©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Flavin Reductase Activity of the Flavoprotein Component of Sulfite Reductase from Escherichia coli
A NEW MODEL FOR THE PROTEIN STRUCTURE (*)

(Received for publication, March 27, 1995; and in revised form, June 20, 1995)

Michel Eschenbrenner Jacques Covès Marc Fontecave (§)

From the Laboratoire d'Etudes Dynamiques et Structurales de la Sélectivité, Unité de Recherche Associée au Centre National de la Recherche Scientifique n332, Université Joseph Fourier, BP 53, 38041 Grenoble Cédex 9, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Sulfite reductase (SiR) from Escherichia coli has a alpha(8)beta(4) subunit structure, where alpha(8) is a flavoprotein (SiR-FP) containing both FAD and FMN as prosthetic groups. It also exhibits a NADPH:flavin oxidoreductase activity with exogenous riboflavin, FMN, and FAD serving as substrates. The flavin reductase activity may function during activation of ribonucleotide reductase or during ferrisiderophore reduction. A plasmid containing cysJ gene, coding for the alpha subunit, overexpresses flavin reductase activity by 100-fold, showing that alpha is the site of free flavin reduction. The overproducer allows a fast and simple preparation of large amounts of the flavoprotein. Kinetic studies of its flavin reductase activity demonstrates a ping-pong bisubstrate-biproduct reaction mechanism. NADP inhibition studies show that both substrates, NADPH and free flavins, bind to the same site. While the FAD cofactor mediates the electron transfer between NADPH and free flavins, the FMN cofactor is not essential since a FMN-depleted SiR-FP retains a large proportion of activity. In contradiction with previous reports, SiR-FP is found to contain 1.6-1.7 flavin per alpha subunit. This result, together with the sequence homology between SiR-FP and NADPH-cytochrome P-450 reductase, suggests a new model for the structure of the protein with one FMN and one FAD prosthetic group per alpha subunit.


INTRODUCTION

NADPH:sulfite reductase, which catalyzes the 6-electron reduction of sulfite to sulfide specifically by NADPH, is one of the most complex soluble enzymes known(1) . Reduction of sulfite is required for the biosynthesis of L-cysteine from sulfate. The enzyme consists of two different types of polypeptide chain, termed alpha and beta, with a alpha(8)beta(4) subunit structure for the holoenzyme(2) . All alpha chains coded for by the cysJ gene are proposed to bind only one flavin prosthetic group (FAD or FMN), and the holoenzyme thus contains four FMN and four FAD. Each beta chain, coded for by the cysI gene, binds one iron-sulfur (Fe(4)S(4)) cluster and one siroheme prosthetic group and is the site of sulfite reduction.

The fact that, in addition to sulfite, sulfite reductase is capable of catalyzing a number of other NADPH-dependent reduction reactions raises the question of its actual physiological function. As an example, we recently rediscovered sulfite reductase from Escherichia coli during our efforts to identify reducing systems involved in the activation of ribonucleotide reductase(3) , a key enzyme absolutely required for biosynthesis of deoxyribonucleotides, the DNA precursors. Moreover, sulfite reductase was shown to be a very efficient system for reducing ferrisiderophores (4) , an essential step for iron uptake by microorganisms. In fact, all of these ``new'' activities of sulfite reductase are dependent on its general flavin reductase activity. Sulfite reductase catalyzes the reduction of riboflavin, FMN, or FAD by NADPH generating reduced flavins that provide the electrons for the activation of ribonucleotide reductase or for the reduction of ferrisiderophores. E. coli contains another flavin reductase of low molecular mass (26 kDa) also involved in the activation of ribonucleotide reductase(5) .

NAD(P)H:Flavin oxidoreductases catalyze the reduction of free flavins by NADPH or NADH. They are members of a poorly studied family of enzymes that, in addition to their role in ribonucleotide reductase activation and in ferric reduction(6, 7) , were discovered as ancilliary enzymes of luciferases in luminescent bacteria(8) . Light is actually emitted during reaction between reduced FMN, a long-chain aliphatic aldehyde, and molecular oxygen. Only two flavin reductases, one from Vibrio harveyi(9) and one from Eubacterium Sp. (10) are known to have a flavin cofactor, and only the first one has been cloned, purified, and crystallized very recently(11) . Sulfite reductase is thus another example of a flavoprotein functioning as a flavin reductase and may serve to study the mechanism of electron transfer from flavin cofactors to flavin substrates. Most flavin reductases are not flavoproteins and probably serve to accommodate the electron donor and acceptor in the same active site for allowing an efficient electron transfer to take place. One example is the 26-kDa flavin reductase of E. coli(5) .

Since reduction of natural substrates such as flavins by sulfite reductase has not been studied in great detail before, despite numerous and brilliant studies of the enzyme in terms of its ``diaphorase'' activities (12, 13, 14) and in view of the physiological importance of such reactions, we investigated the mechanisms of reduction of flavins by sulfite reductase. We suspected the flavoprotein component to be the site of the reaction and the metalloprotein component not to be required. The flavoprotein from E. coli can be obtained during dissociation of the holoenzyme with urea(2) . However, such a treatment generated a protein that was substantially depleted of FMN and had, as a result of partial denaturation, low catalytic activity. That preparation is thus not appropriate for investigating the reactivity of the flavoprotein. On the other hand, an active flavoprotein was purified from a mutant strain of Salmonella typhimurium, which lacks the hemoprotein component of the enzyme (14, 15) .

It is now well accepted that two alpha subunits cooperate to form a single active FAD and FMN pair, where the two flavins play distinct roles in catalysis(12, 15) . The FAD serves as an entry for electrons from NADPH, while FMN serves as a mediator for rapid transfer of these electrons to the hemoprotein component where sulfite is reduced or to artificial acceptors such as cytochrome c. Whether exogenous flavins receive their electrons from FAD and/or from FMN is not established.

We report here (a) the overexpression, purification, and characterization of the flavoprotein component of the sulfite reductase from E. coli, in a fully active non denatured form; (b) a study of its flavin reductase activity; and (c) a new hypothesis about its structure and the mechanisms of electron transfer.


EXPERIMENTAL PROCEDURES

Materials

Riboflavin, FMN, FAD, 3-acetylpyridine adenine dinucleotide phosphate (AcPyADP), (^1)cytochrome c, ferricyanide, MgSO(4), glucose, and p-chloromercuriphenylsulfonic acid were purchased from Sigma. Sephacryl S-400 HR was from Pharmacia Biotech Inc., and hydroxyapatite Bio-Gel HTP was from Bio-Rad. DE52 was from Bio-Rad. All other chemicals were of the purest grade.

Strains, Plasmids, and Growth Conditions

The E. coli LS1312 strain, which lacks an active fre gene, was available in our laboratory. Plasmid pJYW613 carrying the E. coli B cysJI region, including the cysJIH promoter, plus cysG from S. typhimurium was purified from the NM522 (pJYW613) strain kindly provided by Dr N. M. Kredich (Duke University Medical Center, Durham, NC). Plasmid pJYW613 was digested by BamHI and BglII to isolate the 3-kilobase fragment containing the cysJ gene and cysJIH promoter. This fragment was inserted into the BamHI site of pBR322 to obtain the pCYSJ plasmid. The E. coli LS1312 strain was transformed by this plasmid.

LS1312 (pCYSJ) was grown overnight at 37 °C in 100 ml of minimal medium E (16) complemented with glucose (0.5%), thiamin (4 µg/ml), ampicillin (100 µg/ml), and MgSO(4) (2 mM). Then, 4 liters of minimal medium E, where MgSO(4) was omitted, were inoculated with 1% of the overnight culture. Growth was monitored by following the absorbance at 600 nm. Cells were harvested at the stationary phase by centrifugation. 3.3 g of cells were collected.

Purification Procedures

Sulfite reductase holoenzyme was obtained from E. coli LS1312 strain in a pure form as previously described(3) .

A procedure to purify the sulfite reductase flavoprotein component (SiR-FP) was described in (2) . The pure sulfite reductase was incubated with 5 M urea. Then, a DEAE-cellulose (DE52) chromatography was run in the presence of 5 M urea and separated the hemoprotein and the flavoprotein components of sulfite reductase.

The purification of the SiR-FP from its overproducer was as follows. Soluble extracts (16.5 ml, 200 mg of protein) of E. coli LS1312 (pCYSJ) were prepared as previously described(5) . Total extracts were supplemented with 3% streptomycin sulfate under stirring over 1 h and centrifuged for 30 min at 20,000 g. The supernatant was incubated 30 min at 4 °C with DNase. This step was followed by an ammonium sulfate precipitation (60% final saturation). Centrifuged pellets (30 min, 20,000 g) were then dissolved in 50 mM Tris-Cl, pH 7.5, 50 mM NaCl, and 5% glycerol (buffer A) and loaded on a Sephacryl S-400 HR column (2.6 93 cm, 494 ml) previously equilibrated with buffer A. Elution was performed at 1 ml min, and 5-ml fractions were collected. Fractions were assayed for protein (absorbance at 280 nm) and flavin reductase activity. The active fractions (29 ml, 17.4 mg of protein) were pooled. 9.9 mg of protein were fractionated in two runs on a hydroxyapatite column (2.6 1.9 cm, 10 ml) previously equilibrated with 10 mM potassium phosphate buffer, pH 7.0 (buffer B). Proteins were loaded at 0.25 ml min. The column was washed at 1 ml min with buffer B until the base line was recovered. Then, elution was made with a linear gradient from 10 to 500 mM KPO(4) during 60 min. Active fractions (58 ml, 5.8 mg of protein) were desalted by cycles of concentration and dilution in 50 mM Tris-Cl, pH 7.5, 5% glycerol using a Diaflo cell equipped with a YM-30 membrane. All the operations were carried out at 4 °C.

Enzymic Assays

NADPH-dependent reactions were carried out in a final volume of 0.5 ml containing 50 mM Tris-Cl, pH 7.5, 0.25 mM NADPH, an electron acceptor, and an appropriate amount of enzyme. Electron acceptors were present at the following concentrations: 0.1 mM flavin (riboflavin, FMN, or FAD), 0.3 mM ferricyanide, 0.2 mM AcPyADP, and 0.1 mM cytochrome c. Rates were measured at room temperature using a Kontron Uvikon 930 spectrophotometer. Absorbance changes were followed at 340 nm for flavin and ferricyanide as acceptors, at 363 nm for AcPyADP, and at 550 nm for cytochrome c. The following extinction coefficients were used: NADPH, = 6.22 mM cm; AcPyADPH, = 5.6 mM cm; reduced cytochrome c, = 22 mM cm. The reaction was initiated by the addition of the protein solution. 1 unit of activity is defined as the amount of protein catalyzing the oxidation of 1 nmol of NADPH per min or the reduction of 1 nmol of acceptor (AcPyADP or cytochrome c) per min. Specific activity is defined as units per mg of protein.

Other Assays

FAD and FMN extracted from the enzyme were measured fluorimetrically by the method of Faeder and Siegel (17) using a Perkin-Elmer LS 450 fluorimeter. Concentrations of standard flavin solutions were determined spectrophotometrically by means of their absorbances at 450 nm, utilizing reported extinction coefficients (12.2 mM cm for FMN and 11.3 mM cm for FAD)(18, 19) .

Absorption spectra of purified enzymes were recorded at room temperature with a Kontron Uvikon 930 spectrophotometer in a quartz cell (10-mm light path) of 0.1 ml.

Protein concentration was determined routinely by the method of Bradford using bovine serum albumin as standard (20) and the commercial Bio-Rad protein assay solution.

The concentration of the pure protein preparations used for the determination of the flavin:polypeptide ratio was determined from the total amino acid composition of these preparations. Assays were made by Dr. J. Gagnon (Institut de Biologie Structurale, Grenoble, France). Samples were hydrolyzed for 24, 48, 72, and 120 h under reduced pressure at 110 °C in constant boiling HCl containing 1% (w/v) phenol. Analyses were performed on acid hydrolysates with a Beckman 7300 amino acid analyzer using ninhydrin for detection. The amino acid composition is based on the average of triplicate 24-, 48-, and 72-h hydrolysis values, except for threonine and serine, which were extrapolated to zero time, and valine and isoleucine, for which the 120-h values were taken. Half-cysteine and tryptophane were not determined. The number of residues per mol is based on a total number of 585 amino acids, to be compared to 598 derived from the sequence, since the 11 tryptophanes and 2 cysteines have not been determined. A very good fit was found for aspartate/asparagine, alanine, methionine, leucine, histidine, and lysine. From the experimental composition, the mass of protein in the sample has been obtained and the concentration of the protein sample determined with a maximal error of 6%. Additional assays were run in triplicate by Dr. P. Dalbon and L. Bridon (BioMérieux, Lyon, France) using an Applied Biosystems amino acid analyzer model 420 H and were in agreement with results of Dr. J. Gagnon.

Preparation of FMN-depleted Enzyme

The pure sulfite reductase flavoprotein was treated with p-chloromercuriphenylsulfonic acid by the method described in (12) . The protein solution (2 ml, 0.7 mg of protein) was diluted in 46 ml of solution containing 100 mM Tris-Cl, pH 7.7, 0.1 mM EDTA, and 1 µMp-chloromercuriphenylsulfonic acid. The incubation was carried out for 18 h at 4 °C in the dark to release FMN prosthetic groups while FAD moieties remained enzyme bound. The mixture was then washed with 50 mM Tris-Cl, pH 7.5, 5% glycerol and concentrated at 1.67 ml (0.45 mg of protein) on a Diaflo cell equipped with a YM-30 membrane. Aliquots both from the filtrate fraction and the enzyme solution were analyzed for flavin concentration.

Polyacrylamide Gel Electrophoresis

Protein aliquots resulting from the different steps of the purification procedure were analyzed by 0.1% SDS-15% polyacrylamide gel electrophoresis according to (21) . Calibration was obtained with the following protein size markers (Pharmacia kit): phosphorylase b, 94 kDa; bovine serum albumin, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; soybean trypsin inhibitor, 20.1 kDa; and alpha-lactalbumin, 14.4 kDa. The protein bands were visualized with Coomassie Blue staining.

Polymerization of alpha subunits was ascertained by using a Bio-Rad precast 4-20% gradient gel for native electrophoresis. Protein size markers used were sulfite reductase, 780 kDa; thyroglobulin, 669 kDa; ferritin, 440 kDa; catalase, 232 kDa; lactate dehydrogenase, 140 kDa; and albumin, 67 kDa.


RESULTS

SiR-FP Expression from Cloned cysJ

Plasmid pJYW613 is a pBR322 derivative, containing the E. coli cysJI region, including the cysJIH promoter, plus cysG from S. typhimurium, which have been cloned between the EcoRI and BamHI sites of the host vector. The 3-kilobase BamHI-BglII fragment from pJYW613 contains cysJ under the control of the cysJIH promoter and was subcloned into pBR322 to give pCYSJ.

E. coli LS1312 strain, lacking the low molecular mass flavin reductase(22) , was the host for pCYSJ to avoid contamination with extra flavin reductase activity. Overexpression of SiR-FP was best achieved by growing bacteria in minimal medium under sulfur limitation conditions.

Soluble extracts from 3.3-g cells were assayed for flavin reductase activity in the presence of NADPH. A specific activity of 1200 was obtained, to be compared to 12 for the extracts from the plasmid-free LS1312 strain. This then shows both the 100-fold overproduction of SiR-FP and a strong evidence that the flavin reductase of SiR resides in its flavoprotein subunit. A fast and simple two-step purification of SiR-FP from the overproducer (Table 1) led to almost pure preparations of the enzyme. SDS-polyacrylamide gels showed a major band at 66 kDa, the alpha molecular mass, but also a very faint one at about 63 kDa, the beta molecular mass (data not shown). In addition, sulfite reductase activity of such preparations demonstrated that they were contaminated by very low amounts of hemoprotein (SiR-HP) from the host (1% of SiR-FP is complexed to SiR-HP to form the SiR holoenzyme). The gel filtration chromatography is a remarkable purification step allowed by the large molecular weight of the flavoprotein. In the following such a preparation is named recombinant SiR-FP.



Characterization of Recombinant SiR-FP

As previously shown, SiR-FP is a polymer of identical alpha (M(r) 66,000) subunits. This was clear from gel filtration on Sephacryl S-400 and from non-denaturing native gel electrophoresis (data not shown). SiR-FP migrated as a single broad band centered slightly above molecular mass 670 kDa (approximately). In agreement with two previous reports, which showed that SiR-FP derived from S. typhimurium(14) or from urea-dissociated E. coli SiR holoenzyme (2) was an octamer, this value shows that the purified protein is indeed a polymer. Whether it is an octamer or a larger complex cannot be accurately concluded from native gel electrophoresis. The behavior of our preparation on native gels was the same after pretreatment with 8 M urea for 8 h or after removal of the FMN prosthetic group (data not shown).

The absorption spectrum was identical to those previously reported (2) with the bands at 382 and 456 nm (with a shoulder at 480 nm) characteristic of the flavin moieties. By fluorimetry, it was possible to determine the flavin content of SiR-FP. From five measurements, SiR-FP was found to contain 1.6-1.7 mol of flavin/mol of alpha subunit, with comparable contributions of FAD and FMN (0.7-0.9 mol/alpha). This value is significantly larger than that previously reported (1) and would suggest that some alpha subunits contain both FMN and FAD. Measurements on the SiR holoenzyme revealed comparable amounts of FAD (0.8 mol/mol of alpha subunit and smaller amounts of FMN 0.4 mol/mol of alpha). The well established lability of FMN probably explains the lower FMN content in our holoenzyme preparation.

The FMN-depleted form of SiR-FP was obtained by treatment with p-chloromercuriphenylsulfonic acid. It was shown, by fluorimetry, to contain only 0.01 mol of FMN/mol of alpha subunit but a still rather large amount of FAD (0.8-0.9 mol/mol of alpha subunit), confirming that FAD was present in more than 50% of the alpha subunits.

Characterization of the Flavin Reductase Activity of SiR-FP

Flavin reductase activity of SiR-FP was monitored spectrophotometrically with increasing concentrations of both substrates. Kinetic parameters, K and V, for flavins (riboflavin, FMN, and FAD) and for NADPH were obtained from linear Lineweaver-Burk plots (Table 2). These parameters were also determined in the case of the flavoprotein preparation obtained by dissociation from the hemoprotein subunit during treatment of the SiR-holoenzyme with 5 M urea. We verified that the hemoprotein was devoid of flavin reductase activity (data not shown) and that all the flavin reductase activity totally copurified with the flavoprotein. Table 2also shows the values obtained for the SiR holoenzyme. These data show the effect of urea treatment and that of the complexation to the hemoprotein on SiR-FP enzyme activity.



First, urea-treated SiR-FP has much higher K values for all flavins and also lower specific activities than recombinant SiR-FP. The large K value for flavins did not allow the determination of the K value for NADPH, since too large concentrations of absorbing flavins made impossible the spectrophotometric assay. It is thus clear that a significant denaturation of the protein occurs during urea-dependent dissociation of the holoenzyme and that our preparations should be preferred to the ones reported previously for reliable investigations of structure and activity of SiR-FP.

Second, the presence of the hemoprotein subunit greatly affects the enzymatic properties of the flavoprotein, since Kand V values for flavins and NADPH with SiR holoenzyme were significantly different from those with SiR-FP. For example, flavin substrate selectivity of SiR-FP changed upon binding to the hemoprotein, with riboflavin giving higher Kvalue (52 µM) than FMN or FAD with SiR-FP but lower K value (14 µM) with the holoenzyme.

Reaction Mechanisms

Previous studies (12, 13, 14) have clearly established that FAD receives the electrons from NADPH directly and transfers them to the FMN cofactor or to some artificial electron acceptor such as AcPyADP. Reduced FMN cofactor serves for the reduction of the redox centers (iron-sulfur center and siroheme) of the hemoprotein or other artificial electron acceptors such as cytochrome c or ferricyanide. The role of FAD in the reduction of AcPyADP was concluded only from kinetic studies, which showed that NADP was an inhibitor competitive with respect to both substrates. Moreover, the NADPH-AcPyADP transhydrogenase activity of SiR-FP does not involve FMN prosthetic groups since it was largely retained in FMN-depleted SiR-FP preparations and was not stimulated by addition of FMN(14) . The latter observation has been confirmed with the recombinant SiR-FP preparations (Fig. 1). However, both informations do not demonstrate necessarily that FAD has to be reduced first. The polypeptide chain could provide a site for both NADPH and AcPyADP, facilitating thus the direct hydride transfer.


Figure 1: Effects of FMN depletion on various reductase activities of SiR-FP. 100% activity is achieved with recombinant SiR-FP. Specific activities are 19,970 (AcPyADP), 37,590 (Ferricyanide), 88,570 (cytochrome c) and 13,900 (riboflavin). Assays were also carried out, as described under ``Experimental Procedures'' using the same amount of FMN-depleted SiR-FP in each experiment. The residual activity is reported.



The reduction of AcPyADP by NADPH yielding NADP and AcPyADPH is a typical bisubstrate-biproduct reaction. Its mechanism, with respect to orders of substrate addition and product release, can be delineated by steady-state kinetic analysis. Double-reciprocal plots of initial velocities versus substrate concentrations show intersecting patterns for the sequential mechanism and parallel patterns for the ping-pong mechanism. Thus, the transhydrogenase activity of SiR-FP was determined as a function of AcPyADP concentration at several constant levels of NADPH and as a function of NADPH at several constant levels of AcPyADP. Initial velocities followed typical Michaelis-Menten kinetics, and double-reciprocal plots of the results show a series of parallel lines, in agreement with a ping-pong Bi-Bi mechanism in which reduced pyridine nucleotide reduces first the FAD prosthetic group of SiR-FP, which in turn transfers electrons to the pyridine nucleotide (Fig. 2, A and B).


Figure 2: Acetylpyridine-ADP (AcPyADP) transhydrogenase activity mechanism. Panel A, double-reciprocal plots of enzyme activity as a function of NADPH concentration with different AcPyADP concentrations (20 (), 30 (up triangle, filled), 40 (), and 60 µM (bullet)). Panel B, double-reciprocal plots of enzyme activity as a function of AcPyADP concentration with different NADPH concentrations (10 (black square), 20 (), 40 (up triangle, filled), 60 (), and 80 µM (bullet)). Assays were carried out with 1.75 µg of SiR-FP.



The same kinetic analysis was applied to the riboflavin reductase activity of SiR-FP (Fig. 3). Again, the double-reciprocal plots of the results show a series of parallel lines and demonstrate a ping-pong Bi-Bi mechanism in which NADPH reduces the flavin cofactors first, and then reduced SiR-FP transfers electrons to the flavin substrate.


Figure 3: Flavin reductase activity mechanism. Panel A, double-reciprocal plots of enzyme activity as a function of NADPH concentration with different riboflavin concentrations (20 (black square), 40 (), 60 (up triangle, filled), 80 (), and 100 µM (bullet)). Panel B, double-reciprocal plots of enzyme activity as a function of riboflavin concentration with different NADPH concentrations (20 (black square), 40 (), 60 (up triangle, filled), 80 (), and 100 µM (bullet)). Assays were carried out with 1.75 µg of SiR-FP.



That NADPH is able to reduce FAD and FMN prosthetic groups in the absence of electron acceptors has been previously established(12) . Also, it is known that the FAD cofactor of FMN-depleted SiR-FP can be reduced by NADPH. We have confirmed these observations with the recombinant SiR-FP preparations by monitoring by spectrophotometry the bleaching of enzyme solutions during incubation with NADPH. Actually, the absorption at 456 nm, characteristic of the oxidized flavin prosthetic groups, greatly decreases upon reduction (Fig. 4A, compare spectraa and c). During the reaction, a new band appears at 585 nm, which reflects the presence of flavin semiquinone radical (Fig. 4A, spectrumc). The fact that this low energy band could not be detected during reduction of the FMN-depleted form of SiR-FP (Fig. 4B, spectrumc) suggests that the semiquinone radical of reduced SiR-FP was specifically from FMN and not from FAD.


Figure 4: Reduction of SiR-FP and FMN-depleted SiR-FP flavin prosthetic groups by NADPH and NADH. Absorption spectra of 28 µg of SiR-FP (A) and 23 µg of FMN-depleted SiR-FP (B), in 0.1 ml of 50 mM Tris-Cl, pH 7.5, were recorded as described under ``Experimental Procedures'' without (a) or with a reductant (b, 400 µM NADH; c, 400 µM NADPH).



The amazing discovery was that in full disagreement with the total specificity of SiR-FP for NADPH, NADH was able to reduce part of the flavins (Fig. 4A). It was easy to show with the FMN-depleted SiR-FP enzyme that NADH was reducing FMN directly and selectively since no significant change of the visible spectrum could be observed during incubation of the FMN-depleted enzyme, which only contains FAD, with NADH (Fig. 4B, compare spectraa and b), whereas NADPH was still able to reduce FAD even in the absence of FMN (Fig. 4B, spectrumc). That NADH was able to reduce FMN is further shown by the accumulation of the FMN radical, absorbing at 585 nm, also during reaction of SiR-FP with NADH (Fig. 4A, spectrumb).

The puzzling fact is that reduced FMN cofactor seems to be inactive when generated during reduction by NADH while it is active when electrons were provided through the other flavin (FAD) cofactor. We actually confirmed that NADH could not serve as an electron source for the reduction of cytochrome c, ferricyanide, free flavins, and 2,6-dichlorophenol-indophenol. The reason for that is still unclear and requires further studies. We have shown that NADH is not an inhibitor of NADPH-dependent reductions.

Reduction of Flavins by FMN-depleted SiR-FP

In previous studies(12, 13) , depletion of SiR-FP of its FMN content has served to demonstrate that the FMN moiety is required for electron transfer to the site of sulfite reduction on the beta subunit as well as for electron transfer to cytochrome c. We have used this strategy to determine whether free flavins take the electrons from FAD and/or from FMN. As shown in Fig. 1, as previously reported, cytochrome c or ferricyanide reduction was almost totally dependent on the presence of FMN in SiR-FP, and AcPyADP reduction was only slightly affected by the absence of FMN in SiR-FP. Cytochrome c or ferricyanide reduction can be restored by addition of 1 µM FMN but not by 1 µM riboflavin, indicating that riboflavin cannot play the role of the FMN cofactor. Fig. 1shows that a large proportion of the flavin reductase activity is retained when the enzyme is FMN depleted.

Competitive Inhibition of Flavin Reduction by NADP

NADP is known to inhibit all NADPH-dependent reactions catalyzed by sulfite reductase (12) . When the steady-state kinetics of the NADPH:riboflavin reductase activity of SiR-FP was studied as a function of NADP and NADPH concentration, NADP was found to be a competitive inhibitor with respect to NADPH with a K value of 40 µM, comparable to the Kvalue for NADPH. More surprising was the observation from similar kinetic studies that inhibition by NADP was also competitive with respect to flavins (FMN, K = 39 µM; riboflavin, K = 38 µM). This shows that both substrates (NADPH and free flavin) interact with SiR-FP to the same binding site. A common binding site for riboflavin, NADPH, and NADP was also demonstrated in the case of FMN-depleted SiR-FP (data not shown) by similar steady-state kinetic studies.


DISCUSSION

We have shown here that the flavin reductase activity of sulfite reductase resides in its flavoprotein component, exclusively: (a) overexpression of the alpha subunit in E. coli leads to an overexpression of the flavin reductase activity in bacterial soluble extracts; (b) during dissociation of the holoenzyme into separate alpha and beta subunits, the flavin reductase came along with alpha, and no activity was found in fractions containing beta; and (c) catalytic activities of the SiR holoenzyme were even lower than those of the flavoprotein, demonstrating that the alpha-beta association partly affects the recognition of the flavins by the flavoprotein and/or the efficiency of the electron transfers from NADPH to the substrates.

For the first time, large amounts of flavoprotein (SiR-FP) from E. coli can be obtained in a pure and active form. The pure enzyme is in fact a large molecule because of the spontaneous aggregation of alpha subunits. Because of the excellent expression of alpha in LS1312(pCYSJ) strain and of the large size of the alpha polymer, the flavoprotein is very easily purified. A single gel filtration step provides the enzyme more than 90% pure. An extra hydroxyapatite step is used for a further purification. Previously described preparations of the flavoprotein from E. coli (obtained from dissociative treatment of the holoenzyme by urea) were partially denatured. Indeed, kinetic parameters of the latter preparations were very different from those of the recombinant SiR-FP preparations reported here, with much higher K values for flavins and much lower V values.

Several structural and functional aspects of this enzyme are intriguing. The absence of three-dimensional structure leaves the door open for speculation. The first problem concerns the flavin:alpha polypeptide ratio. In the current model, (a) each alpha subunit contains only one flavin prosthetic group (either 1 FAD or 1 FMN), (b) alpha-FAD and alpha-FMN cooperate to form a pair required for electron transfer to the beta subunit, and (c) each flavin has a distinct role in electron transfer with alpha-FAD being the ``entry port'' for electrons from NADPH. Thus, while alpha polypeptide chain has the ability to bind both cofactors, it would only bind one. This would indicate that the binding of one flavin precludes the binding of a second flavin to the same polypeptide chain. Moreover, this model implies that the binding of FAD to one alpha polypeptide chain is the signal for the selective binding of FMN to a second alpha chain. Finally, NADPH would bind exclusively to alpha-FAD and not to alpha-FMN, while each one contains a NADPH binding domain (see below). Even though all this is conceivable, the validity of the present model is based on a number of assumptions, whose structural and functional implications have not been investigated. How the binding selectivity is achieved is not understood.

Such a model needs that in any preparation of SiR-FP the FAD (or FMN):alpha polypeptide ratio be not larger than 0.5. However, in all our preparations, we found a ratio between 0.7 and 0.9. In most previous papers, 0.5 was the maximum value reported. However, there is also one example in which higher values were reported(2) . We thus would like to suggest another model for SiR-FP protein structure with 2 flavins (1 FAD + 1 FMN) per alpha subunit (Fig. SI). This is also in good agreement with the sequence homology of alpha to NADPH:cytochrome P-450 reductase(14, 23) , in which a single polypeptide chain of 77 kDa binds 1 FAD and 1 FMN at distinct binding sites.


Figure SI: Scheme I.



The microsomal flavoprotein NADPH:cytochrome P-450 reductase shuttles electrons from NADPH via its FMN and FAD prosthetic groups to cytochrome P-450 but also to other electron acceptors, including cytochrome c and ferricyanide. Another group of proteins, the NO-synthases, has been recently shown to contain these flavins as prosthetic groups and to share significant sequence identity, particularly within the suggested functional domains(24) .

Alignment of the amino acid sequences of SiR-FP and NADPH-cytochrome P-450 reductase with sequences of one FMN binding protein, the flavodoxin from Desulfovibrio vulgaris, and one FAD-binding protein, the spinach ferredoxin-NADP oxidoreductase, has clearly revealed a FMN- and a FAD-binding domain in the N-terminal and C-terminal regions, respectively, of the alpha polypeptide chain(14) . A binding domain for NADPH can also be found from these sequence alignments. The similarity between alpha and NADPH-cytochrome P-450 reductase has actually led to the hypothesis that the two proteins evolved from a common precursor, which contained binding regions for both FMN and FAD.

Variations of the FAD (or FMN) content of SiR-FP could partly be explained by partial losses of flavins and partial denaturation of the protein during protein extraction and purification. The well established greater lability of FMN would then explain why the FMN:FAD ratio is frequently lower than 1. It is possible that the overexpression of alpha and the rapid purification procedures reported here provide conditions for limited denaturation and loss of flavin prosthetic groups.

The kinetic studies of the flavin reductase activity show that (a) there is only one site for NADPH, NADP and flavin substrates, NADP acting as a competitive inhibitor; and (b) the reduction reaction proceeds by a ping-pong mechanism. NADPH has been shown spectrophotometrically to transfer its electrons to the FAD prosthetic group in the absence of substrates. Our data thus suggest that the flavin substrates mainly receive the electrons directly from reduced FAD in the same site (Fig. SI). From that point of view, flavins and AcPyADP as substrates behave similarly. Cytochrome c, ferricyanide, or subunit beta belong to a second class of substrates, which receive their electrons from FMN and not from FAD, FAD then serving as a mediator between NADPH and FMN.

According to such a hypothesis, depletion of SiR-FP of its FMN moiety should not have affected its flavin reductase activity. This is clearly not the case. Nevertheless, a large proportion of that activity (60%) is retained. If we assume that removal of FMN may have significant harmful effects on the structure of the protein and on the efficiency of FAD to catalyze the electron transfer from NADPH to free flavins, thus explaining the 40% loss, we may still conclude that our proposed model is correct.


FOOTNOTES

*
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§
To whom correspondence should be addressed. Tel.: 33-76-51-44-67; Fax: 33-76-51-43-82.

(^1)
The abbreviations used are: AcPyADP, 3-acetylpyridine adenine dinucleotide phosphate; AcPyADPH, reduced 3-acetylpyridine adenine dinucleotide phosphate; SiR, sulfite reductase; SiR-FP, sulfite reductase flavoprotein moiety.


ACKNOWLEDGEMENTS

We thank Dr. Pierre Michon for fluorimetric technical support, Dr. Vincent Nivière for assistance during molecular biology experiments, and Drs. Jean Gagnon and Pascal Dalbon for performing amino acid compositions.


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