Biochimica et Biophysica Acta, 1038 (1990) 253-259

253

Elsevier BBAPRO 33611

Iron-reductases in the yeast Saccharomyces cerevisiae E. Lesuisse 1, R.R. C r i c h t o n 2 a n d P. L a b b e 1 1 Laboratoire de Biochimie des Porphyrines, Unioersite Paris 7, lnstitut J. Monod, Tour 43, Paris (France) and 2 Laboratoire de Biochimie, Universite Catholique de Louvain, Louvain-La-Neuve (Belgium)

(Received 30 March 1989) (Revised manuscript received13 December1989)

Key words: Iron transport; Iron reductase; Yeast; (S. cerevisiae) Several NAD(P)H-dependent ferri-reductase activities were detected in sub-cellular extracts of the yeast Saccharomyces cerevisiae. Some were induced in cells grown under iron-deficient conditions. At least two cytosolic iron-reducing enzymes having different substrate Specificities could contribute to iron assimilation in vivo. One enzyme was purified to homogeneity: it is a flavoprotein (FAD) of 40 kDa that uses N A D P H as electron donor and Fe(III)-EDTA as artificial electron acceptor. Isolated mitochondria reduced a variety of ferric chelates, probably via an 'external' N A D H dehydrogenase, but not the siderophore ferrioxamine B. A plasma membrane-bound ferri-reductase system functioning with N A D P H as electron donor and F M N as prosthetic group was purified 100-fold from isolated plasma membranes. This system may be involved in the reductive uptake of iron in vivo.

Introduction Iron reductases catalyse the movement of iron from various ferric chelates to ferrous acceptors. They play a central role in iron assimilation by microorganisms, as a consequence of the solution chemistry of iron, which is dominated at physiological p H by the autoxidation of Fe(II) and by the hydrolysis and polymerisation of aqueous Fe(III) to insoluble ferric hydroxides and oxyhydroxides. Many microorganisms synthesize and excrete low molecular weight compounds, siderophores, which chelate ferric iron, making it available for transport (for a review, see Ref. 1). One of the strategies commonly used by microorganisms for assimilating chelated iron once it has entered the cell involves the reduction of the iron to Fe(II), for which the siderophore has little affinity [2]. NAD(P)H : ferrisiderophore oxidoreductase activities have been reported in cell extracts of several microorganisms [3-8].

Abbreviations: T/G/Tween buffer, 25 mM Tris-acetic acid (pH 7.3) containing 10% (v/v) glycerol and 0.6% (w/v) Tween 80; BPS bathophenantroline suifonate; BP, bathophenantroline; PMSF, phenylrnethylsulfonylfluoride; HMP, hexose monophosphate. Correspondence: E. Lesuisse, Laboratoire de Biochimie des Porphyfines, Universit6 Paris 7, Institut J. Monod, Tour 43, 2 Place Jussieu, 75251 Paris cedex 05, France.

There are no reports of a siderophore secreted by the yeast S a c c h a r o m y c e s cerevisiae [9]. However, we have shown that this yeast can reduce extracellular ferric chelates - including siderophores - by a plasma membrane-bound redox system that is dramatically induced in iron deficient conditions. Iron is then taken up by the cells as Fe 2+ ions [10]. A similar reductive mechanism of iron assimilation has been described in bacteria [11], fungi [12,13], higher plants [14] and animal cells [15] (for a review, see Ref. 16). We have shown that S. cerevisiae is also able to assimilate iron from the siderophore ferrioxamine B by a non-reductive process, involving internalization of the siderophore without prior extracellular dissociation [17]. In that case, iron is released inside the cell, probably via a reduction step. lntraceUular iron is stored mainly in vacuoles, from which it may be redistributed to other cellular compartments for iron-requiring processes [18]. One of these processes takes place at the M-side of the inner mitochondrial membrane, where the enzyme ferrochelatase catalyzes the insertion of iron into protoporphyrin to make heme. Since Fe E+, but not Fe 3+, is the metal substrate of the ferrochelatase, a membrane-bound iron-reducing process may also be expected to operate at the mitochondrial level, as has been described for mammalian mitochondria [19]. This study analyses subcellular fractions of S. cerev i s i a e in order to identify and characterize the ferri-reductases involved in iron assimilation by the yeast.

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254 Special attention was focussed on the plasma membrane-bound ferri-reductase system, since the plasma membrane components involved in reductive iron assimilation by higher plants and yeasts have not, as yet, been characterized. Materials and Methods

Yeast strains and growth conditions Intact mitochondria were obtained from the laboratory diploid strain D261. It was grown aerobically at 30 ° C in a medium containing (in 1 liter of water) 20 g yeast extract, 2 g KH2PO4, 2 g (NH4)2SO4, 1 g glucose and 30 g ethanol. This strain was also used for measuring changes in the ferri-reductase activity of whole cells and of the glucose-6-phosphate dehydrogenase activity during growth in iron-deficient or iron-rich media. The iron-rich and iron-deficient media were as previously described [10]. The haploid strain G204 [20] was used for all other experiments. It was grown in a complete medium containing 1% yeast extract, 1% bactopeptone, 2% glucose, 0.2% Tween 80, 20 mg per liter ergosterol and 50 mg per liter 5-aminolevulinic acid. Cultures of this strain in iron-rich or in iron-deficient conditions were prepared in synthetic media supplemented with 50 mg per liter 5-aminolevulinic acid as previously described [17]. The cells were harvested in the exponential growth phase and washed twice with distilled water. The commercially available baker's yeast S. cereoisiae used for purifying the plasma membrane ferri-reductase was from Fould Springer (Maison Alfort, France).

Cellular fractionations and enzyme assays Intact mitochondria were prepared according to Briquet et al. [21] from the D261 strain. The crude membrane and the cytosolic and plasma membrane fractions were prepared from the G204 strain and from commercial baker's yeast as described by Dufour et al. [22]. The crude membrane fraction was the pellet from the 600 000 x gm~n centrifugation, while the cytosolic fraction was the supernatant. The plasma membrane fraction was prepared by centrifugation (20 000 x gmin) after acid precipitation of the mitochondrial membranes. Azide-insensitive ATPase assayed at pH 6 was used as a, plasma membrane marker, while azide-sensitive ATPase assayed at pH 9 was used as a mitochondrial marker in estimating the mitochondrial contamination of the plasma membrane fraction. The assays were carried out according to published methods [22]. Cytochrome oxidase and glucose-6-phosphate dehydrogenase activities were measured according to Jauniaux et al. [23]. Mitochondrial respiratory control was measured as described by Briquet et al. [21]. The specific activity of azide-insensitive ATPase in the plasma membrane fraction was about 2/~mol phosphate per rnin per mg protein at 35 ° C (pH 6), which

corresponds to a 12-fold increase with respect to the whole homogenate. In a typical experiment, the ratio of the azide-insensitive ATPase activity (pH 6) to the azide-sensitive ATPase activity (pH 9) was 0.8 in the crude membrane fraction and 4.5 in the plasma membrane fraction. The specific activity of cytochrome oxidase in the purified mitochondria was about 1.1 ~tmol per min per mg protein, which corresponds to a 4-fold increase with respect to protoplasts. The respiratory control with succinate was higher than 2.

Iron reduction assays The ferri-reductase activities of whole cells and subcellular fractions was measured as previously described [10] with either ferric-EDTA, ferric citrate, ferric mannitol or ferrioxamine B (180-360 /~M) as iron sources, and bathophenantroline sulfonate (1 mM) as the irontrapping reagent. All the assays were performed at 3 0 ° C in 50 mM Tris buffer (pH 7.5) except for the assays with the semi-purified plasma membrane ferri-reductase which were done in the same buffer containing 10% ( v / v ) glycerol and 0.6% (w/v) Tween 80, and for the assays with intact mitochondria which were done in 10 mM imidazole buffer (pH 6.4) containing 0.6 M mannitol.

Purification of the ferri-reductases from cytosolic and plasmamembrane fractions (a) cytosolic ferri-reductase. All operations were carried out at 4 ° C. The cytosolic fraction obtained from laboratory-grown cells (about 75 ml of a 20 mg protein per ml solution) was dialysed overnight against 25 mM Tris-acetic acid (pH 8.3) and loaded onto a 1 x 15 cm column packed with Polybuffer exchanger 94 equilibrated in the same buffer. Proteins were eluted with a linear p H gradient produced by a 210 ml solution of 7% ( v / v ) Polybuffer 74 and 3% (v/v) Polybuffer 96 adjusted to p H 5 with acetic acid. The flow rate was 20 ml per h, and 3.5 ml fractions were collected. Aliquots were assayed for ferri-reductase activity with ferric EDTA a n d / o r ferrioxamine B (180 /~M) as substrate plus 50 /~M N A D P H as electron donor. Active fractions were pooled, loaded onto a 1 x 20 cm column packed with Blue Sepharose CL-6B equilibrated in 25 mM Tris-acetic acid (pH 7.5) and eluted with 120 ml linear NaCI gradient (0-2 M in the same buffer) at a flow rate of 20 ml per h. The fractions containing ferri-reductase activity were pooled, saturated with ammonium sulfate and loaded onto a phenyl-Sepharose column equilibrated with a saturated ammonium sulfate solution containing 25 mM Tris-acetic acid (pH 7.5) and 2 M NaCI. The column was eluted with 25 mM Tris-acetic acid (pH 7.5) at a flow rate of 20 ml per h, and 2.5 ml fractions were collected. Active fractions were pooled and the resulting enzyme preparation was homogeneous when analysed by SDS-PAGE.

255

(b) Plasma membrane ferri-reductase. Plasma membranes, purified from either commercial or laboratorygrown cells, were suspended at about 10 mg protein per ml in solubilization medium [22]. Membranes were solubilized by adding 0.6% ( w / v ) Tween 80 to the suspension (laboratory-grown cells) or by adding 0.8% ( w / v ) Tween 80 to the suspension which was then sonicated (3 × 30 s) at 100 kHz (commercial baker's yeast). The final detergent/protein ratio was 0.8 in both cases, and the mixtures were stirred magnetically for I h at 0 o C. Insoluble material was removed by centrifugation for I h at 75 000 × g. The supernatant containing most of the ferri-reductase activity was saved and diluted 10% (v/v) in glycerol. The purification procedures described below gave identical results for both commercially available yeast cells and laboratory-grown cells. In a typical experiment, about 100 ml of the solubilized membranes were loaded onto a 2.5 × 20 cm column packed with D E A E Sepharose equilibrated in 25 mM Tris-acetic acid (pH 7.3) containing 10% ( v / v ) glycerol and 0.6% ( w / v ) Tween 80 ( T / G / T w e e n buffer). The NADPH-dependent ferri-reductase was eluted with a linear gradient of NaC1 (0-0.35 M, 400 ml) in the same buffer. The flow rate was 35 ml per h, and 4 ml fractions were collected. Aliquots of these fractions were assayed for ferri-reductase activity with ferric E D T A (180 /~M) as substrate and N A D P H (50 /tM) as electron donor. Active fractions were pooled and concentrated to about 20 ml on an Amicon PM-10 filter. The concentrated solution was loaded onto a 2.5 × 20 Sephadex G-25 column and the enzyme was eluted with the T / G - T w e e n buffer (pH 8.3). The desalted solution was then chromatofocussed under the same conditions as those described for the purification of cytosolic ferri-reductase (see above), except that all buffers contained 10% ( v / v ) glycerol and 0.6% (w/v) Tween 80. When the pH had reached 5, the ferri-reductase was eluted with a linear gradient of NaC1 (0-0.3 M, 120 ml) in T / G / T w e e n buffer (pH 5, flow rate 30 ml per h). The pH of the collected fractions (4 ml) was brought to 7.5 with Tris base (1 M, pH 9), and aliquots were assayed for ferri-reductase activity. The active fractions were pooled and concentrated to about 5 ml on Amicon PM-10 filter. Two cycles of dilution with 25 mM Tris (pH 7.5) followed by concentration were performed to decrease the concentrations of salts and Tween 80. The resulting enzyme preparation was stored at - 20 ° C. Results and Discussion

Overview Subcellular fractions of S. cerevisiae grown in ironrich or iron-deficient media were assayed for ferri-reductase activity with Fe(III)-EDTA or ferrioxamine B

TABLE I

Ferri-reductase activities of subcellular fractions of S. cerevisiae The cells were grown in iron-rich media ( ' + Fe', iron added as 180 /~M ferric citrate) or in iron-deficient media ( ' - F e ' , media treated as described in Materials and Methods). The assays were performed as described in Materials and Methods; the reaction was initiated by adding 50 /tg protein per ml to the sample cuvette. The ferric substrates were either ferric EDTA or ferrioxamine B (values in parentheses), both at 180 /~M. N A D H and N A D P H concentrations were 50 p M (The data are from a single representative experiment). Fraction

Yield (% protein)

Ferri-reductase-specific activities ( n m o l - m i n - l . m g - 1)

Electron donor: N A D H

Homogenate

100

NADPH

Growth cond.: + Fe

- Fe

+ Fe

- Fe

(0.4) 3.5

(0.4) 3.6

(1.75) 3.3

(3.9) 7.15

Crude membrane

19.7

(0.3) 11.3

(0.5) 11.3

(1.5) 4.7

(4) 8.47

Cytosol

64.4

(0.45) 3.1

(0.4) 4.35

(0.7) 1.9

(1.9) 4.4

0.7

(0.2) 3.8

(0.55) 4.6

(3.2) 8.2

(7.6) 18.5

Plasmamembrane

as ferric substrates, and N A D H or N A D P H as electron donors. As shown in Table I, the specific ferri-reductase activities measured in the cell homogenates were similar to those reported for other microorganisms [3-8,24-27]. Both N A D H - and NADPH-dependent activities were detectable when Fe(III)-EDTA was used as electron acceptor. The highest NADPH-dependent activity was detected in the plasma membrane fraction, while the NADH-dependent activity was found in the crude membrane fraction (which includes a majority of mitochondrial membranes, more than 90% of the total proteins). This distribution was not found when ferrioxamine B was used as the ferric substrate. In that case, the NADH-dependent ferri-reductase activity was low in all fractions assayed, N A D P H being obviously the best electron donor (Table I). Iron deprivation during growth also resulted in a significant increase in the N A D P H dependent ferri-reductase activity in all fractions, with both Fe(III)-EDTA and ferrioxamine B as electron acceptor. In contrast, the extent to which iron deprivation induced NADH-dependent activity varied with the cellular fraction tested and the ferric substrate used (Table I). The relationships between the N A D ( P ) H : Fe 3+ oxidoreductase activities measured in vitro in different subcellular fractions and the iron content of the culture medium strongly indicate that several ironreleasing enzymes are required for iron assimilation in vivo. This is also consistent with the findings of

256 T A B L E II

Rate of iron reduction by isolated yeast mitochondria Iron reduction was assayed as described in Materials and Methods, with 360 ttM Fe(III)-mannitol as the iron source. W h e n present, N A D H , N A D P H , succinate, ATP, MgCl 2 and K C N were 1 m M each, while dinitrophenol (DNP) was 20 /tM. The mitochondria (0.15-0.2 mg protein per ml) were preincubated for 2 min at 30 ° C with the substrates and salts before addition of iron. (The data are from two experiments). Addition

NADH NADPH Succinate N A D H + A T P + MgCI 2 A T P + MgC12 NADH + KCN KCN NADH + DNP

Rate of iron reduction (nmol - m i n - 1. m g - 1 ) 0.8, 1.7 31.1, 35.7 2.9, 3 1.1, 1.4 68.1, 75 0.8, 0.95 36.25, 38.75 2.45, 3 29.7, 34.7

The very poor iron-donor properties of sideraminetype iron chelates have been previously demonstrated in Neurospora crassa [8]. It was suggested that acids of the citric acid cycle could mediate iron transfer from siderophores into mitochondria in this fungus. Independent of the iron chelate(s) used in vivo by the mitochondria, our results suggest that a reduction step could occur at the C-side of the inner membrane by interaction between the iron chelate and an 'external' NADH-dehydrogenase. The presence of such an NADH-dehydrogenase in the yeast mitochondria is well documented [28]. Our results disagree with those obtained with mammalian mitochondria, where iron reduction seems to occur at lipophilic sites on the inner membrane (poor accessibility of the reduced iron to hydrophilic chelators; see Refs. 29, 30). Either the Cside, via the respiratory chain at the level of cytochrome c [19], or the M-side, via the succinate dehydrogenase complex [31] could be involved in iron reduction in these mitochondria.

Cytosolic fraction Arcenaux and Byers [3], who postulated that several ferri-reductases could be associated with various ironrequiring metabolic systems, allowing direct delivery of iron to these systems.

Mitochondrial fraction The iron-reducing properties of intact, functional yeast mitochondria were assayed in the presence of different electron donors (Table II). The maximal reduction rate was obtained with exogenous N A D H as electron donor. N A D P H or succinate had little or no effect. ATP, and to some extent KCN, stimulated iron reduction in the presence of N A D H (Table II). This could be due, at least in part, to inhibition of the electron flow from exogenous N A D H to oxygen. The reduced iron was more accessible to the hydrophilic chelator bathophenantroline sulfonate (BPS) than to the lipophilic chelator bathophenantroline (BP). The apparent reduction rate of iron, as measured by the formation of the (BP)3Fe(II) or (BPS)3Fe(II) complexes, was 3-times higher with BPS than with BP (data not shown). The ferric chelate~ Fe(III)-mannitol, Fe(III)-EDTA and Fe(III)-citrate were efficient electron acceptors for the mitochondria (reduction rates of 100, 106 and 280%, respectively). However, the siderophore ferrioxamine B was not an electron acceptor (reduction rate of 6%). This suggests that ferrioxamine B, which can be accumulated by the cells in vivo [17], cannot be used directly by mitochondria as an iron source, so that dissociation of that chelate must occur either outside the cells via the plasma membrane-bound reductase [17], or inside the cells probably via a cytosolic ferri-reductase, as in other microorganisms [4].

As shown in Table I, both N A D H - and NADPH-dependent ferri-reductase activities were present in the soluble fraction. However, the low NADH-dependent reduction of ferrioxamine B was not induced by iron deprivation. Two fractions with NADPH-dependent ferri-reductase activity were obtained by chromatofocusing. The first (fraction A) did not bind to the Polybuffer exchanger gel at p H 8.3 and was recovered in the dead volume. The other (fraction B) was eluted at p H 6. Fraction A efficiently reduced both Fe(III)-EDTA and ferrioxamine B, while fraction B reduced ferrioxamine B very slowly (Fig. 1). The ferri-reductase activity of fraction A was unstable. It was completely lost after 2 days at 4 ° C or after overnight dialysis against fresh buffer, even in the presence of PMSF (1 mM), gluta02 E O

8

tO O

7

0.1 tO r~

I

6 0.0 0

20

40 F r a c t i o n number

60

5 80

Fig. 1. Chromatofocusing profile of the cytosol fraction of laboratory-grown cells. The procedure was as described in Materials and Methods, except that the cytosol was not dialysed before loading onto the column. Ferri-reductase activity of ehited fractions was assayed with N A D P H as electron donor and either Fe(III)-EDTA (D) or ferrioxamine B (It) as iron sources; p H gradient ( . . . . . . ).

257 TABLE III

Purification of cytosolic ferri-reductase from the cytosol fraction of laboratory-grown cells The ferri-reductase activity was assayed as described in Materials and Methods, with 180 ttM ferric EDTA and 50 ttM NADPH as substrates. The reaction was initiated by adding 50 ttg protein per ml to the sample cuvette. (The data are from a single representative experiment). Fraction

Cytosol Chromatofocusing Blue Sepharose Phenyl-Sepharose

Total protein (mg)

Total activity (tt mol •min -1)

1500 550 2 0.47

2.3 0.5 0.09 0.09

Spec. act. (nmol. min.mg -1) 1.55 15.5 46 195

Yield (%) 1

100 23 4 4

thione (1 mM), F M N or F A D (5 #M). Fraction B was stable for several days at 4 ° C and was used for further purification (Table III). The protein was tightly b o u n d to Blue Sepharose and was eluted by 2 M NaC1. It was eluted f r o m phenyl-Sepharose with Tris buffer (25 m M , p H 7.5). The enzyme, which gave a single b a n d on S D S - P A G E , was purified 126-fold with respect to the cytosol fraction (final yield = 4%). The purified protein (molecular mass of 40 kDa) reduced F e ( I I I ) - E D T A ( g m = 50 gM), but not ferrioxamine B, in the presence of N A D P H (Km = 3 #M). N A D H was totally ineffective as electron donor, even at concentrations greater than 1 mM. Addition of F A D or F M N did not enhance the reduction rates. However, the purified protein had a fluorescence 2

Plasma membrane fraction A plasma m e m b r a n e - b o u n d ferri-reductase activity has been reported to be involved in iron uptake b y whole cells of S. cereoisiae in vivo [10,17]. This enzymic activity was strongly induced in cells grown in iron-deficient conditions [10]. Several factors indicate that N A D P H , rather than N A D H , is the physiological electron d o n o r of this redox system. The N A D P H - d e p e n dent ferri-reductase of isolated plasma m e m b r a n e was increased (2-fold) when the cells were grown in iron-deficient conditions (Table I). The apparent affinities of the plasma m e m b r a n e - b o u n d reductase for N A D H and N A D P H were quite different: the calculated values of the K m for N A D H and N A D P H with purified plasma membranes and F e ( I I I ) - E D T A as substrate were 32 and 3 # M , respectively. The activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase - the first enzyme of H M P pathway, which furnishes most of the N A D P H was increased in iron-deficient cells, and its m a x i m u m activity during growth was correlated with the peak of ferri-reductase activity of whole cells (Fig. 2). -

1.0

A

>

c

io "~

excitation spectrum typical of a flavoprotein with F A D as c o e n z y m e (Xexc = 466, 450 and 375 n m ; hem = 530 nm). O u r data demonstrate that S. cerevisiae has several different cytosolic enzymes having ferri-reductase activity, as do other microorganisms [3,5,24,25]. However, more information is needed before it can be stated that the cytosolic ferri-reductase we have purified actually plays a role in iron assimilation in vivo.

L

~X

g

A

.l::

,

0.5

I

I1.

E E

t v

." N ~= I/ o U. 0

(D

0.0

0 0

10

Time (h)

20

,

0

I 10

,

I

20

T i m e (h)

Fig. 2. Changes in the ferri-reductase activity of whole cells (B, I ; A) and in the cytosolic glucose-6-phosphate dehydrogenase activity (n, I ; B) during growth (o, e) in iron-rich or in iron-deficient media. Open symbols refer to the Fe-deficient condition and closed symbols to the Fe-rich condition. Iron reduction by whole cells was measured as described in Materials and Methods, with 360/tM ferric citrate as iron source and 5% (w/v) glucose as energy source. Glucose-6-phosphate dehydrogenase was assayed as described in Methods. (Mean+S.D. for two experiments.)

258 T A B L E IV

Purification of plasma membrane-bound ferri-reductase from the plasma membrane fraction of commercial baker's yeast The ferri-reductase activity was assayed as described in Materials and Methods, with 180 # M ferric E D T A and either 50 ptM N A D H , N A D P H or N A D P H plus 2.5 # M F M N . The reaction was initiated by adding 50 #g protein per ml to the sample cuvette. (The data are from a single representative experiment). Total protein (mg) Electron donor

Total activity ( # mol- min - 1)

Spec. act. (nmol. min - 1. m g - l )

NADPH + FMN

NADH

NADPH

Yield (%) NADPH + FMN

NADPH + FMN

Plasma membrane

1020

6.8

2.5

6.7

6.7

100

Solubilized plasma membrane

777

6.9

5

8.8

8.8

101

48

5.3

3.7

81.7

111.7

79

2

1.4

0

65.3

720

20

DEAE Chromatofocusing

The NADPH-dependent ferri-reductase was purified from isolated plasma membranes of commercial and laboratory-grown cells; similar results were obtained in both cases (see Methods). A typical purification procedure from commercial baker's yeast is summarized in Table IV. 2 kg (wet wt.) of cells yielded about 30 g (protein) of crude membranes, which, after acid precipitation, gave I g (protein) of purified plasma membranes. After solubilization and addition of glycerol, the ferri-reductase activity remained stable for days at 4 ° C and for months at - 2 0 o C. However, an irreversible loss of the enzymatic activity occurred after overnight dialysis against fresh buffer. The active extract eluted from the DEAE-Sepharose column by 0.3 M NaC1 was therefore desalted on a Sephadex G-25 column. The ferri-reductase activity was eluted at pH 5 and 0.2 M NaC1 in the subsequent chromatofocusing step. The reducing activity of this preparation was recovered by adding FMN (Table IV). FAD was less than 50% as effective as FMN. These results would suggest that the plasma membrane-bound reductase is a flavoprotein with a weakly bound F M N prosthetic group. Non-covalently bound flavins have been previously detected in purified plasma membranes of S. cerevisiae [32]. The chromatofocussed enzyme was purified about 100-fold with respect to plasma membranes, with a yield of 20%, but it did not migrate as a single band on SDS-PAGE. All the attempts to purify the protein to homogeneity resulted in the complete loss of activity. However, preliminary results (data not shown) indicate that several components, rather than a single protein, could be involved in the plasma membrane-bound ferri-reductase activity, and this could account for the loss of activity during purification. The semi-purified protein had a high affinity for NADPH (Km = 1.5/~M), while N A D H was not used as electron donor. The enzyme exhibited a fairly sharp pH optimum at 7.5, and no N A D ( P ) H : O 2 oxidoreductase

activity (data not shown). We therefore conclude that the semi-purified enzyme is not the NAD(P)H oxidoreductase purified by Ainsworth et al. [33,34] from a non-mitochondrial membrane fraction of S. cerevisiae. This flavoprotein was shown to use both N A D H and N A D P H as electron donor, and oxygen as electron acceptor [34]. The activity of the semi-purified enzyme was measured with different electrons acceptors, in the presence of various inhibitors (Table V). The enzyme had a low substrate specificity. The apparent affinity of the protein for the siderophore

TABLE V

Actioity of the plasmamembrane ferri-reductase with different electron acceptors and effect of various inhibitors The assays were performed as described in Materials and Methods. The reactions were initiated by adding the semi-purified enzyme (about 0.5 # g m1-1) to the sample cuvette. The final concentration of the different electron acceptors was 180 #M. The reaction mixtures contained 50 # M N A D P H and 2.5 # M F M N . The 100% activity corresponds to a specific activity of 650 nmol min -1. m g - 1 . FOB, ferrioxamine B; DFOB, desferrioxamine B; DCIP, dichlorophenolindophenol; and PCMB, para-chloromercuribenzoate. (Data are from a single representative experiment.) Substrate

Addition

Reduction rate (%)

FOB Fe(III)-EDTA Fe(III)-citrate (1 : 20) Ferricyanide DCIP FOB FOB FOB FOB FOB FOB FOB

HgCI 2 50/xM lodoacetamide 1 m M PCMB 200 # M N-ethylmaleimide 2 m M N a N 31 m M Ga(III)-DFOB 0.5 m M Ga(III)-DFOB 1 m M

100 110 7 1140 200 0 75 8 87 100 43 21

259 ferrioxamine B was higher (K m = 50/~M) than that for Fe(III)-EDTA (Km = 100/~M). Such a lack of specificity has been previously reported for the plasma membrane-bound ferri-reductase functioning in vivo. Whole cells reduced ferric citrate and ferric EDTA, as well as the siderophores ferrioxamine B, ferricrocin [10], rhodotorulic acid [17] and ferrichrome A (unpublished data). A similar low specificity was also described for the ferri-reductase systems in the roots of higher plants [14] and could be a general feature of reductive iron uptake [16]. Nevertheless, the high inhibitory effect of the nonreducible substrate analogue Ga(III)-desferrioxamine B on the reduction rate of ferrioxamine B indicates that the enzyme has some specificity toward the ferric substrate (Table V). It is not known why ferric citrate, which was previously shown to be rapidly reduced by whole cells [10], was reduced very slowly by the semi-purified protein (Table V). This paradox could mean that iron must be displaced from its citrate ligands prior to reduction in vivo. Such a displacement could occur in the cell wall, which bears many binding sites for di- and trivalent cations [35]. Fe(III) was previously shown to bind reversibly and in amounts at the cell surface when the cells are incubated with ferric citrate [10]. The sulfhydryl reagents HgCI 2, para-chloromercuribenzoate and to a lesser extent, iodoacetamide, all inhibited the enzyme activity (Table V). Sulfhydryl groups could therefore be involved in the active site of the enzyme, as has been shown for the ferrichrome reductase of Ustilago sphaerogena [6]. Studies are in progress to confirm this latter point, to characterize the enzyme further and to define the factors involved in the regulation of its expression.

Acknowledgments E. Lesuisse is a grantee of the Commission of the European Communities (Brussels, Belgium). This work was supported by grants from CNRS, Universit6 Paris 7 and the Ministrre de la Recherche et de l'Enseignement suprrieur (MRT 510069). We thank Dr. C.O. Parkes for his help in the preparation of the manuscript.

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Iron-reductases in the yeast Saccharomyces cerevisiae.

Several NAD(P)H-dependent ferri-reductase activities were detected in sub-cellular extracts of the yeast Saccharomyces cerevisiae. Some were induced i...
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