(Received for publication, March 27, 1995; and in revised form, June 20, 1995)
From the
Sulfite reductase (SiR) from Escherichia coli has a
subunit structure, where
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
subunit,
overexpresses flavin reductase activity by 100-fold, showing that
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
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
subunit.
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
and
, with a
subunit
structure for the holoenzyme(2) . All
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
chain, coded for by the cysI gene, binds one
iron-sulfur (Fe
S
) 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 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.
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 (2 mM). Then,
4 liters of minimal medium E, where MgSO
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.
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
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.
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.
Polymerization of 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.
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 molecular mass, but also a
very faint one at about 63 kDa, the
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.
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 subunit, with
comparable contributions of FAD and FMN (0.7-0.9 mol/
). This
value is significantly larger than that previously reported (1) and would suggest that some
subunits contain both FMN
and FAD. Measurements on the SiR holoenzyme revealed comparable amounts
of FAD (0.8 mol/mol of
subunit and smaller amounts of FMN 0.4
mol/mol of
). 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 subunit but a
still rather large amount of FAD (0.8-0.9 mol/mol of
subunit), confirming that FAD was present in more than 50% of the
subunits.
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 K
value (52 µM) than FMN or FAD with SiR-FP but lower K
value (14 µM) with the
holoenzyme.
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 (
), 40 (
), and 60
µM (
)). Panel B, double-reciprocal plots of
enzyme activity as a function of AcPyADP
concentration
with different NADPH concentrations (10 (
), 20 (
), 40
(
), 60 (
), and 80 µM (
)). 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 (), 40 (
), 60 (
), 80 (
), and
100 µM (
)). Panel B, double-reciprocal
plots of enzyme activity as a function of riboflavin concentration with
different NADPH concentrations (20 (
), 40 (
), 60 (
),
80 (
), and 100 µM (
)). 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.
We have shown here that the flavin reductase activity of
sulfite reductase resides in its flavoprotein component, exclusively: (a) overexpression of the 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
and
subunits, the flavin reductase
came along with
, and no activity was found in fractions
containing
; and (c) catalytic activities of the SiR
holoenzyme were even lower than those of the flavoprotein,
demonstrating that the
-
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
subunits. Because of the excellent expression of
in LS1312(pCYSJ)
strain and of the large size of the
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: polypeptide ratio. In the
current model, (a) each
subunit contains only one flavin
prosthetic group (either 1 FAD or 1 FMN), (b)
-FAD and
-FMN cooperate to form a pair required for electron transfer to
the
subunit, and (c) each flavin has a distinct role in
electron transfer with
-FAD being the ``entry port'' for
electrons from NADPH. Thus, while
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
polypeptide chain is the
signal for the selective binding of FMN to a second
chain.
Finally, NADPH would bind exclusively to
-FAD and not to
-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): 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
subunit (Fig. SI). This is also in good agreement with the
sequence homology of
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
polypeptide
chain(14) . A binding domain for NADPH can also be found from
these sequence alignments. The similarity between
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 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
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.