diphenylether-type * This work was supported by grants from the Centre Nationalde la Recherche Scientifique, the Institut National de la Recherche herbicides are very potent inhibitorsof the protoporphyrinogen Agronomique, and Universite Paris 7. The costs of publication of this oxidase activities of yeast, mammalian, and plant mitochondria article were defrayed in part by the payment of page charges. This and plant chloroplasts in vitro (18, 19). Paradoxically, diphenarticle must thereforebe hereby marked "aduertzsenent" in accordance ylether-type herbicides can induce the accumulation of massive with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence shouldbe addressed. Tel.: 33-44-27-63-56; amounts of protoporphyrin M in vivo in both plants (20-24) Fax: 33-44-27-57-16; E-mail: [email protected]. and mammals (25, 26). This mimics the symptoms of human

32085

Purification and Properties

32086 w,

HC=CH,

w ,

of ~C=CH,

Yeast Protoporphyrinogen Oxidase fractions were further purified by differential centrifugation followed by sedimentation on isokinetic continuous sucrose gradient. Published procedures were used to assay marker enzymes: antimycin-sensitive NADH cytochrome c reductase (31) and cytochrome c oxidase (32) for the inner membrane and antimycin-insensitive NADH cytochromec reductase (31) for the outer membrane. Protoporphyrinogen oxidase wasassayed as described in this paper, and ferrochelatase assayed as in Ref. 33.

Immunodetection of Protoporphyrinogen Oxidase Published procedures were used to prepare extracts from trichloroacetic acid-treated cells (341, for SDS-polyacrylamide gelelectrophoreProtoporphyrinogen IX (A) Protoporphyrin1X (B) sis (35), and for electrophoretictransfer of the proteins to nitrocellulose FIG.1. Structuresof protoporphyrinogen M (A) and protopor- sheets (36).Preparations were incubated with the anti-serum (IgG fraction) and visualized with alkaline phosphatase-conjugated anti-rabbit phyrin M ( B ) . IgG-secondary antibodies as recommended by the manufacturer (Promega Biotec). patients with the inherited disease porphyriavariegata in in vivo was immuProtoporphyrinogenoxidase metabolically labeled which there is adeficit of protoporphyrinogen oxidase activity noprecipitated according to Maccechini et al. (37) as modified byUrban(27). A clear understanding of the interactions between the Grimal et al. (38). Yeast cells grown on a semi-synthetic medium demembrane-bound enzyme and the inhibitors could wellhelp to prived of sulfate ions (34) were harvested during the early exponential elucidate the molecular basis of protoporphyrinogen oxidase growth phase and resuspended in 25 m sodium citrate buffer, pH 6.5, containing 10% glucose, [35Slmethionine(500 mCi/ml), and 10 &ml function and hence the mechanisms underlying the patho- tyrosine to increase methionine uptake efficiency (39). Aliquots were physiology associated with the enzyme defect (human porphy- taken after incubation times from 15 s to 1h and were diluted with cold ria) or inhibition (herbicidal effects of diphenylethers). methionine. The cells were broken with glass beads, the proteins preAs a first step inour ongoing study of the function of proto- cipitated with 20% trichloroacetic acid, and samples were analyzed for porphyrinogen oxidase and the molecular basis of its interac- immunoprecipitable protoporphyrinogenoxidase.Sinceprotoporphytions with diphenylethers, we have determined the intracellu- rinogen oxidase represents only 0.005%of the total yeast proteins, two consecutive immunoprecipitations were required for analysis of the lalar location of yeast protoporphyrinogen oxidase and purified beled material. The first immunoprecipitated pellet was dissolved in the the enzyme. Immunochemical studies showed that protopor- SDS-PAGE sample buffer, and labeled proteins were precipitated with phyrinogenoxidase is an inner mitochondrial membrane- trichloroacetic acid (20% final concentration). The precipitate was rebound enzymesynthesized as a largerprecursor. It is activated dissolved with 50 mM Tris-HC1, pH 8.0, containing 150 mM NaC1,5 mM during its incorporation into the mitochondria asis yeast EDTA, and 1%Triton X-100, processed again for immunoprecipitation and analyzed by SDS-PAGE.

ferrochelatase (28).

MATERIALS AND METHODS

Chemicals

Protein Assays Protein concentrations were determined by the method of Lowry, as modified by Wangand Smith(40)for the initial steps of purification. For the last steps of the purification, proteins were quantified by total amino acid composition using a Waters Associates Pico-Tag" analysis and high performance liquid chromatography separation system after acid hydrolysis of proteins in uacuo.

Phenyl-Sepharose CL-GB, Sephadex G25,DEAE Sepharose CL-GB Fast flow, Polybuffer Exchanger PBE 94, Polybuffer 96, and molecular mass standard proteins for Coomassie Blue staining of SDS-PAGE' were fromPharmacia Fine Chemicals, Les Ulis,France; labeled molecular mass standard proteins, [35Slmethionine,and rabbit reticulocyte Flavin Determination lysate were from AmershamFrance SA, Les Ulis,France. ProtoporphyFlavins were analyzed spectrofluorimetrically(41,42) by measuring rin JX hydrochloride was fromPorphyrin Products, Logan, UT; PMSF, leupeptin, and pepstatin were from Sigma;dithiothreitol, CHAPS, and the difference in the fluorescence intensities of the cofactor in enzyme Tween 80 were from Serva, Heidelberg, Germany. Anti-rabbit IgG-al- preparations at pH 3 and pH 7 (Aexc = 450 nm; emissionspectra recorded kaline phosphatase-conjugated antibodies and alkaline phosphatase from 480 to 600 nm). substrates were fromPromega Biotec, Madison, WI; acifluorfen sodium Protoporphyrinogen Oxidase Assay salt was fromChemservice,West Chester, PA. Acifluorfen-methyl Protoporphyrinogen oxidase was assayed by measuring the rate of (AFM) wasprepared by esterification of acifluorfen with diazomethane. appearance of protoporphyrin fluorescence (4,43).Enzyme assays were carried out at 30 "C.The incubation mixture was 0.1 M potassium phosYeast Strains and Growth Conditions phate buffer, pH 7.2, saturated with air, containing 2 p~ protoporphyThe commercially available bakers' yeast S. cereuisiae, from which rinogen E,3 m~ palmitic acid (in dimethyl sulfoxide 0.5% (v/v) final protoporphyrinogenoxidasewas purified, was fromFould Springer, concentration),5 m~ DTT, 1mM EDTA, and 0.3 mg/ml (final concentraMaisons Alfort,France. tion) Tween 80to ensure maximum fluorescencesignal of protoporphyAlaboratory haploid strain D273-10B (Mat a, ATCC 25657)was used rin E.Protoporphyrinogen was prepared by reducing protoporphyrin for subcellular localization,immuno-characterization, and in uiuo labelM hydrochloride dissolvedin KOHiEtOH (0.04N, 20%)with 3% sodium ing of protoporphyrinogen oxidase. Cells were grown in a complete amalgam ( 2 ) . medium containing 1%yeast extract, 1%Bactopeptone,2%glucose Protoporphyrinogenoxidase activity was initiated by the addition of (autoclaved separately), and 1 g/liter Tween 80 plus 20 mgfliter ergosthe enzymatic fraction to the assay medium and followed by (i)continuterol (29). Cells were harvested during the early exponential phase of ously recording the increase in protoporphyrin fluorescence in 3-ml growth and processed immediately. glass cuvettes with magnetic stirring (method used for all kinetic studies) or, (ii) time-dependent measurement of fluorescence in 1-ml glass Subcellular Distribution of Yeast Protoporphyrinogen Oxidase tubes (method used for monitoring protoporphyrinogen oxidaseactivity Mitochondriawere prepared from protoplasts of galactose-grown in columns effluents). yeast cells (strain D273-10B) and purified on a discontinuous Percoll The Michaelis constant for molecularoxygen was estimated by using gradient. Inner and outer membranes were then prepared as described an oxygen-depletedassay medium. This was prepared by degassing and by Riezman et al. (30). The purified mitochondria were allowedto swal- then flushing with nitrogen the incubation medium without the enlow, shrunk inthe presence ofATP, and thenwere sonicated. Membrane zyme. To remove traces of dissolved oxygen, we added to the assay an enzymatic oxygen trap consisting of a glucose/glucose oxidase/catalase The abbreviations used are: PAGE, polyacrylamide gelelectrophore- mixture (10md10 units/lunit). Any contact with air was prevented by sis; PMSF, phenylmethylsulfonyl fluoride; AFM, acifluorfen-methyl; overlaying the assay medium with mineral oil. The efficiency of the DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammoniol- anaerobiosis-promotingsystems was checked in parallel assays by OXYpolarographic measurements of oxygen consumption in the incubation 1-propanesulfonate.

32087

Purification and Properties of Yeast Protoporphyrinogen Oxidase medium with a Clark-type oxygen electrode. Protoporphyrinogen oxidase reaction was initiatedby the additionof the enzyme. Theconcentration of dissolved oxygen in the incubation medium was then varied from 0.5 to 20 p~ by the addition to the anaerobic medium of known volumes of 0.1 M potassium phosphatebuffer, pH 7.2, saturated withair (the concentrationof dissolved oxygen in this buffer is 236 p~ at 30 "C). One unit of protoporphyrinogen oxidase activity is the amount of enzyme needed to catalyze the formation of 1 nmol of protoporphyrin M per hour at 30 "C in the standard assay system.

TABLEI Distribution of yeast protoporphyrinogen oxidase activity between inner a n d outer mitochondrial membrane Enzyme

199 Cytochrome c oxidase NADH cytochrome c reductase (antimycine sensitive) NADH cytochrome c reductase (antimycine insensitive) Ferrochelatase" Protoporphyrinogen oxidase"

Inner membrane

Outer membrane

nmollminlmgprotein 1157 814 42

93 328 Purification of Protoporphyrinogen Oxidasefrom Yeast Cells All operations were carried out at 4 "C. The purification procedures 14 0.01 described below gave identical results when used with commercially 27 Nondetectable produced yeast cells (kg of starting material) and laboratory grown Activities are expressed in nmoWmg of protein. strains (gof starting material). Membrane fractions enriched in mitochondrial membranes were prepared as described (33). Protoporphyrinogen oxidase activity was solubilized by diluting the Purification and Properties of Yeast Protoporphyrinogen membrane fraction to a protein concentration of 20 mglml in 0.1 M Oxidase-Preliminary experiments showed that the enzyme potassium phosphate buffer, pH 7.2, containing 5 mM CHAPS, 1 mM DTT, 0.1 mM EDTA, and 0.5 m~ PMSF. The membrane suspension was cannot be extracted from the membranes by alkali (sodium is gently homogenized with a Potter homogenizer and stirred for 1 h; the carbonate 0.1 M) or chaotropic reagents but that itsolubilized insoluble material was removed by centrifugation for 45 min at 100,000 by detergents. Several neutral or ionic detergents were tried x g. Protoporphyrinogen oxidase activity was recovered in the soluble (Triton X-100, Tween 80, sodium cholate, CHAPS). Triton protein fraction. X-100 was themost efficient at solubilizing protoporphyrinogen precipiStep 1: Ammonium Sulfate Fractionation-Proteins were oxidase. However, for unknown reasons, this detergentdid not tated by the addition of solid ammonium sulfate to45%saturation and allow the furtherpurification of the enzyme by ionic interaction stirring for 30 min at 4 "C. The precipitated proteins were pelletedby chromatography. We therefore used the ionic detergent centrifugation for 30 min at 40,000 x g. Protoporphyrinogen oxidase activity remained in the supernatant. Tween 80 (0.2%(w/v)) was added CHAPS. This solubilized protoporphyrinogen oxidase with a to the enzyme solution, and the mixture was dialyzed against phenyl-good yield (60%,Table 11),but it has themajor disadvantage of Sepharose equilibration buffer until theTween 80 was dissolved. rapidly denaturing theenzyme. This was overcome by an iniStep 2: Hydrophobic InteractionsChromatography-The dialyzed ential protein fractionation with ammonium sulfate and the rapid flow column zyme solution was passed through a phenyl-Sepharose fast exchange of CHAPS for Tween 80 by a short dialysis. Tween 80 (2.6 x 40 cm) equilibrated with100 mM potassium phosphatebuffer, pH was added to the protein solution in the dialysis tubing. The 7.2, containing 1 M KCl, 20% (wh) glycerol, 1 m~ DTT, 0.1 mM EDTA, and 0.5 mM PMSF. The column was washed with the same buffer fol- Tween 80 did not dissolve initially because of the high ionic lowed by the same buf€er without KCl. The enzyme was eluted by strength. But the salt concentration decreased during dialysis, adding 2% (w/v) Tween 80 in 10 mM potassium phosphatebuffer, pH 7.2, allowing Tween 80 to dissolve. The process was stopped as soon containing 20% (w/v) glycerol, 1 mM DTT, 0.1 mM EDTA, and 0.5 mM completely dissolved. Protoporphyrinogen PMSF. Active fractions werepooled and concentratedon Amicon YM 30 as the detergent was oxidase was further purified by loading the protein solution filters. onto a phenyl-Sepharose chromatography column. The enzyme Step 3: Zon Exchange Chromatography-The concentrated enzyme a DEAE-Sepharose fastflow column (2.6 x solution was passed through a low ionic strength buffer in the activity was eluted with 20 cm) equilibrated with 10 mM potassium phosphate buffer, pH 7.2, presence of2%of the neutral detergent Tween 80. This step 0.2%(w/v) Tween 80,20%(w/v) glycerol, 1 m~ DTT, 0.1 mM EDTA, and was very efficient for purifying yeast protoporphyrinogen oxi0.5 mM PMSF. Protoporphyrinogen oxidase did not bind toDEAE, and active fractions were recovered in thevoid volume of the column. Pro- dase, but at least 20 other polypeptides were eluted with the protoporphyrinogen oxidase when analyzed by SDS-PAGE. teins were concentrated witha Amicon YM 30 filters. The high PI of yeast protoporphyrinogen oxidase (8.5) was Step 4: Chromatofocusing-The active fractions were loaded onto a Mono P HR 5/20 fast protein liquid chromatography column equili- the key to further purification. Exclusion chromatography on brated in 25 mM ethanolamine-HC1,pH 9.4, containing 0.2% (wh) DEAE-Sepharose (pH 7.25) ensured the adsorption of most Tween 8 0 , 20%(wh) glycerol, 1 mM DTT, 0.1 mM EDTA. Protoporphy- proteins except protoporphyrinogen oxidase. The breakrinogen oxidase was eluted witha linear gradient of pH produced by a solution of 10%polybuffer 96, 0.2%(w/v) Tween 80,20%(w/v) glycerol, through was loaded onto a fast protein liquid chromatography 1 mM DTT, 0.1 m~ EDTA, adjusted t o pH 6.0 with acetic acid. Theflow chromatofocusing column equilibrated at pH 9.4. ProtoporphypH 8.5 rate was1 mumin, and2-ml fractions werecollected in tubes containing rinogen oxidase bound to thecolumn and was eluted at 0.2 ml of 1 M potassium phosphate buffer, pH 7.2, to minimize the time by a linear gradientof pH. The enzyme appeared tobe purified that the enzyme was at alkaline pH. Active fractions were pooled and to homogeneity and displayed a singleband on SDS-PAGE with concentrated on Centricon PM30 filters (Amicon). an apparent molecular mass of 55 kDa (Fig. 2). The resulting enzyme preparation was apparently homogeneous on The fluorescence spectrum of the purified enzyme revealed SDS-PAGE and was stored at -80 "C as 0.1-mg protein samples. The purified enzyme was usedto raise antibodies in rabbits, IgG fractions were the presence of a flavin cofactor; the difference in fluorescence intensities at neutral andacidic pHs indicated that the flavin as described earlier (28). prepared from the sera with the higher titers

was probably FAD (Fig. 3). The fluorescence of the flavin was abolished by reducing the enzyme preparation with dithionite S u b c e l l u l a r L o c a t i o nof Yeast Protoporphyrinogen Oxidaseand wasrecovered by reoxidation with air. Despite the presence Table I shows the distribution of several mitochondrial en- of D l T i n t h all e buffers used for protoporphyrinogen oxidase zymes for the wild type strainD273-10B. The subcellular pat- purification and storage, the fluorescence signal of the flavin tern of protoporphyrinogen oxidase distribution appears to be was not increased by extensive aeration of the enzyme prepaidentical to that of cytochrome c oxidase and NADH cytochrome ration, suggestingthat the flavin remained in an oxidized form. c reductase-antimycin A sensitive,but completely different Quantitative analysis of the FAD content of purified enzyme from that of NADH cytochrome c reductase-antimycin A-insen- preparations indicateda flavin-to-protein ratio of 1(0.95 2 0.1, sitive, a marker enzymeof the outermitochondrial membrane. n = 4). There was, however, a strong correlation between the Protoporphyrinogen oxidase has the same distribution as fer- loss of the flavin cofactor and theloss of enzyme activity esperochelatase. Thus both enzymes are located in the innermem- cially during chromatofocusing when the enzyme was at alkabrane of yeast mitochondria. line pH. RESULTS

32088

Purification and

Properties of Yeast Protoporphyrinogen Oxidase TABLEI1 Purification of yeast protoporphyrinogen oxidase

proteins Total activity Total fraction Enzyme

Specific Purificution activity

membranes 7.5 Mitochondrial 2,250 (from 1 kg wet weight cells) CI"S solubilized proteins fractionation Ammonium sulfate Phenyl-Sepharose 4.5 0 DEAE-Sepharose 0.8 Mono P Sepharose Blue-Sepharose 25

A

B

nmollh 16,800

mi?

10,080 13,440 8,400 6,720 4,200 25 4,200

1,010

10

6103

22

factor

Recovery

nmollhlmg

lo

1

100

1.3

60 (80) 50 40

250

1,130 5,470 0.11 5,600

39,000 42,000

0.1

C

946743-

30B 550

20.4-

460

500

600

EMIISSION WAVRWGTH (nm)

FIG.3. Emission fluorescence spectra of purified protoporphyrinogen oxidase. ( A exc = 430 nm) recorded a t acidic pH (A) and neutral pH ( B).

rinogen oxidase had a higher molecular mass (58 kDa). This higher molecular mass polypeptide was in vivo only with labeling times shorter than 1 min (Fig. 5, lanes B and C); longer labeling times (5 min) led to immunoprecipitation of the mature form of the enzyme (55 kDa) (Fig. 5,lane D).The half-life of the mature form of protoporphyrinogen oxidase was estiZmmunocharacterization of Yeast ProtoporphyrinogenOximated to 2 h from chase experiments (Fig. 5, lane E). dase-The purified enzyme and the enzyme detected by the Catalytic Properties of Yeast Protoporphyrinogen Oxidaseimmune replica method in total protein extracts prepared from The purified yeast protoporphyrinogen oxidase required fatty a wild type laboratory strain had the same molecular mass acids to befully active in vitro. As shown in Fig. 6, the activity (Fig. 4 lanes E and F)).Thus, the relative molecular mass of was lost much faster in the absence of palmitic acid (Fig. 6A) native yeast protoporphyrinogen oxidase is 55 kDa. Protopor- than in assays run with3 mM palmitic acid (Fig. 6B). phyrinogen oxidase appeared to be extremely susceptible i n The optimum pHfor purified yeast protoporphyrinogen oxivitro to degradation by endogenous proteases. This was par- dase activity was 7.2. This value is close to that previously tially overcome by using the proteases inhibitors EDTA and reported for the partially purified enzyme (2). The Michaelis PMSF. Addition of leupeptin or pepstatin did not significantly constant for protoporphyrinogen IX was 0.1 PM, and the maximprove the enzyme protection., Some catalytically active deg- imum velocity was 40,000 nmoWmg enzyme. During the last radation products of protoporphyrinogen oxidase were more steps of the purification, protein determinationby conventional acidic and smaller (35-40 kDa) than the mature protein and methods was not satisfactory, a s i tgave abnormally high proremained bound to the DEAE-Sepharose (Fig. 4, lane D ) . The tein concentrations incomplete disagreement with theelectroantibodies toprotoporphyrinogen oxidase wereused to monitor phoretic profiles or enzyme activities. This wasprobably due to the enzyme integrity inmitochondrial membranes. As shown in the binding of detergents (which interfere with most protein Fig. 4, lane A, no degradation of protoporphyrinogen oxidase colorimetric assays)to protoporphyrinogen oxidase. Itwas was observed in freshmitochondrial membranes. Storageof the overcome by calculating proteinconcentrations from total membrane fractions a t -80 "C led to limited proteolysis of the amino acid compositions. enzyme (Fig. 4, lane B ) that was much more important upon The membrane-boundprotoporphyrinogenoxidase is very storage a t -20 "C (Fig. 4, lane C). Under allof these conditions, efficiently inhibited i n vitro by diphenylethers-type herbicides. the specific activity of protoporphyrinogen oxidase and K , for These compounds were equally active on the purified enzyme. protoporphyrinogen were not significantly affected. However, Kinetic studies of the inhibition of the purified enzymeby AFM the reactivity of the enzyme toward acifluorfen-methyl was showed that this inhibitor competes with protoporphyrinogen greatly modified (see below). IX (Fig. 7) with a Kiof 10 nM, as determined by the secondary When protoporphyrinogen oxidase was immunoprecipitated plots of the inhibition data. Thetype of inhibition determined from pulse labeled cells, the newly synthesized protoporphy- on the purified enzyme differed from that determined on the

FIG.2. SDS-PAGE of protoporphyrinogen oxidase purified fromcommercially available bakers' yeast (Fould Springer). Lane A, molecular mass standards; lane B , 0.5 pg of purified yeast protoporphyrinogen oxidase;lane C, 5 pg of purified yeast protoporphyrinogen oxidase.

Purification and Properties of Yeast Protoporphyrinogen Oxidase

A 9467-

B

C

D

32089

E F A

+

43-

3020-

E

FIG.4. Western blot analysis of protoporphyrinogen oxidase from proteins were electrophoretically separated in 1070polyacrylamide gel, transferred to nitrocellulose sheets, and processed for immunodecoration. Anti-yeastprotoporphyrinogen oxidase antisera 1 I& fraction) wereused a t a dilution of 1/3000. IAne A, 100 pg of proteins from freshly prepared mitochondrial membranes; lane 8,100 pg of proteins from the same membranes stored1 month at -80 "C; lane C, 100 pg of proteins from the same membranes stored 1 month a t -20 "C; lane D,10 pg of the active protoporphyrinogen oxidase hound to DEAE-Sepharose, eluted with 0.5 M NaCI; lane E, 50 pg of total yeast TCA-precipitated proteins;lane F , 0.1pg of purified protoporphyrinogen oxidase.

2

I

2

I

TIME (mnl

FIG.6. Effect of palmitic acid on purified yeast protoporphyrinogen oxidase: recordingsofprotoporphyrin M formation (A,,, = 410 nm,A,, = 632 nm) during protoporphyrinogen oxidase M palmitic acidin the assay assays (A) without and ( R )with 3 m medium.

-69

M -1 0

46 FIG.5. Autoradiography of labeled protoporphyrinogen oxidase immunoprecipitated from in vivo metabolic labeling at to (lane A ) and after 1.5 s (lane B ) , 30 s (lane C), 5 min (lane D ) of labeling and 5 min of labeling followed by a 2-h chase (lane E). M , , "'C-labeled molecular mass markers.

0

-5

515

1 / [ Prot oporphyrlnogen

10

Iw

( p W 1)

FIG.7. Lineweaver-Burk plot of purified yeast protoporphyrinogen oxidase activity versus protoporphyrinogenM concentration at various concentrationsofAFM. Values on each curve are AFM concentrations in n x 7-

40

membrane-bound enzyme (competitiveversus mixed). We reinvestigated the mechanism of inhibition of the enzyme by aci20 fluorfen-methyl in preparations of intact (as in Fig. 4A) or 10 proteolyzed (as inFig. 4C) membrane-bound protoporphyrino5 0 gen oxidase. As shown in Fig. 8 intact protoporphyrinogen oxidase exhibited a competitive inhibition (K, = 9 nM), while the proteolyzed enzyme was inhibited according to a mixed mechanism (K, = 10 nM, K', = 20 nM) (data not shown). -1 0 -5 0 5 10 Preliminary experiments in which protoporphyrinogen oxil/[hotoporphyrlnogen Iw ( p W 1 ) dase activity was measured after depletion of dissolved oxygen by degassing all buffers and reagents and nitrogen flushing FIG.8. Lineweaver-Burk plotof membrane-bound yeast protoshowed than the residual activity was stillclose to the maximum porphyrinogen oxidase activity (measured on fresh membrane velocity. This suggested that theenzyme had a high affinity for preparations) uernun protoporphyrinogen M concentration at various concentrations of AFM. Values on each curve are AFM conmolecular oxygen, limiting the possibility of experimentally centrations in m . determining the kinetic parametersfor oxygen. Enzymatic oxygen trapping systems such as glucose-glucose oxidase-cata- However, the initialvelocity of protoporphyrinogen oxidase aclase or xanthine-xanthine oxidase-catalase were much more tivity was used to calculate an approximate apparent K, 5 1 efficient in depleting the incubation mixture of oxygen. These p~ at saturating concentration of protoporphyrinogen. systems stopped the protoporphyrinogen oxidase reaction once DISCUSSION anaerobiosis was reached. Preliminary measurements of the We have purified yeast protoporphyrinogen oxidaseto homoK,,, for oxygen were made by measuring protoporphyrinogen oxidase activity in the presence of known concentrations of geneity and measuredsome of its properties. The purification dissolved oxygen after the removal of dissolved oxygen by en- was hampered by the small amount of the enzyme in yeast zymatic oxygen trapping systems. The oxygen traps caused mitochondrial membranes (3 pmoVmg of total proteins) asdecompetition between the variousoxidases present in theassay. termined by Scatchard analysisof a tritiated inhibitor([3Hlaci-

32090

Purification and Properties of Yeast Protoporphyrinogen Oxidase

fluorfen) binding t o yeast mitochondrial membranes,' and its extreme susceptibility to proteolytic degradation and rapid loss of activity at alkaline pH. The enzyme was purified 5200-fold with a 25% overall recovery, and the specific activity was 40,000 nmol protoporphyrinogen formedper h/mg of protein at 30 "C. This value is of the same order of magnitude as that calculated from the binding studiesof [3H]acifluorfen to membrane-bound protoporphyrinogen oxidase.' This specific activthat reported for the bovine liver ( l l ) , ity is much higher than mouse liver (12), or barley enzymes (14) where the specific activities were in the 900-6900 units range at 37 "C.The relative molecular mass of yeast protoporphyrinogen oxidase was 55 kDa when measured under denaturating conditions. This value is identical to the mass of the polypeptide specifically photolabeled with a diazoketone derivative of tritiated acifluorfen.' It is also close to that reported for the purified bovine liver enzyme (11).The same 55-kDa polypeptide was detected by immunodecoration in total trichloroacetic acid-precipitated proteins after SDS-PAGE and transfer tonitrocellulose. The in vivo labeled enzyme immunoprecipitated with anti-protoporphyrinogen oxidaseantibodies revealed that a polypeptide with a relative molecular mass of 58 kDa rapidly matureda stable to 55-kDa form,suggesting that the enzyme was synthesizedas a larger precursor. As reported for the partiallypurified barley protoporphyrinogen oxidase (441, the purified yeast protoporphyrinogen oxidase was activatedby palmitic acid, while the membrane-bound enzyme was not affected by exogenous fattyacids(datanot shown). The molecular basis for such activation remain unclear. The detergents present during purification and during the enzyme assay did not activate the protein. Perhaps fatty acids affect the conformation or stability of the solubilized protein or the solubilities of the substrate(protoporphyrinogen) or the product (protoporphyrin IX)of the reaction. The K, of the yeastenzyme for protoporphyrinogen ( K , = 0.1 PM) is muchlower than thatof the purified bovine (K, = 17 p; (11))and mouse (12 PM (12)) enzymes but is comparable with that measured on the membrane-bound enzyme from human liver (17), mouse liver, and corn and yeast mitochondria (45), probably due to the high sensitivityof our enzyme assay. Diphenylether-type herbicides are very potent inhibitors of the membrane-bound protoporphyrinogen oxidase (191, and of the purified enzyme, as shown in thispaper. The inhibition is strictly competitive for the purified enzyme and for the membrane-bound enzyme, provided it remains structurally intact. The mixed inhibition of the membrane-boundenzyme (45) has now been traced back to the fact that mitochondrial membranes may carryproteolytic fragments of protoporphyrinogen oxidase (generated during preparation) that are active. Proteolysis may have a complex effect with regard to inhibitorbinding, generating a binding site that affects activity but that cannot be displaced by substrate. The reaction catalyzed by protoporphyrinogen oxidase involves two substrates: protoporphyrinogen IX and molecular oxygen. Poulson and Polglase (2) reported that the partially purified yeast protoporphyrinogenoxidasefunctionedonly with molecular oxygen as electron acceptor. Our resultsfor the highlypurified yeast enzyme support this assumptionand show that protoporphyrinogen oxidase has a very high affinity This value is considerably for oxygen (apparent K, < 1 p). lower than that reported by Ferreira and Dailey (46) for the mouse enzyme,where theK , for oxygen was 120 p ~ i.e., half of the saturation concentration of dissolved oxygen in aqueous medium at 37 "C. The technical difficulties involved in preJ.-M. Camadro, M. Matringe, F. "home, N. Brouillet, R. Mornet, and P. Labbe, submitted for publication.

cisely controlling low concentrations of dissolved oxygen made it impossible to fully investigate the mechanism of action of the enzyme, with special reference to the order of binding of the substrates to theenzyme and thestoichiometry of oxygen consumption during thereaction. Protoporphyrinogen oxidase contains 1 mol ofFAD/molof enzyme. The occurrence of a flavin at the active site of protoporphyrinogen oxidase may explain some of its catalytic properties. Jones et al. (471, studied the oxidation of protoporphyrinogen stereospecifically tritiated at the methylene bridges. They found that three hydrogen atoms from the methylene bridgesare removedfromone side of protoporphyrinogen, while the fourth is removed from the other side of the cyclic tetrapyrrole. The protons from bridges a and b (Fig. 1) also appeared t o be removed by two distinct mechanisms. A model was proposed for protoporphyrinogen oxidation that involved the removing of three hydrides and one proton. Thus, it is reasonable to suggestthat, while the flavin may be involved in hydride removal, another functional groupon the protein,possibly a basic amino acid may be involved in theremoval of the fourth proton. Such a model is supported by the molecular dynamics quantitative structure-activity relationship of the interactions of protoporphyrinogen oxidase with inhibitors (48, 49). They indicate the potential role of charge transfer and electrostatic interactions in the action of protoporphyrinogen oxidase. The presence of a flavin in the active site of protoporphyrinogen oxidase raises the question of a n alternative electron acceptor(s)for protoporphyrinogen oxidasefunctioning in anaerobically grown yeast cells. This question also applies to other facultative or obligate anaerobic organismsthat can synthesize some heme. Although molecular oxygen appears t o be the only electron acceptor for the mammalianenzyme (161, fumarate or nitrate can support the anaerobic oxidation of protoporphyrinogen by cell-free extracts of Escherichia coli grown anaerobically on fumarate or nitrate, respectively (50-52). The mechanismof anaerobic protoporphyrinogen oxidation in E. coli seems complex, since the fumarate-dependentactivity requires quinones as hydrogen acceptors and/or part of the electron transport chain t o be efficient (53, 54). An even broaderelectron acceptor specificity was described for the anaerobicreaction catalyzedby extracts from the obligate anaerobic bacteriumD. gigas (15,551. Our invitro studies on the purified enzymesuggest that the high affinity of yeast protoporphyrinogen oxidasefor oxygen may provide the basis for the enzyme to function under microaerobic conditions, but they do not exclude the possibility that other electron acceptors may be involved in heme synthesis. The precise intramitochondrial location of yeast protoporphyrinogen oxidase was investigated relative to the cytosolic localization of yeast coproporphyrinogen oxidase, the preceding enzyme in the heme biosynthesis pathway. Protoporphyrinogen mitochondrial membrane in yeast. oxidase is bound to the inner Ferrochelatase, the next enzyme in the heme biosynthesis pathway, is also located in the innermitochondrial membrane. The location of coproporphyrinogen oxidase in the cytosol of yeast cells raises theproblem of the supply of protoporphyrinogen to the inner membrane-bound protoporphyrinogen oxidase. Rat liver protoporphyrinogen oxidase was shown to be an integral protein of the inner mitochondrial membrane (56). Its kinetic properties are dependent on the location of coproporphyrinogen oxidase (56). When assayed in a coupled system where protoporphyrinogen M is generated from coproporphyrinogen 111 through coproporphyrinogen oxidase activity, rat liver protoporphyrinogen oxidase activity is higher in intact mitochondria (where coproporphyrinogen oxidase is confined to the intermembrane space of mitochondria) than in mito-

Purification and Properties

of

Yeast Protoporphyrinogen Oxidase

plasts (where coproporphyrinogen oxidase is released into the assay medium), despitethere being equal amounts of coproporphyrinogenoxidase activityinboth cases. Theseresults strongly suggest that a limited accessibility of the substrate protoporphyrinogen to the active site of protoporphyrinogen oxidase could modulate the activity of the membrane-bound enzyme (56). Recent measurements of the partition coefficients for protoporphyrinogen M between an aqueous and an organic phase showed that it is much more hydrophilic than generally assumed and is 3 orders of magnitude less lipophilic than protoporphyrin IX (57). This supports the idea that protoporphyrinogen M may diffuse i n a n aqueousenvironment.Inyeast, protoporphyrinogen is synthesized in the cytosol and must cross the outer membrane of mitochondria to reachits final site of oxidation. The apparent absence of diffusion of protoporphyrinogen in the cytosol of the cells suggests some channeling of protoporphyrinogen to protoporphyrinogen oxidase by transitory interaction of coproporphyrinogen oxidase with the mitochondrial membranes.Theactivation of purified yeast coproporphyrinogen oxidase by neutral detergents and phospholipids ( 5 ) points in this direction, but further studies are clearly required. The fact that protoporphyrinogen is made in the cytosol of yeast cells is importantfor understanding themode of action of diphenylether-type inhibitors of protoporphyrinogen oxidase. The basic assumption required to explain the cytotoxicity of these inhibitors is that, when protoporphyrinogen oxidase is blocked, the substrate of the inhibited enzyme, protoporphyrinogen IX, diffuses out of its metabolic site and is nonspecifically oxidized into protoporphyrinM (19).Jacobs et al. (58) and Lee et al. (57) recently reported the characterization of several protoporphyrinogen-oxidizing enzyme activities lying outside plant plastids. These enzymatic systems were not inhibited by diphenylethers, suggestinga mechanism of reaction similar to that described for the protoporphyrinogen oxidaseactivity from E. coli (59). These nonspecific oxidases may be involved in the rapid accumulation of protoporphyrin in plants after diphenylether treatment. Protoporphyrinmislocation within thecells wasdemonstrated by fluorescence studies of acifluorfentreated cucumber cotyledons (60). Protoporphyrin is thus no longer a substrate for ferrochelatase and may accumulate and mediate theperoxidative effects of the herbicides treatment. In yeast cells, protoporphyrinogen diffusion from the cytoplasm to the mitochondria must be tightly controled. This emphasizes the role of cellular compartmentation of both metabolites and enzymes in the proper functioning of the heme biosynthesis pathway. The purified yeast protoporphyrinogen oxidase, antibodies to the protein, and specific radioligands will be used in further protoinvestigations on the structureffunction relationships in porphyrinogen oxidase and to characterize some of the topological elements in the active site of the enzyme. Our present results provide evidences that yeast protoporphyrinogen oxidase represents an adequatemodel for these studies. Acknowledgments-We thank H. Chambon and Dr. C. Jomary for help during the initial phases of this work, Dr. R. Scalla and Dr. M. Matringe for many helpful discussions, and Dr. 0. Parkes for help in preparing the manuscript.

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