The glycyl radical (Gly-734) contained in the
active form of pyruvate formate-lyase (PFL) of Escherichia
coli is generated by the
S-adenosylmethionine-dependent pyruvate
formate-lyase-activating enzyme (PFL activase). A 5'-deoxyadenosyl
radical intermediate produced by the activase has been suggested as the
species that abstracts the pro-S hydrogen of the glycine
734 residue in PFL (Frey, M., Rothe, M., Wagner, A. F. V.,
and Knappe, J. (1994) J. Biol. Chem. 269, 12432-12437). To enable mechanistic investigations of this system we
have worked out a convenient large scale preparation of functionally
competent PFL activase from its apoform. The previously inferred
metallic cofactor was identified as redox-interconvertible polynuclear
iron-sulfur cluster, most probably of the [4Fe-4S] type, according to
UV-visible and EPR spectroscopic information. Cys
Ser replacements
by site-directed mutagenesis determined Cys-29, Cys-33, and Cys-36 to
be essential to yield active holoenzyme. Gel filtration chromatography
showed a monomeric structure (28 kDa) for both the apoenzyme and
holoenzyme form. The iron-sulfur cluster complement proved to be a
prerequisite for effective binding of adenosylmethionine, which induces
a characteristic shift of the EPR signal shape of the reduced enzyme
form ([4Fe-4S]+) from axial to rhombic symmetry.
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INTRODUCTION |
Pyruvate formate-lyase
(PFL)1 is a key enzyme of the
anaerobic glucose fermentation in Escherichia coli and other
microorganisms, catalyzing the CoA-dependent cleavage of
pyruvate to acetyl-CoA and formate (1). In its active form, this enzyme
contains a stable glycyl radical (Gly-734) required for catalysis (2). Studies of mutants and substrate analogs propose that on substrate binding, the spin is transferred from Gly-734 to the reaction center
(Cys-418/Cys-419), where a thiyl radical initiates the homolytic
cleavage of the pyruvate C-C bond (3, 4).
The PFL radical is produced post-translationally by abstraction of the
Hsi atom from the Gly-734 methylene group (5). This occurs
by the action of pyruvate formate-lyase-activating enzyme (PFL
activase), which employs adenosylmethionine (AdoMet) and dihydroflavodoxin (or artificial 1e
donors) as
co-substrates, yielding 5'-deoxyadenosine and methionine as
stoichiometric co-products (Equation 1).
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(Eq. 1)
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Because the abstracted H atom is recovered in the
5'-CH3 of deoxyadenosine, a 5'-deoxyadenosyl radical
intermediate has been proposed as the actual H abstracting species in
this system. How this nucleoside radical is generated from AdoMet is an
intriguing problem, requiring in the first place PFL activase to be
fully characterized at the molecular level.
Previous work has identified PFL activase as a monomer of 28 kDa, and
its primary structure, as deduced from the DNA-nucleotide sequence (246 amino acids), has been established by N- and C-terminal amino acid
sequencing (6, 7). The enzyme has long since been recognized to require
Fe2+ for catalytic activity (6), and iron contents in the
order of one iron/polypeptide chain were determined in purified
preparations (3, 8). Only more recently, an iron-sulfur cluster has
been reported in anaerobically purified enzyme (9). Previously, a
[4Fe-4S] cluster had been identified in the analogous activating protein of anaerobic ribonucleotide reductase (RNR) (10).
We report here a new procedure for the isolation of larger quantities
of PFL activase from overproducing E. coli cells. The enzyme
was carried through the apoform and reconstituted in vitro with Fe2+ and sulfide. The holoenzyme form obtained has
full catalytic activity under modified assay conditions that omit the
previous free Fe2+ supplement. UV-visible and EPR
spectroscopic data indicate that the metal center comprises a
redox-interconvertible [4Fe-4S] cluster, which is essential for the
interaction of the enzyme with AdoMet. In addition, single Cys
Ser
mutations were performed that corroborate that the cysteinyl residues
in the Cys-Xaa3-Cys-Xaa2-Cys motif of the
N-terminal polypeptide section, proposed previously as metal
interaction site (7), are essential for reconstituting enzyme activity.
Our data place PFL activase, together with the AdoMet-dependent activating protein of anaerobic RNR (11),
into a new subclass of iron-sulfur proteins that generate glycyl
radicals in free radical enzymes.
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EXPERIMENTAL PROCEDURES |
Materials--
Pyruvate formate-lyase (nonradical form) was
prepared by previous procedures (2, 6). High potential iron-sulfur
protein from Ectothiorhodospira halophila was available in
the laboratory. [8-14C]Adenosylmethionine was prepared
enzymatically as described in Ref. 5 using [8-14C]ATP
from Amersham-Buchler. AdoMet, S-adenosylhomocysteine,
catalase, DNase I, and RNase were from Boehringer Mannheim; Q-Sepharose FF, Sephadex G-25 fine, Superdex 75 HR, and Ultrogel AcA44 were from
Pharmacia Biotech Inc.; Polymin G35 was from BASF AG (Ludwigshafen, Germany).
Bacterial Strain, Plasmids, and Site-directed
Mutagenesis--
E. coli strain 234M1 (2), a MC4100
derivative carrying the Cmr gene inserted into the
chromosomal act gene, was used as host for the
overproduction of activase proteins (wild type or mutants). The
parental expression plasmid pXA-1 contained the act gene
encompassing the 1-kilobase pair DNA fragment comprising nucleotides
2529-3541 in the sequence (7) linked to the Tac promotor of pKK223-3 (Pharmacia). This construct differs from the earlier pKE-1 (5) by
deleting 0.4 kilobase pairs of the act downstream
region.
Site-directed mutagenesis was performed with the phosphorothioate
method (12) using the SculptorTM in vitro mutagenesis
system (RPN 1526, Amersham) according to the supplier's manual. This comprised single Ser substitutions of the six Cys residues: TGT codons
(for Cys in amino acid positions 12, 36, and 102) and TGC codons (for
Cys in amino acid positions 29, 33, and 94) were mutated to TCT and TCC
codons, respectively, by using 24-30-mer antisense mutagenic
oligonucleotides that were synthesized by R. Frank (Zentrum für
Molekulave Biologie, Heidelberg, Germany). All mutant genes were proof
sequenced.
Activase Assays--
The reaction mixtures (0.5 ml) for PFL
conversion to the radical form contained in general: 0.15 M
Tris-HCl, pH 7.5, 0.1 M KCl, 5 mM DTT, 10 mM potassium oxamate, 0.2 mM AdoMet, 50 µM 5-deazariboflavin, 1 µM riboflavin, 10 µg of catalase, 60 µg (340 pmol) of PFL, and 0.1-2 µg of the
activase sample. A slide projector light was used for continuous
photoreduction of deazaflavin, and reactions were run at 30 °C under
argon, usually for 10 min. In the assay version termed "holoactivase
assay" (see "Results"), the reaction mixture contained
additionally 10 µM EDTA, and the reaction was started with the activase sample. In the "standard assay," which was used to determine intact activase together with iron center defective enzyme
forms, the reaction mixture contained additionally 0.2 mM
Fe(NH4)2(SO4)2 (and
optionally 0.1 mM Na2S) and was preincubated, in the presence of activase, in the dark for 20 min; the reaction was
started by illumination. The produced radical form of PFL was
determined by activity measurement (coupled optical assay with a
5-10-µl aliquot); 35 IU correspond to 1 nmol of PFL dimer containing
one Gly radical.
One unit of activase activity was defined to produce 1 nmol of the PFL
radical/min. (Activity data reported here are lower by a factor of 700 when compared with those in previous work (6), which used a different
unit definition.
Chemical Analyses--
Protein concentrations of purified
apoenzyme solutions were determined by the 280-nm absorption using a 1 mg/ml value of 1.38 (as calculated from the Trp and Tyr residues in the
polypeptide and verified by amino acid analysis). On reconstituted
enzyme and other enzyme fractions, the Bradford assay (13) or a biuret procedure, preceded by trichloroacetic acid precipitation, was employed; these assays were standardized against apoenzyme. For quantitation of polypeptides or enzyme concentrations we used the
protein values and the molecular mass of 28.1 kDa. Iron was determined
according to Ref. 14, and sulfide was determined according to Ref.
15.
Bacterial Growth and Isolation of the Apoenzyme--
E.
coli 234M1/pXA-1 cells were grown in LB medium with 50 µg/ml
ampicillin and 30 µg/ml chloramphenicol at 30 °C under aeration, maintaining pH 7 by addition of 5 N KOH. Cells were
harvested at the transition to the stationary growth phase
(A550 = 4.8) and frozen in liquid nitrogen
(yield = 5 g/liter).
Purification of the enzyme was made at 0-6 °C, except for the
Q-Sepharose step. Buffer solutions were previously degassed and flushed
with argon, but column operations were not strictly anoxic. Enzyme
fractions collected were stored under argon.
A 50-g portion of cell paste was suspended in 100 ml of 50 mM Mops/KOH, pH 7.5, 5 mM DTT, 0.1 mM EDTA, and 0.2 mM phenylmethanesulfonyl fluoride. After sonication, DNase I (1 mg) and RNase (1 mg) were added,
and the sample was centrifuged at 100,000 × g for 60 min. The supernatant (138 ml, 45 mg of protein/ml) was adjusted to pH
7.5 and 31 ml of 1% (w/v) PolyminG35 (neutralized with
H2SO4) were added during 60 min of stirring;
the precipitate was discarded. The enzyme solution was then
chromatographed on a column of Ultrogel AcA44 (20 cm2 × 85 cm) with 50 mM Mops/KOH, pH 7.5, 0.1 M KCl, 5 mM DTT, and 0.1 mM EDTA (flow rate = 5 cm/h). The brown-red fractions containing activase activity, which
eluted between 0.75 and 0.85 column volumes, were concentrated by
ultrafiltration on a Amicon PM-10 membrane (32 ml, 25 mg/ml). For final
chromatographic purification with simultaneous removal of protein-bound
iron, we used Q-Sepharose FF (20 cm2 × 9 cm). The column
was equilibrated with 50 mM Tris-phosphate (KH2PO4 titrated with Tris base), pH 8.0, containing 5 mM DTT and 1 mM EDTA and was
operated at 25 °C. A 490-mg portion of the AcA44 fraction,
previously gel filtered into the same buffer and warmed to 25 °C,
was applied at a flow rate of 12 cm/h. After a wash with 450 ml of the
starting buffer, 90 mM Tris-phosphate pH 8.0 (with DTT and
EDTA as before) was applied, which eluted the apoactivase as a
symmetric protein peak (275 ml), whereas the iron-based color remained
column-bound. The colorless solution was concentrated (in the cold)
over a PM-10 membrane, then gel filtered into 50 mM
Mops/KOH, pH 7.5, containing 0.1 M KCl, 5 mM
DTT, and 0.1 mM ETDA (18 ml, 8.7 mg/ml). Long term storage was at
70 °C. Analysis by SDS-polyacrylamide gel electrophoresis (12% gel) showed the 28-kDa band of PFL activase (6, 7) in addition to
a few minor bands comprising
5% of the total protein.
The same protocols (but down-scaled) of bacterial growth and isolation
as apoenzyme were applied for C12S, C29S, C33S, C36S, and C102S mutant
enzymes. The C94S mutant was carried only to the Ultrogel AcA44 stage
(protein purity = 85%).
Reconstitution of Holoenzyme--
Samples of holoenzyme were
prepared in batches up to 20 ml composed of 50 mM Mops/KOH,
pH 7.5, 0.1 M KCl, 0.1 M mercaptoethanol, 1 mg
(36 nmol) of apoprotein/ml, 0.2 mM Na2S, and
0.25 mM
Fe(NH4)2(SO4)2. Prior
to the addition of iron, the mixture was purged with argon and cooled
to 0 °C; Fe2+ was admixed from an anaerobic 10 mM stock solution. Incubation was performed routinely for
10 h. With protein concentrations of
0.1 mg/ml (see Fig. 1),
reconstitution was complete within 5 min.
For analyses of the iron center, reconstitution mixtures were gel
filtered anaerobically at 2 °C through Sephadex G-25 columns into 50 mM Mops/KOH, pH 7.5, containing 0.1 M KCl, 10 mM mercaptoethanol, and 10 µg/ml of catalase. The sample
size was 0.1 column vol. and the protein fraction collected between
0.38 and 0.51 column vol.; excessive free iron appeared at >0.65
column vol. Concentrations up to 15 mg/ml (for EPR spectra) were made
by ultrafiltration. The greenish brown colored solutions were stored in
closed tubes under argon at 0 or
70 °C, conditions under which the
holoactivase activity was stable for days and several weeks,
respectively.
Spectroscopy--
UV-visible spectra were recorded anoxically,
using septum-sealed cells (type 117, Hellma, Müllheim, Germany).
EPR first derivative spectra (5-10 accumulations) were recorded with a
Bruker ESP 300 X-band spectrometer (9.50 GHz), using a continuous flow
helium cryostat (ESR 900, Oxford Instruments) in a temperature range from 4 to 80 K. The microwave frequency and the magnetic field were
measured with a frequency counter and a Gaussmeter, respectively. Recording conditions are indicated in the legends to Figs. 6 and 8.
Samples (0.2 ml) with 0.5 mM enzyme were filled into
0.36-cm quartz tubes under argon and stored in liquid nitrogen. Spin
quantification was performed by comparing the double integrals of the
activase sample and the [4Fe-4S]3+ cluster of E. halophila of known concentration determined for nonsaturating
conditions.
Molecular Size Determination--
To determine the apparent
molecular weight of holoactivase, a 100-µl sample (0.1 mg) of the
reconstitution mixture was gel chromatographed on a Superdex 75 HR
10/30 that was preequilibrated and operated (at ambient temperature)
with anoxic buffer composed of 50 mM Mops/KOH, pH 7.5, 0.15 M KCl, 10 mM mercaptoethanol, 0.2 mM Fe2+, and 0.05 mM sulfide (flow
rate = 0.4 ml/min). The eluate was monitored for protein (280 nm)
and holoactivase activity (see Fig. 3). For the apoenzyme sample
monitored at 280 nm, the column was run with anoxic buffer that lacked
Fe2+ and sulfide. Proteins for column calibration were
bovine serum albumin, ovalbumin, carbonic anhydrase, soybean
trypsin inhibitor, and cytochrome c.
AdoMet Binding Assays with Penefsky Columns--
The centrifuged
column procedure (16) for separating enzyme-bound and free small
molecules was used to monitor binding of [14C]AdoMet to
various activase forms. Bio-Spin columns (Bio-Rad) in which the plastic
support was replaced by a glass fiber disc (GF-C, Whatman) were
employed with a 1-ml filling of Sephadex G-25 fine (Pharmacia), which
was equilibrated with anaerobic buffer composed of 50 mM
Mops/KOH, pH 7.5, 0.15 M KCl, and 30 mM
mercaptoethanol. The experiments were carried out in the cold room
(4 °C), using a swing bucket table top centrifuge. After
precentrifugation at 380 × g for 30 s, which
displaced 400 µl of buffer, a 150-µl sample of the
enzyme-[14C]AdoMet mixture was applied, and the
centrifugation was run as before. The effluent (155-160 µl) was
analyzed for radioactivity and protein content. The total amount of
enzyme-bound AdoMet (see Fig. 7) was estimated from the effluent
radioactivity with accountancy of the protein recovery, which was
typically 42%.
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RESULTS |
Overproduction and Functional Assay of PFL Activase--
To study
the molecular properties of wild-type and mutant forms of PFL activase,
we overproduced the protein in the E. coli act null strain
234M1 carrying the expression vector pXA-1, wherein the act
gene is under control of the Tac promoter. Optimal yields of activase
protein, amounting to 15% of the soluble protein fraction, were
obtained by aerobic cell growth in LB medium at 30 °C.
Glucose/minimal medium, at aerobic or anaerobic conditions, proved
unsuitable because this produced the enzyme chiefly deposited in
inclusion bodies.
Two protocols were used to assay PFL activase activity via the
production of the radical form of PFL. In the first protocol, which has
been used in previous work (6, 8) and will be termed now standard
assay, free Fe2+ (0.2 mM) is contained in the
DTT-based anaerobic reaction mixture, which is preincubated (20 min)
before initiating the reaction by photoreduced 5-deazaflavin. We
recognized during our present work that both the intact enzyme and iron
center-defective or -deficient enzyme forms are measured by this
procedure. The native iron center is readily restored during
preincubation, with the DTT reagent furnishing the sulfide source. To
assay specifically the intact enzyme, we turned to a modified protocol,
termed holoactivase assay, that omits free Fe2+ from the
reaction mixture and starts the reaction by the activase sample.
In both assay versions, oxamate, replacing the physiological pyruvate,
was present as the effector compound required for the PFL activation
process (17). It should be noted that oxamate or pyruvate are
allosteric ligands to the nonradical form of PFL, not to the activating
enzyme.
Isolation of Homogenous Apoenzyme and Reconstitution of the Fe-S
Center--
Table I summarizes our
experimental strategy that led finally to 20-mg quantities of
homogeneous PFL activase with a virtually full complement of the native
iron center. As in previous work (5, 6), the high speed centrifuged
cell extract was first subjected to molecular sieve chromatography on
Ultrogel AcA44, where the enzyme elutes, after 0.8 column volume, in
fractions displaying a red-brown color. At this stage, the protein
purity, estimated by SDS-polyacrylamide gel electrophoresis analysis, is about 50%. The fraction of intact enzyme molecules, determined by
the two activase assays, is only about 0.1, compared with a value of
0.5 at the cell extract stage. EDTA (0.1 mM) was routinely included in the column buffer, which was not rigorously made anoxic; this probably caused substantial degradation of the native iron center.
The spectral properties (see below) suggest that the iron content of
the AcA44 prep is largely due to inactive [3Fe-4S] clusters.
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Table I
Preparation of functionally competent PFL activase by isolation of
apoenzyme and chemical Fe-S center reconstitution
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Various standard anion-exchange columns, which we tested for further
purification, proved unsatisfactory because of low recoveries and
separation into subfractions. Therefore we turned to removal of any
protein-bound iron as a first step. Although standard metal chelators
proved generally suitable, the optimal method was finally found in
using a Q-Sepharose column that is operated with 50 mM phosphate buffer, pH 8, at a temperature of 25 °C. The iron center is disintegrated upon enzyme adsorption to this column, and the virtually homogeneous apoenzyme can be eluted subsequently by a single
elution step with 90 mM phosphate. The isolation protocol is described under "Experimental Procedures," and the purification analysis is shown in Fig. 1.

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Fig. 1.
SDS-polyacrylamide gel electrophoresis
analysis of enzyme purification steps. Protein samples (see Table
I) were applied to polyacrylamide gel electrophoresis (12%) in the
presence of sodium dodecyl sulfate (0.1%) as follows: lane
1, centrifuged extract (56 µg); lane 2, Ultrogel
AcA44 fraction (20 µg); lanes 3A and 3B,
Q-Sepharose fraction (4 and 18 µg, respectively). Commassie Blue was
used for staining. Numbers (in kDa) indicate marker protein migrations.
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Through monitoring the appearance of holoactivase activity, the native
iron center was then found to become rapidly reconstructed by anaerobic
incubation of the apoenzyme, at pH 7.5 and 0 °C, with
Fe2+ and S2
. With 2-mercaptoethanol in place
of dithiothreitol as protective thiol reagent in the reconstitution
mixture, the sulfide requirement was total (Fig.
2), thus immediately demonstrating a
polynuclear Fe-S cluster in the catalytically competent enzyme
form.

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Fig. 2.
Sulfide requirement for holoenzyme
reconstitution. Reaction mixtures (0.4 ml) containing 50 mM Mops/KOH, pH 7.5, 100 mM KCl, 20 mM mercaptoethanol, 3.1 µM (87 µg/ml)
apoenzyme, 250 µM
Fe(NH4)2(SO4)2, and
0-50 µM Na2S were incubated at 0 °C for 10 min. Activity measurements were with the holoactivase assay, using
2-5-µl aliquots.
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The specific activity values of reconstituted enzyme were in the range
of 60 ± 5 units/mg. This calculates the turnover number to be 1.7 min
1 at assay conditions with 0.7 µM PFL
(Km = 1.4 µM) or 5 min
1
at a saturating PFL concentration. It is interesting to note that the
presence of up to 5 mM EDTA in the holoactivase assay mixture does not impair the catalytic efficiency within the usual reaction period of 10 min. Reconstituted enzyme alone, however, is
quite labile against EDTA (see below).
Molecular Size, Iron-Sulfur Content, and Stability Properties of
Reconstituted Holoenzyme--
To structurally characterize the
functional competent enzyme form, we determined in the first place its
molecular size using a calibrated Superdex-75 column. Holoactivase
activity migrated as a single band, together with the principal protein
band, according to 25 ± 1 kDa (Fig.
3). For the apoenzyme (data not shown)
the molecular size was found to be 23 ± 1 kDa. These values
compare with the sequence-deduced molecular mass of the polypeptide of 28.1 kDa (7), indicating that activase is monomeric with or without the
Fe-S complement.

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Fig. 3.
Molecular size determination of reconstituted
PFL activase. A 0.1-ml sample of reconstitution mixture (0.1 mg; 5 units) was applied to Superdex 75 HR 10/30 operated anoxically at
25 °C with reconstitution medium as described under "Experimental
Procedures"; enzyme activity was measured by the holoactivase assay.
The arrows indicate the migration of standard
proteins.
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On Superdex-75 chromatography, which was operated with anaerobic
reconstitution medium containing iron and sulfide, the recovery of
holoactivase activity was routinely
95%. However, yields dropped to
10% when free Fe2+ and sulfide was omitted from the
running buffer, even at 4 °C column operation. The residual activity
was again found exclusively in the fractions corresponding to a
monomeric size.
Removal of excessive ligand components contained in the reconstitution
mixture without damaging the Fe-S center proved technically difficult
and has as yet been accomplished only partially. With Sephadex G25
columns run at 4 °C with anaerobic Mops buffer, pH 7.5 (see
"Experimental Procedures"), activase preparations were obtained
that showed activity values from 40 to 50 units/mg in the holoactivase
assay and about 60 units/mg in the standard assay. Iron and sulfide
contents were determined in such samples to be 2.6 ± 0.2 iron/polypeptide chain and 2.5 ± 0.2 sulfur/polypeptide chain.
Taken that the deficiency of holoactivase activity is due to partial
loss of the iron-sulfur center, a content of 3.3 ± 0.3 iron and
3.2 ± 0.3 sulfide/polypeptide would result for the fully
complemented enzyme.
In SG25 gel filtered enzyme samples stored under argon at 0 °C, the
functionally competent Fe-S cluster, as monitored by the holoactivase
activity, proved to be totally stable for at least several days.
However, exposure to air under slight agitation destroyed the activity
with a half-time of about 2 min. Likewise, incubation with metal
chelators under anaerobic conditions caused rapid inactivation, the
half-life with 0.3 mM EDTA being 5 min.
Spectral Characteristics--
Gel filtered holoenzyme samples,
which are greenish brown in color, showed a UV-visible spectrum (Fig.
4) comprising a prominent 280 nm peak
followed by a shoulder at 320 nm and a broad peak at 370-420 nm, with
extensions of the absorbance to wavelengths >600 nm, which is typical
for Fe-S proteins containing [4Fe-4S]2+ clusters. The
difference spectrum of holo versus apoenzyme calculates absorption coefficients for iron at maximum wavelengths of 310 and 420 nm to be 5.3 and 3.8 mM
1 cm
1,
respectively. These spectral data closely match those reported for
[Fe4S4(SEt)4]2
(18)
but are different from those for
[Fe2S2(SEt)4]2
(19). Addition of
0.1 mM dithionite (
5-fold excess)
reduced the 420-nm absorption by 45% (Fig. 4, inset), and a
subsequent short exposure (1-2 min) to air restored the original
spectrum, which, however, became bleached upon prolonged admission of
air.

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Fig. 4.
Electronic spectra of apoenzyme and
holoenzyme. The spectra are from solutions of 0.72 mg/ml (25 µM) enzyme. The holoenzyme sample, anaerobically gel
filtered through Sephadex G25 into Mops/KOH buffer, pH 7.5, 10 mM mercaptoethanol, 0.1 M KCl contained 2.3 iron/polypeptide and 2.3 sulfur/polypeptide (holoactivase activity, 45 units/mg). The inset shows the spectral change upon
treatment (5 min) with 0.1 or 1.5 mM dithionite.
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For comparison, Fig. 5 shows the optical
spectrum of the Ultrogel AcA44 fraction of the enzyme purification
scheme, which is largely defective as to holoenzyme activity (Table I).
The prominent feature is a pronounced peak at 416 nm (estimated
absorption coefficient for iron = 6 mM
1
cm
1), followed by shoulders at 470 and 580 nm, giving
such samples a characteristic red-brown color appearance. These
spectral data suggest the presence of 3Fe-4S clusters, possibly a
mixture of linear and cubic forms, when the spectrum is compared with
the optical spectra of the 3Fe-4S forms of aconitase (20). This is
supported by the EPR spectrum of the Ultrogel fraction (not shown),
which displays a strong nearly isotropic signal at
gav = 2.026 similar to the g = 2.01 signal of the (cubic) [3Fe-4S]+ cluster form of
aconitase (20, 21). It should be noted here that the functionally
defective Fe-S core is usually retained on carrying the Ultrogel
fraction through conventional protein purification protocols. This
occurred most probably also with the first isolation of chromosomally
encoded PFL activase from wild-type E. coli, where an
associated color had been ascribed to an organic factor (6). However,
this possibility was dismissed later on by studies of overproduced or
heterologically expressed enzyme (3, 8).

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Fig. 5.
Electronic spectrum of the Ultrogel AcA44
fraction. The Ultrogel fraction of the enzyme purification scheme
(see "Experimental Procedures" and Table I) containing 25 mg/ml and
16 nmol Fe/mg, was recorded in a 0.1-cm cuvette.
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EPR spectroscopy of reconstituted holoenzyme samples as obtained after
gel filtration showed a very weak, nearly isotropic signal at
g = 2.01, which we ascribe to a small content of
[3Fe-4S]+ species. On reduction with dithionite, an
intense EPR signal of axial symmetry, g
= 2.029, and g
= 1.925, was obtained suggestive
of an abundant S = 1/2 [4Fe-4S]+ cluster
(Fig. 6). (A small shoulder at
g = 1.95, registered on various samples, could arise
from a minor component, the origin of which is presently unknown).
Signal intensity was maximal in the temperature range 15-20 K and
virtually undetectable above 45 K. Spectral saturation effects were
observed only for microwave powers above 20 mW (at 20 K). Using a HiPIP
(high potential iron-sulfur protein) reference sample for spin
quantification, we estimated that the axial resonance accounts for
~0.4 spins/four-iron atoms in the enzyme preparations.

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Fig. 6.
X-band first derivative EPR spectrum of PFL
activase (reduced enzyme form). The spectra are from a sample of
holoenzyme (15 mg/ml or 0.5 mM; 2.6 iron/polypeptide and
2.5 sulfur/polypeptide; holoactivase activity, 46 units/mg) contained
in gel filtration buffer as indicated in Fig. 4 and incubated with 10 mM dithionite (5 min). The EPR conditions were: temperature
as indicated; microwave power, 2 mW; modulation of 0.5 mT at 100 kHz.
The g values are shown in the 20 K spectrum.
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From these electronic and EPR spectroscopic characteristics we conclude
that the metal content in reconstituted holoenzyme preparations
comprises predominantly a [4Fe-4S]2+ cluster that is
readily redox-interconvertible into the [4Fe-4S]+
state.
Essential Cysteine Residues of PFL Activase--
Because cysteine
residues are the prominent ligands of Fe-S cores in iron-sulfur
proteins, we investigated the effect of Cys mutations as a first,
simple approach toward the coordination chemistry of the metal cluster
in PFL activase. Each of the six cysteine residues (Cys-12, Cys-29,
Cys-33, Cys-36, Cys-94, and Cys-102) in the polypeptide chain was
replaced separately by a serine residue via site-directed mutagenesis.
The mutant proteins, which were overproduced in the E. coli
act
host strain in soluble form to about the same
extent as the wild-type enzyme, were purified, and their catalytic
activities were determined after subjection to Fe-S cluster
reconstitution conditions. Results are compiled in Table
II.
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Table II
Catalytic properties of Cys Ser mutants
Mutant proteins C12S, C29S, C33S, C36S, and C102S were purified to the
apoenzyme stage, and C94S was purified to the Ultrogel AcA44 stage (see
text). Each enzyme sample was incubated for 12 h in Fe-S cluster
reconstitution medium (see "Experimental Procedures") prior to
activity determination.
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C12S, C94S, and C102S were reddish brown colored proteins on
purification to the Ultrogel AcA44 stage, with spectral features resembling that of the wild-type enzyme at this stage (Fig. 5). Although C12S and C102S were readily convertible to the apoenzyme form,
the C94S mutant protein became insoluble upon removing the iron center
by the Q-Sepharose step and therefore was analyzed with the Ultrogel
sample. All three mutants displayed full holoactivase activity (as
compared with standard assay activities), albeit absolute values were
slightly lower, by a factor of
2, than the wild-type value. In
contrast, C29S, C33S, and C36S mutants proved catalytically
incompetent. These proteins, which were colorless at the Ultrogel
purification stage, behaved normally throughout the isolation
procedure, suggesting that they are unimpaired with respect to global
protein-folding. These observations suggest that C29, C33, and C36
constitute cysteine sulfur ligands of the Fe-S core in PFL
activase.
Studies of Adenosylmethionine Interaction with PFL
Activase--
The Fe-S cluster in PFL activase is a prerequisite for
effective binding of AdoMet, the crucial co-substrate of this enzyme. This was demonstrated by assays using 14C-labeled AdoMet
and the convenient column centrifugation technique (16) as shown in
Fig. 7. No interaction was detected with
the apoenzyme sample or with the critical Cys
Ser mutants (C29S, C33S, and C36S) previously subjected to Fe-S cluster reconstitution conditions. The data for the holoenzyme sample yielded a tentative Kd value of 3 ± 1 µM, which
compares to reported Km values of 2.8-7
µM (8, 22). (The maximal quantity of 0.55 AdoMet bound
per polypeptide, as calculated for this experiment, is lower than
expected, but our analytical technique is insufficient to give accurate
data.) The holoenzyme-AdoMet complex was also detectable by
conventional gel filtration at 4 °C on Sephadex G-25 run with anoxic
Mops buffer, pH 7.5. It should be noted that the 14C label
associated with the protein fraction in these binding experiments was
verified by high pressure liquid chromatography analysis to be due to
intact AdoMet.

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Fig. 7.
Binding of 14C-AdoMet. A
2-ml batch of holoenzyme (18 µM) was prepared as in Fig.
2, using 0.2 mM sulfide, 30 mM mercaptoethanol, and apoenzyme at a concentration of 0.5 mg/ml in the reconstitution mix. After 10 h, 200-µl aliquots were incubated with up to 8 µl [8-14C]AdoMet (7050 dpm/nmol) at final
concentrations as indicated for 5 min (0 °C) and then passed through
Penefsky columns to determine protein-bound AdoMet (see "Experimental
Procedures"). Control reactions were run without enzyme protein ( )
or without sulfide ( ) in the reconstitution mix.
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Spectroscopic examinations found that binding of AdoMet slightly
decreased the holoenzyme absorbance between 320 and 440 nm (maximally
by 5% at 375 nm). Drastic effects, however, were found with the EPR
spectrum as shown in Fig. 8A.
On incubation with AdoMet, the axial signal of dithionite-reduced
enzyme (Fig. 6) changed to a distinctive rhombic symmetry
(g1 = 2.009, g2 = 1.921, and g3 = 1.886). A similar but subtly different
signal change to rhombicity, with g-factors of 2.038, 1.930, and 1.899, was obtained with S-adenosylhomocysteine (Fig.
8B). In the activity assays, this compound is a competitive
inhibitor of PFL activase with respect to
AdoMet.2 (In both samples,
weak resonances with g factors of 1.91 and 1.87 (Fig.
8A) or 2.014 and 1.998 (Fig. 8B) again indicated
the presence of a minor, second component.)

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Fig. 8.
EPR spectra of dithionite-reduced PFL
activase incubated with AdoMet (A) or
S-adenosylhomocysteine (B).
Dithionite-reduced holoenzyme (Fig. 6) was preincubated for 3 min with
AdoMet (1 mM) for spectrum A or
S-adenosylhomocysteine (1 mM) for spectrum B. The recording conditions were: temperature, 20 K; microwave power, 20 mW; other parameters as in Fig. 6.
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It is apparent that binding of either nucleoside significantly affects
the environment of the iron-sulfur core, inducing modifications of its
electronic and magnetic properties. In the sample of Fig. 8A, containing dithionite (10 mM), AdoMet (1 mM) and holoenzyme (0.5 mM), we determined
after the EPR measurement a 5'-deoxyadenosine content of about 30 µM, i.e. a small fraction of AdoMet underwent reductive cleavage.
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DISCUSSION |
Through isolation as apoenzyme and chemical reconstitution of the
iron-sulfur cluster, substantial quantities of PFL activase in its
catalytically competent form have now been made available enabling
closer structure-function analyses of this radical-generating enzyme.
The reconstituted enzyme has been determined to be monomeric (28 kDa),
and data reported here are strongly indicative of a 4Fe-4S cluster
content (rather than a 2Fe-2S type). The electronic spectrum of the
greenish brown colored solution shows features (shoulder at 320 nm and
broad absorption in the 420 nm region) typical of a
[4Fe-4S]2+ cluster type, which is reducible by
dithionite. Accordingly, enzyme samples as obtained are largely EPR
silent but on reduction yield an axial S = 1/2 signal
(g = 2.03, 1.92) indicative of a [4Fe-4S]+ cluster. Integration of the fast relaxing EPR
signal, observed optimally at 20 K, estimated
0.4 spin/four-iron
cluster as calculated from the protein-bound iron. Iron and sulfide
analyses regularly found 2.6 ± 0.2 iron/polypeptide and 2.5 ± 0.2 sulfur/polypeptide, values that are too low for a 4Fe-4S
stoichiometry/monomer. However, allowance has to be made for the fact
that the samples of reconstituted enzyme, on which all cluster
characterizations were made, were previously gel filtered, an operation
that looses between 15 and 30% of the catalytic holoenzyme
activity.
For PFL activase samples prepared by anaerobic enzyme isolation from
E. coli extracts, Broderick et al. (9) have
recently described the iron center content as a mixture of
[4Fe-4S]2+ and [2Fe-2S]2+ clusters, the
latter form representing an oxidative degradation product. Neither
enzyme samples as obtained nor dithionite-treated enzyme contained a
paramagnetic Fe-S center. Cluster reduction to the
[4Fe-4S]+ state required the presence of AdoMet, yielding
an almost axial EPR resonance (g = 2.01, 1.89, and
1.88). This behavior is strikingly different from what we observe for
reconstituted PFL activase (ready reduction to the 1+ state without
AdoMet and shift of the axial S = 1/2 EPR signal
to rhombicity (g = 2.01, 1.92, and 1.88) in the
presence of AdoMet). The difference remains to be resolved.
It should be noted that the anaerobically isolated enzyme had been
obtained via protein expression in E. coli from a different vector (pMG-AE) at the increased temperature of 42 °C (9). The
sample under study might have contained functionally damaged iron
center(s). It is reported as red-brown colored (not greenish brown as
with the reconstituted holoenzyme), showing a specific activity of only
5.5 units/mg (recalculated for our unit definition) under assay
conditions equivalent to our standard assay (an assay version that is
unable to report the actual amount of catalytically competent enzyme
form contained in activase samples).
A close analog of PFL activase is the activating protein of anaerobic
ribonucleotide reductase, which also uses AdoMet and reduced flavodoxin
for generating a protein-based glycyl radical (11, 23, 24). Its 4Fe-4S
center (10) has a spectroscopic signature (UV-visible and EPR)
resembling closely the signature that we have found here for PFL
activase. The two activases are different in molecular size and
operational terms, however. RNR activase (
, 17 kDa) is dimeric, with
the [4Fe-4S] cluster placed between the two polypeptides and is
permanently associated with the reductase enzyme proper in a
2
2 complex (10, 11). In contrast, PFL
activase is a monomeric protein (28 kDa) interacting with its target
protein pyruvate formate-lyase only transiently, i.e. as a
catalytically operating converter enzyme. This property is reflected
also in the separate transcriptional control of PFL and PFL activase
genes (1).
At the amino acid sequence level, the two activases are strongly
homologous in the N-terminal section harboring the characteristic Cys-Xaa3-Cys-Xaa2-Cys pattern that we had
previously proposed as iron interaction site (7). Mutagenesis studies
reported here have now demonstrated that each of these three Cys
residues is essential, whereas single Ser replacements of other
cysteines in the polypeptide chain do not affect the reconstitution of
active PFL activase. The sequence similarity between the two activases is strongest for a stretch of 26 amino acid residues as shown in Fig.
9, wherein alignments with additional
sequences for organisms other than E. coli have been
integrated. The consensus comprises 11 amino acid residues. (Other
homologous sequences in the current data bases that refer to proteins
of different or nonestablished functions were not rated. It should be
noted that biotin synthase, another Fe-S protein employing AdoMet for a
radical reaction (25, 26), shares the
Cys-Xaa3-Cys-Xaa2-Cys pattern but no other
significant homology.)

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Fig. 9.
Three-cysteine cluster region in PFL activase
and the -subunit of RNR class III. Shown are conserved amino
acid residues in the sequences of PFL activase from E. coli
(), C. pasteurianum (), and H. influenzae () (A) and in the sequences of the
-subunit of anaerobic ribonucleotide reductase from E. coli (), H. influenzae (), and phage T4
() (B). SwissProt accession numbers are given in
brackets. The numbers in A and B refer
to the proteins from E. coli. The consensus is in bold
type.
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The unique utilization of AdoMet as a radical generating cofactor had
been recognized at first in 1984 by studies of the pyruvate formate-lyase system, when we identified 5'-deoxyadenosine and methionine as co-products of the post-translational radical
introduction into PFL (27). The chemical mechanism of this process,
proposed to involve intermediary formation of a H abstracting
5'-deoxyadenosine radical (3, 5), should be approachable now more
directly because the metal center of PFL activase has been
characterized, and functionally correct enzyme samples are available in
abundance. The ligands of the Fe-S core must still be identified. It is
tempting to speculate that the metal cluster is not completely
cysteinyl coordinated, thus enabling close interaction with the AdoMet
co-substrate. Its effective binding to PFL activase has been
demonstrated to require the Fe-S complement. Reductive cleavage of
AdoMet occurs presumably through the Fe-S cluster; however, this
process is largely suppressed when the PFL substrate or Gly-734 site
analogous peptide substrates are not present simultaneously (5). The essential role of the metal center for AdoMet reduction has recently been shown also for the parallel case of anaerobic RNR (28).
We thank Dr. A. F. Volker Wagner for
helpful discussions, Christine Sessler for plasmid constructions, and
Dr. Rainer Frank (Zentrum für Molekulare Biologie) for
oligonucleotide syntheses.