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Purification and Characterization of Protocatechuate 2,3 Dioxygenase

Vol. 175, No. 14
JOURNAL OF BACTERIOLOGY, July 1993, p. 4414-4426
Copyright © 1993, American Society for Microbiology
Purification and Characterization of Protocatechuate
2,3-Dioxygenase from Bacillus macerans: a New
Extradiol Catecholic Dioxygenase
Department of Biochemistry, University of Minnesota, Minneapolis, Minnesota 554551; Gray Freshwater
Biological Institute, University of Minnesota, Navarre, Minnesota 553922; Department of Bacteriology
and Biochemistry, University of Idaho, Moscow, Idaho 838433; and Department of Chemistry,
Carnegie Mellon University, Pittsburgh, Pennsylvania 152134
Protocatechuate 2,3-dioxygenase (2,3-PCD) from Bacillus macerans JJlb has been purified to homogeneity
for the first time. The enzyme catalyzes proximal extradiol ring cleavage of protocatechuate (PCA) with the
attendant incorporation of both atoms of oxygen from 02. The holoenzyme has a mass of 143 7 kDa as
determined by ultracentrifugation and other techniques. It is composed of four apparently identical subunits
with Mrs of 35,500, each containing one iron atom. Mossbauer spectroscopy of "7Fe-enriched enzyme showed
that the irons are indistinguishable and are high spin (S = 2) Fe2+ in both the uncomplexed and
substrate-bound enzyme. However, the quadrupole splitting, AEQ, and isomer shift, 8, of the Mossbauer
spectrum changed from AEQ = 2.57 mm/s and 8 = 1.29 mm/s to AEQ = 2.73 mm/s and 8 = 1.19 mm/s upon
PCA binding to the enzyme, showing that the iron environment is altered when substrate is present. The
enzyme was also found to bind variable and substoichiometric amounts of Mn2", but this metal could be
removed without loss of activity or stability. The inherently electron paramagnetic resonance (EPR)-silent Fe2+
of the enzyme reversibly bound nitric oxide to produce an EPR-active species (g = 4.11, 3.95; S = 3/2). The
specific activity of the enzyme was found to be correlated with the amount of the S = 3/2 species formed, showing
that activity is dependent on Fe2'. Anaerobic addition of substrates to the enzyme-nitric oxide complex
significantly altered the EPR spectrum, suggesting that substrates bind to or near the iron. The enzyme was
inactivated by reagents that oxidize the Fe2 such as H202 and K3Fe(CN)6; full activity was restored after
reduction of the iron by ascorbate. Steady-state kinetic data were found to be consistent with an ordered bi-uni
mechanism in which the organic substrate must add to 2,3-PCD before 02. The enzyme has the broadest
substrate range of any of the well-studied catecholic dioxygenases. All substrates have vicinal hydroxyl groups on
the aromatic ring except 4-NH2-3-hydroxybenzoate. This is the first substrate lacking vicinal hydroxyl groups
reported for catecholic extradiol dioxygenases. 2,3-PCD is the final member of the PCA dioxygenase family to be
purified. It is compared with other members of this family as well as other catecholic dioxygenases.
Protocatechuate (PCA) is one of a relatively small number
of single-ring aromatic compounds that are found at the
points of confluence of bacterial pathways for metabolism of
complex aromatics (12, 17, 28, 37). The aromatic ring of PCA
is opened in reactions catalyzed by dioxygenase enzymes
that result in the incorporation of both atoms of 02 into the
open chain products (14, 19, 25, 46, 65). Two of these
enzymes, protocatechuate 3,4-dioxygenase (3,4-PCD) (10,
21, 25, 59, 65, 70) and 4,5-PCD (5, 18, 19, 53) were among the
first of this class of enzyme to be recognized. They continue
to serve as prototypical enzymes for the two major subclasses of catecholic dioxygenases termed intradiol and
extradiol on the basis of the site of ring cleavage (for reviews
see references 37 and 46). Intradiol dioxygenases like 3,4PCD contain Fe3" (26, 59, 70) and open the aromatic ring
between the vicinal hydroxyl groups (65). In contrast, extradiol dioxygenases such as 4,5-PCD invariably contain Fe2+
(5, 67) and open the ring adjacent to one of the hydroxyl
groups to
form highly colored muconic semialdehyde prod-
ucts (19). The analogous set of reactions also occurs in the
oxidative cleavage of catechol. The relevant intradiol and
extradiol dioxygenases are, respectively, catechol 1,2-dioxygenase (pyrocatechase, or 1,2-CTD) (34) and catechol 2,3dioxygenase (metapyrocatechase, or 2,3-CTD) (35, 44, 47).
The intradiol and extradiol nomenclature cannot be applied
to enzymes that catalyze ring cleavage of key intermediates,
such as gentisate, that do not have vicinal hydroxyl groups.
Nevertheless, we have shown recently that gentisate 1,2dioxygenase (1,2-GTD) contains Fe2' and behaves mechanistically like an extradiol dioxygenase (29, 30).
The cleavage site of the PCA ring by dioxygenases is
completely specific; i.e., extradiol dioxygenases do not
cleave in an intradiol manner and vice versa. Moreover, the
PCA ring cleavage site in pseudomonads is species specific
and is used as a taxonomic characteristic (66). To determine
whether this specificity was present in Bacillus species,
Crawford investigated the pathways of 4-OH benzoate degradation in various bacilli (14, 15). Interestingly, strains of
aerobic spore-forming Bacillus circulans and Bacillus macerans were identified that contained a novel extradiol PCA
dioxygenase that catalyzed opening of the aromatic ring
Corresponding author.
t Present address: Conta Luna Foods, Grand Forks, ND 58203.
t Present address: Eastman Chemical Co., Kingsport, TN 376625150.
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Received 10 March 1993/Accepted 3 May 1993
VOL. 175, 1993
& OH
a-Hydroxy-8-carboxymuconic semialdehyde
f3-Carboxy-cis,cismuconic acid
FIG. 1. Known positions of ring cleavage of PCA.
Chemicals. All chemicals were of the best quality available
as supplied by Aldrich or Sigma Chemical companies and
were used without further purification. 3-Chlorophenol was
the generous gift of Peter Chapman (Environmental Protection Agency, Gulf Breeze, Fla.). Water was deionized and
glass distilled.
Enzymes. Pseudomonas testosteroni 4,5-PCD (5) and
Pseudomonasputida (arvilla) 2,3-CTD (35) were prepared as
previously described (5, 6) and had specific activities of 200
and 320 U/mg, respectively.
Purification of 2,3-PCD. B. macerans JJ1b (ATCC 35889
[16]) was grown as previously described (71). The culture
procedure is straightforward, except for the fact that there is
a critical time window during which the cells can be harvested or transferred. It begins when the growth substrate,
4-hydroxybenzoate, is depleted, and it lasts for less than 1 h.
The purification procedures reported here were adapted
from preliminary studies (16, 71). All procedures were
performed at 40C, and the buffer was 50 mM MOPS (morpholine propanesulfonic acid [pH 6.9]) with 100 pM
Fe(NH4)2(SO4)2. 6H20 and 2 mM cysteine (10x stock prepared daily) unless otherwise indicated. All centrifugations
were at 35,000 x g for 20 min. Approximately 100 g of cell
paste was suspended in 300 ml of buffer and broken by
sonication (Branson Inc., model 350) at 15'C. After treatment with -3 mg each of RNase, DNase, and MgCl2, the
mixture was centrifuged. The supernatant was then diluted
with buffer to bring the ionic strength below that of 0.025 M
NaCl in buffer and was loaded onto a DEAE-Sepharose Fast
Flow (Pharmacia/LKB) column (5 by 25 cm) equilibrated
with buffer. The column was washed with 1 liter of buffer,
and then 2,3-PCD was eluted with a linear gradient (1 by 1
liter) from buffer to 0.6 M NaCl in buffer. The fractions
exhibiting >50 and >10% of the activity of the peak fraction
on the low- and high-salt sides of the peak, respectively,
were pooled and concentrated to 70 ml by ultrafiltration
under N2 on a YM-10 membrane (Amicon Inc.). This solution was made 1.5 M NaCl in buffer and was then applied to
a phenyl-Sepharose (Pharmacia/LKB) column (4 by 10.5 cm)
preequilibrated in 1.5 M NaCl in buffer. The column was
washed with 250 ml of 1.5 M NaCl in buffer. Apparently
homogeneous enzyme was eluted as a broad peak near the
middle of a linear gradient (250 by 250 ml) from 1.5 M NaCl
in buffer to buffer with no added NaCl. Fractions with >5%
of the peak activity were concentrated under pressure as
described above to 15 ml (>7.5 mg/ml). Enzyme was stored
in liquid N2 and was stable for >2 years.
Incorporation of 57Fe. The 2,3-PCD used in Mossbauer
experiments was prepared from cells grown in media containing 57Fe (Icon Inc.). All glassware used in culturing the
bacteria and purification of the enzyme was acid washed
before use. With the exception of the culture plate growth
media and the soil extract media, all of the growth media
contained 1.4 mg of s7Fe metal dissolved in 6 N HCl as the
only source of iron per liter. The purification of 2,3-PCD was
identical to that described above, except that s7Fe dissolved
in 6 N HCl was substituted for Fe(NH4)2(SO4)2. 6H20.
Enzyme assay. Routine assays of 2,3-PCD activity were
made by monitoring the rate of 02 uptake with a Clarke-type
oxygen electrode (YSI). Enzyme was added last to the
reaction chamber containing 1.3 mM PCA in air-saturated 50
mM MOPS buffer (pH 7.0) at 23°C. Calculations and electrode calibration were performed as previously described
(70, 71).
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between the 2 and 3 carbons (2,3-PCD) (14, 15). The enzyme
partially purified from B. macerans (16). With the
discovery of this enzyme, the known positions of ring
cleavage of PCA can be depicted as shown in Fig. 1.
Studies of 3,4-PCD and 4,5-PCD have contributed significantly to our understanding of the structures and mechanisms of intradiol and extradiol dioxygenases. Both have
been crystallized (5, 25, 51, 62, 70), and the three-dimensional structure of 3,4-PCD has been solved (50). This is the
only structure of a dioxygenase currently available. A wide
range of spectroscopic techniques has been applied to each
enzyme that revealed differences in the ligation spheres of
the active-site irons as well as the manner in which they
react with organic substrates and oxygen analogs (37). For
example, the technique of forming the nitrosyl complex of
the active site Fe2+ of mononuclear iron-containing enzymes
and proteins was developed, in part, during the spectroscopic investigation of 4,5-PCD (4-6). This technique converts the normally electron paramagnetic resonance (EPR)silent Fe2+ to an EPR-active form, thus allowing the number
and type of iron ligands to be assessed by transferred
hyperfine coupling effects from isotopically labeled ligands.
On the basis of these spectroscopic and crystallographic
studies, molecular mechanisms for both intradiol and extradiol dioxygenases have been proposed (4, 37, 58, 69). For
each type of enzyme, PCA is proposed to initially bind
directly to the iron through both hydroxyl groups to form a
chelate complex (4, 56). In the case of 4,5-PCD, 02 is then
proposed to bind directly to and be activated by the iron
before insertion into the PCA ring. In contrast, the Fe3+ of
3,4-PCD is not proposed to bind 02. Rather, it is thought to
activate the bound PCA so that 02 can attack the substrate
Because of the pivotal role 3,4-PCD and 4,5-PCD have
played in our understanding of the structure and mechanism
of ring cleavage dioxygenases, it is important to completely
characterize the one remaining PCA dioxygenase, 2,3-PCD,
for comparative studies. This is particularly important because the extradiol mechanism is much less firmly established than the intradiol mechanism because of the difficulties of studying the inherently EPR-silent and colorless Fe2+
of the extradiol enzyme class. The availability of 2,3-PCD as
another extradiol enzyme that utilizes the same substrate as
4,5-PCD offers the best approach to determining which
aspects of the structure and spectroscopy are essential to
extradiol catalysis. We describe here the first purification of
this enzyme to homogeneity as well as its physical and
spectroscopic characterization.
2H 3 PCD
61 rh
The Stokes radius, rh in equation 2, is obtained directly from
the gel filtration data, and the other terms are the usual
constants (see reference 11). Do was calculated for water at
The Perrin shape factor, F, is the ratio of the translational
frictional coefficients, f/fo, wheref is obtained from equation
2 and fo is that of an unhydrated spherical protein given by
equation 3 (24):
fo = 67
The molecular mass, m, is obtained by dividing the MW
(from sedimentation velocity measurements) by 6 x 102.
The partial specific volume, v, is approximately 0.72 cm3 g-1
Calibrated gel filtration chromatography. MW was also
estimated by calibrated gel filtration chromatography with a
Bio-Gel A-0.5m column (1 by 90 cm) and appropriate protein
standards. The total column volume was determined by
locating NaCl added with the sample by using a refractometer. Plots to determine MW were made with the Stokes
radius of the standard proteins calculated from the known
molecular masses and diffusion coefficients. The MW of
2,3-PCD was then estimated from the determined Stokes
radius and the diffusion coefficient obtained from the ultra-
centrifugation experiment described above with the Einstein-Sutherland equation.
SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) was performed according to
the procedure of Laemmli (36). Protein was stained overnight with 50% CH30H-10 CH3COOH-0.25% Coomassie
brilliant blue and was destained with 5% CH30H-10%
Amino acid analysis and amino-terminal sequence. The
holoenzyme was reduced and S carboxymethylated according to the method of Lundell and Howard (39). Hydrolysis
and amino acid analysis procedures were performed as
previously described (5, 70). Cysteine was measured as
carboxymethylcysteine. Tryptophan was measured from extinction coefficients at 280 and 288 nm according to the
method of Edelhoch (22). Analysis of the NH2-terminal
sequence of S-carboxymethylated holoenzyme was performed with a Beckman System 890 sequenator. Highperformance liquid chromatography analyses of the phenylthiohydantoin derivatives of amino acids were performed
with an Altex C-18 reverse-phase column (Ultrasphere ODS)
with authentic standards.
Spectra. EPR spectra were recorded on a Varian E-109
spectrometer equipped with an Oxford Instruments ESR-910
liquid helium cryostat. Data were recorded for analysis with
a computer interfaced directly to the spectrometer. The
Mossbauer spectrometer was of the constant acceleration
type (70). All isomer shifts, bFe' are quoted relative to iron
metal at room temperature. Optical spectra were recorded
on a Hewlett Packard 8451A diode array spectrophotometer.
Analysis of EPR and Mossbauer spectra. The EPR spectra
of proteins with mononuclear iron sites are largely determined by the spin of the electronic ground state (64). The
spin depends upon the oxidation state of the iron as well as
the nature of the coordination environment. For Fe3+, the 5
d-electrons can combine to yield ground state spins of S =
5/2, 3/2, or 1/2. Fe2 , on the other hand, has an even number
of d-electrons which combine to yield states with S = 2 or 0.
Most frequently, high-spin (S = 2) ferrous ions do not permit
the observation of EPR because the five spin levels exhibit
splittings in a zero magnetic field that exceed the energy of
the EPR microwave quantum. However, more recently
some Fe2+ sites have been shown to be amenable to EPR
(31). By complexing a ligand containing an unpaired electron
to a site with integer spin, one can transform the site into an
EPR-active form with half-integral electronic spin. It has
been shown for a variety of Fe + systems that addition of
nitric oxide (NO) gives a complex with S = 3/2 (5, 37). The
spectra of such complexes can be interpreted in terms of the
spin Hamiltonian (where S = 3/2) (42):
He = D ['
- 1/3 (S (S + 1))
+ EID (S2 - S2 )] + goj3eS
The first term in brackets describes the splitting of the spin
quartet in zero magnetic field; D and E are the axial and
rhombic zero-field splitting parameters, respectively. By
appropriate choice of coordinate system, one can keep E/D
constrained to 0 c E/D < 1/3. In a zero magnetic field,
solution of equation 4 yields two degenerate doublets separated in energy by A = 2D[1 + 3(E/D)2]1/2. For S = 3/2 NO
complexes studied thus far (5, 57), D is positive, D is + 10
to 15 cm-1, and E/D is <0.10, implying that the ground
doublet has magnetic quantum number M = + 1/2. The
Zeeman (second) term in equation 4 describes the interaction
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Steady-state kinetics. The initial velocity of 2,3-PCD
steady-state reactions was determined as described in "Enzyme assay," with the following modifications. All assays
were performed at 250C. The electrode was calibrated with
buffer which had been equilibrated with known primary gas
standards. The concentration of PCA in stock solutions was
determined as E290 = 0.389 M-1 cm-' (65). The 02 concentration in assay mixtures was altered by equilibrating assay
solutions with 02-N2 gas mixtures established by a precision
dual flowmeter (Air Products). The solubility of 100% 02
was taken to be equal to 1.4 mM at 250C. The apparent Km
and Vm. values were determined by directly fitting the
Michaelis-Menten equation with a nonlinear regression program (7).
Sedimentation velocity ultracentrifugation. Sedimentation
coefficients were measured with a Beckman model E analytical ultracentrifuge equipped with a mechanical speed control, the RTIC temperature control, Schlieren optics, and an
An-D rotor. Schlieren boundary positions were measured
with a Nikon model 6C comparator. Protein solutions of 2.5
to 3.5 mg/ml in 50 mM MOPS (pH 6.9) were run in singlesector cells at 29,500 rpm and 8.3YC and were photographed
at 16-min intervals. Values for sedimentation and diffusion
coefficients extrapolated to standard conditions (s20.W and
D°20 ,) were determined by standard procedures (13). The
molecular weight (MW) of 2,3-PCD was determined from
these values by the Svedberg equation (13).
Calculation of diffusion coefficient and Perrin shape factor.
The diffusion coefficient at infinite dilution of a two-component system is given by the Einstein-Sutherland equation
(equation 1). For a sphere in "stick" boundary conditions,
the translational frictional coefficient, f, in equation 1 is
given by Stokes law (equation 2).
VOL. 175, 1993
TABLE 1. Purification of B. macerans 2,3-PCD
Purification step
Vol Activity
Sp act Yield
(U/mlr Protein
(mg) (U/mg)
(%) fication
Cell supernatant"
DEAE-Sepharose Fast 70
Flowc plus pressure
Phenyl-Sepharose plus 15
pressure dialysis
a 23C, air saturated buffer.
b On the basis of 100 g (wet weight) of cells.
c After concentration by pressure dialysis.
Protein concentration. Protein concentrations of three homogeneous 2,3-PCD samples of known A220 were independently measured by quantitative amino acid analysis as
previously described (29) and were used to calibrate a
Bradford protein assay (8), from which subsequent protein
concentrations were measured. The data from the accurately
determined samples were also used to determine an extinction coefficient for the enzyme at 280 nm.
Purification and characterization of 2,3-PCD. The purification of 2,3-PCD is summarized in Table 1. The apparently
homogeneous enzyme has a specific activity approximately
sixfold greater than previously reported for a partial purification (16). This improvement in specific activity was due to
altered growth procedures and the use of the stabilizing
reagents ferrous ion and cysteine, which we have found
useful in the purification of many nonheme iron-dependent
enzymes (5, 23, 29).
The amino acid composition of reduced S-carboxymethylated holoenzyme is shown in Table 2. On the basis of
quantitative amino acid analysis, the extinction coefficient of
the holoenzyme in cysteine-free, 50 mM MOPS (pH 7.0) was
found to be C(280) = 1.4 mg- ml cm-.
The physical properties of 2,3-PCD determined as described in Materials and Methods are summarized in Table 3.
The holoenzyme has an MW of approximately 143,000 and a
slightly nonglobular shape as indicated by the Perrin shape
factor of 1.3. SDS-PAGE shows a single Coomassie bluestaining protein band with an Mr of 35,500 ± 800 (Fig. 2),
suggesting that the enzyme is a tetramer with four identical
subunits. To support this conclusion, automated Edman
degradation was performed for nine cycles on the reduced,
S-carboxymethylated holoenzyme. A single amino acid was
obtained in each cycle, suggesting that the protein is homogeneous and that the subunits are identical. The sequence
obtained is shown in Table 2. Over this limited sequence
interval, no homology with the sequences of other dioxygenases is noted. Omitting the 2-mercaptoethanol from the
incubation buffer prior to electrophoresis had no effect on
the apparent MW of the subunit, suggesting that the quaternary structure is not stabilized by disulfide bonds.
Steady-state kinetics. Double reciprocal plots of initial
velocities observed when one substrate was varied at several
fixed concentrations of the other substrate intersect, showing that the mechanism of 2,3-PCD is sequential (Fig. 3).
Previous studies have indicated that dioxygenases have
ordered mechanisms in which the organic substrate binds
first (32). Because the mechanism is sequential and PCA
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of the applied magnetic field with the electronic magnetic
moment. In all cases studied thus far for iron-nitrosyl
complexes, this interaction has been found to be isotropic,
with go = 2.0 (55). For EID = 0, equation 4 predicts for the
ground doublet effective g values at g, = 4.0, g = 4.0, and
= 2. For small values of EID, the two g = 4fresonances
split symmetrically around g = 4, while gz drops slightly
below 2.0. The EID values for the 2,3-PCD nitrosyl complexes were calculated from the observed effective g values.
Mossbauer spectra can be analyzed with the same spin
Hamiltonian augmented by nuclear hyperfine and Zeeman
terms (see reference 41). Detailed analysis of Mossbauer
spectra can yield values for all of the Hamiltonian parameters. In the current application, Mossbauer spectra are used
only to determine the redox and spin state of the active site
iron and to provide values for the characteristic absolute
value of the quadrupole splitting and isomer shift parameters. This information can be obtained directly from the
spectra with no further analysis.
Quantitation of iron. Total iron was measured in a multielement analysis by inductively coupled plasma emission
spectrometry (ICPES) in an Applied Research Laboratories
model QA 137 spectrometer at the Soil Sciences Center,
University of Minnesota. All samples were prepared from
enzyme-NO complexes which were evacuated to remove the
NO and then acidified with 2 N HC1 to release the iron. The
total Fe2+ of the enzyme-NO complexes was determined by
single integration of the S = 3/2 EPR spectrum of the
complex for comparison with a standard solution of 1 mM
Fe2+-EDTA-NO complex, which gives a similar spectrum
and was standardized by ICPES. Corrections for g value
anisotropy were made according to the method of Aasa and
Vanngard (1).
Specific removal of adventitiously bound ferric ion and
manganese. Adventitiously bound Fe3' and Mn2+ were
removed by two different methods. A 1-mg/ml solution of
2,3-PCD in 50 mM MOPS (pH 6.9) (buffer) was anaerobically
incubated with 0.3 mM 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron) (Sigma) for 1 h at 4°C. The solution was
made 1.5 M in NaCl and loaded onto a phenyl-Sepharose
column (1 by 5 cm) preequilibrated with a buffer solution of
1.5 M NaCl and 2 mM cysteine. The Tiron adventitiously
bound metal complex was removed by extensively washing
the column with the same solution. The enzyme was eluted
by buffer plus 2 mM cysteine. All column steps were
performed at 4°C with N2 gas bubbling through degassed
buffers. In the second method, a 1.8-mg/ml solution of
2,3-PCD in buffer was added to a slurry of Chelex resin
(Bio-Rad) preequilibrated in buffer. This was centrifuged in a
tabletop microcentrifuge for 2 min, and the enzyme-supernatant solution was removed for metal analysis.
Anaerobic procedures and nitric oxide addition. Samples
were made anaerobic by repeated cycles of evacuation and
flushing with argon which had been passed over a column of
BASF Inc. copper catalyst at 150°C. Transfers of anaerobic
solutions were by gas-tight syringe. NO was added by slow
bubbling (saturated solution, 3.3 mM) (Matheson Inc.). An
Ar flush was maintained above the sample to remove excess
NO and to protect the sample from 2. Approximately 3 min
of bubbling (80 bubbles from 24-gauge tubing) was used and
was sufficient to saturate the sample (4, 6). Enzyme-substrate-NO complexes could be made by initially adding
either substrate or NO to the enzyme. The order of addition
did not change the EPR spectrum of the resulting complex.
NO is very toxic and should be handled in a well-ventilated
TABLE 2. Amino acid
composition of B.
Total residues
Total MW
Total enzyme
Physical properties and kinetic constants
Physical properties
Sedimentation velocity .................... 143,000 ± 5%
Gel filtration ............................
149,900 ± 10%
Estimated from SDS-PAGE ............. 142,000 ± 10%
Subunit MW ............................
35,500 ± 10%
7.63 x 10-13 s ± 3%
D 20,w-............................................ 4.64 x 10-7cm2/s ± 3%
Perrin shape factor (f/fo)...................1.3 ± 5%
Subunit structure ...
Fe2+ content (mol/mol) .
0 ± 0.15
pI ..........................
a N-terminal sequence: Ser-Leu-Glu-Met-Ala-Leu-Leu-Ala-Ala.
b Values normalized to the yield of carboxymethylcysteine (absolute yield,
2.00 nmol).
c On the basis of an estimated MW of 35,500. Values rounded to the nearest
Assumes an a4 subunit structure.
e Determined as carboxymethylcysteine.
f Extrapolated to
appears to be able to bind to the iron in the absence of 02 but
vice versa (see below), an ordered bi-uni mechanism is
likely to pertain to 2,3-PCD. Unfortunately, the appropriate
product inhibition studies to confirm this conclusion cannot
be completed in the case of the ring cleavage dioxygenases
because the products spontaneously isomerize after they
leave the active site.
The kinetic constants and some rate constants for the
2,3-PCD-catalyzed reaction can be determined by analyzing
the kinetic plots and replots shown in Fig. 3. Values of Km
for 02 and PCA at saturating concentrations of the nonvaried
substrate are given in Table 3. A limiting Vm~, cited in terms
of a turnover number per iron at saturating concentrations of
both substrates is also shown. For the case in which PCA
was varied at several fixed 02 concentrations, the double
reciprocal plots intersect very close to the horizontal axis,
indicating that KmpCA is =KipcA. The value of KPCA obtained from the replots is given in Table 3. Because Ki = Kd
for the first substrate to bind to the enzyme in any ordered
mechanism (63), the Kd value for PCA (24 pM) can be
determined. Solution of the net velocity equation for a bi-uni
mechanism shows that KmpCA = KipCA if PCA and the
product dissociate from the enzyme at approximately equal
rates (see the following equations).
Steady-state kinetic constantsa'b
142 FM
24 PM
KmPCAC ..........................
20 LM
Turnover numbe( .......................... 210 s-1 active site-1
k1 (PCA association rate constant) .......8.75 x 106 M-1 S-1
k-, (PCA dissociation rate constant) .... 175 s-'
k3 (product dissociation rate constant) ....210 s-1
KmO2- ...........................
a Kinetic and rate constants are determined assuming an ordered bi-uni
reaction sequence (see text).
b Approximate error in values is +10%. Rate constants k, and k-1 are
determined as ratios of kinetic constants and therefore have a larger potential
c Value determined for the condition in which both substrates are extrapolated to infinite concentration.
g Extrapolated from a timed hydrolysis.
h Determined from extinction coefficients at 280 and 288 nm.
i Includes the weight of four iron atoms.
TABLE 3. Summary of characterization of 2,3-PCD
E + PCA z E PCA + 02 t [E * PCA 02
k-E kP2
E + Product
Vmax. KiPCA
KmPCA .Etotal
KmPCA Etotal
14,400FIG. 2. SDS-PAGE of 2,3-PCD. The 2,3-PCD was denatured in
SDS and 2-mercaptoethanol, subjected to electrophoresis, and
stained as described in Materials and Methods. (A) MW standards
(Bio-Rad): phosphorylase B (92,500), bovine serum albumin
(66,000), ovalbumin (45,000), carbonic anhydrase (31,000), soybean
trypsin inhibitor (21,500), lysozyme (14,400). (B) Approximately 5
pg of 2,3-PCD. (C) Incubation buffer alone (same volume as used in
lanes A and B). Minor bands appear in each lane because of an
impurity in the incubation buffer.
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VOL. 175, 1993
TABLE 4. Comparison of substrate turnover by
extradiol dioxygenases
3-Substituted catechols
0.01~~~~~~~~~~~~~~-50 40 -30 -20 0
The product dissociation rate constant, k3, is defined as the
turnover number (VmaiEtotal = 210 sol). The value for the
second-order rate constant for PCA association can also be
estimated [k1 = Vmjj(KmpcA Etoj) = 8.75 x 106 M s-].
Together with the Kd value for PCA, the calculated k1 value
allows the dissociation rate constant for PCA to be estimated
as k1 = 175 sSubstrate specificity. Sixteen compounds other than PCA
were observed to be turned over (Table 4). Compounds that
serve as substrates include 3-substituted and 4-substituted
catechols, 2-substituted and 5-substituted PCAs, esters of
PCA, 1-substituted PCAs with extended side chains, and a
novel substrate analog, 4-NH2-3-OH benzoate (4-AHB). The
absorbance maxima of most of the products were observed
to be 350 to 382 nm, suggesting that the substrates are
cleaved in a proximal extradiol manner between C-2 and
C-3. Distal extradiol cleavage would be expected to yield
products with A 4w (18, 19), whereas the products of
intradiol cleavage would be expected to absorb in the UV
range (25, 65). The cleavage of homo-PCA (HPCA) and
3,4-dihydroxyphenylpropionic acid (DHPP) each gave reaction products with two absorption maxima, potentially indicating two different ring cleavage products for these compounds. However, the optical spectra of the reaction
products observed here are identical to published spectra of
the reaction products of extradiol cleavage of HPCA and
DHPP catalyzed by DHPP 2,3-dioxygenase from Pseudomonas ovalis (2, 54). This comparison strongly suggests that
these two compounds are cleaved in an extradiol manner
between the 2 and 3 carbons by 2,3-PCD. Thus, despite a
very large substrate range, no evidence for cleavage other
than proximal extradiol has been observed for 2,3-PCD.
The common feature of all alternate substrates, with the
4,5-PCD 2,3-CTD
4-Substituted 3-OH-benzoic
acid (3-OH-4-NH2-benzoate)
4-Substituted catechols
Simple substituents
PCA methylester
PCA ethylester
Homoprotocatechuate analogs
3,4-(OH)2-mandelic acid
2-Substituted PCA [2,3,4(OH)3-benzoic acid]
5-Substituted PCAs
a The average of three trials by polarographic assay (see Materials and
Methods). Values have an average error of approximately ±10%. Substrates
were 1.5 mM.
b Represents a single optical assay. Enzyme (0.1 pM), substrates (100 PM),
50 mM MOPS, pH 7.0.
c From reference 3.
d From reference 48.
¢ ND, not determined.
f This work.
exception of 4-AHB, is the presence of vicinal hydroxyl
groups on the benzenoid ring. The unique aspects of the
4-AHB reaction will be examined in detail in a subsequent
EPR spectra of 2,3-PCD. The EPR spectra of 2,3-PCD are
shown in Fig. 4. The enzyme as isolated (Fig. 4A) displayed
signals from several distinct species: resonances near g =
4.3 reflect a high-spin (S = 5/2) Fe3+ in a rhombically
distorted (EID = 1/3) electronic environment, resonances
between g = 5.6 and 8.2 are characteristic of multiple,
high-spin Fe31 species in more axial electronic environments, and the multiple resonances nearg = 2.0 result from
Mn2". Quantification of the latter spectrum showed that the
concentration of Mn2+ was only a few percent of the total
subunit concentration (see Table 6 below). Direct quantitation of the iron by spectral integration was difficult because
the spectra are very broad. Nevertheless, approximate quantitation indicated that the amount of EPR-active Fe3+ was
much less than the amount of total iron in the sample,
suggesting that much of the iron may have been in the
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1 / [PCA] (mM l)
FIG. 3. Steady-state kinetics of 2,3-PCD turnover. Initial velocity of PCA turnover was measured at several fixed
concentrations: 0, 290 PM; *, 150 PM; +, 90 PM; A, 35 p.M. Inset plot is a
replot of the slope and intercept values from the main plot versus the
reciprocal of the concentration. Solid lines are nonlinear regression fits to the original data converted to double reciprocal form.
The observed initial velocities (measured in units of micromolar
concentration per second) were divided by the enzyme active site
concentration to convert the units of the y axis into those of the
reciprocal turnover number (1/s-5). Conditions: enzyme, 0.016 P.M
(0.064 pM sites); temperature, 250C; pH 7.0; MOPS buffer, 50 mM.
Other conditions are given in Materials and Methods.
Relative Product
c'W1 -T
4)~ ~ ~~41
Magnetic Field (Gauss)
FIG. 4. EPR spectra of 2,3-PCD. X-band EPR spectra of 2,3PCD were recorded at 6.5 K. (A) Enzyme as isolated (70 A~M); (B)
enzyme from A plus 0.5 mM Na ascorbate added anaerobically; (C)
enzyme prepared as in B, treated with 8 mM Tiron for 5 min, and
then chromatographed on phenyl-Sepharose to remove the Tironmetal chelates as described in Materials and Methods; (D) enzyme
from B plus NO gas added anaerobically by bubbling with NO gas
for 3 mmn Instrumental conditions: field center, 2,100 G; field
sweep, 4,000 G; scan rate, 1,000 G/min; modulation amplitude, 10
G; modulation frequency, 100 kHz; microwave frequency, 9.22
GHz; microwave power, 0.2 mW. Relative scale factors are shown
on the figure.
(usually EPR-silent) Fe2+ state. Ascorbate diminished some
of the Fe3+ signals, whereas the Mn2+ signal remained
virtually unchanged (Fig. 4B). Concomitant with ascorbate
reduction, the enzyme activity increased 5 to 10% depending
upon the amount of Fe3+ in the preparation. We speculate
that the Fe2+ of the enzyme is partially oxidized during
purification and handling and that these factors determine
the extent of EPR-active iron and ascorbate reactivation.
EPR spectra of NO complexes of 2,3-PCD. Previous studies
have shown that the EPR-silent Fe2+ of many enzymes can
be made EPR active by forming the nitrosyl complex (5, 6,
29, 37, 61). Accordingly, upon anaerobic addition of NO to
2,3-PCD, a new and much more intense EPR spectrum with
resonances at g = 4.11 and 3.95 was observed (Fig. 4D).
Resonances with such features are characteristic of a species
with an electronic ground state spin of S = 3/2. Because this
type of signal has only been observed for the NO complex of
bound in proteins and organic chelate complexes, this
constitutes an unambiguous demonstration that 2,3-PCD
contains Fe2s. The spin concentration of this highly resolved spectrum could be accurately determined; it represents >95% of the iron in the ascorbate-reduced enzyme that
had been treated with a chelator to remove adventitiously
bound iron (see below). The low field portion of the spectrum is shown in a narrower magnetic field sweep range in
Fig. 5A. Only one species is apparent. The positions of the
Magnetic Field (Gauss)
FIG. 5. EPR spectra of 2,3-PCD-nitric oxide complexes. Samples of 2,3-PCD were made anaerobic and exposed to NO gas (3.3
mM dissolved concentration) for 3 min prior to freezing. (A)
2,3-PCD (52 ,uM) plus 1 mM Na ascorbate. (B) Enzyme (26 ,uM) plus
1 mM Na ascorbate plus 2 mM PCA. (C) Enzyme (23 pM) plus 2 mM
Na ascorbate plus 2 mM 2,3,4-(OH)3 benzoic acid. Instrumental
conditions: field center, 1,660 G; field sweep, 400 G; scan rate, 100
G/min; modulation amplitude, 1 G; modulation frequency, 100 kHz;
microwave frequency, 9.22 GHz; microwave power, 0.2 mW;
temperature, 10.1 K. Relative scale factors are given on the figure
after correction for enwyme concentration.
resonances show that the iron was in a nearly axial electronic environment with an EID value of 0.014. Upon the
addition of substrate, the splitting of the resonances around
g = 4 increased, indicating that the electronic environment
of the Fe2" had changed, becoming more rhombic, with an
EID of 0.034 (Fig. 5B). A single species was observed,
indicating that the complex was homogeneous. Quantitative
analysis showed that nearly all of the enzyme had bound
substrate. The weak resonances near g = 4.3 in the spectra
from the NO-treated samples are due to the small amount of
Fe3+, as was also seen in the spectra shown in Fig. 4A and
B. Addition of alternate substrates such as 2,3,4-trihydroxybenzoate to the enzyme-NO complex elicited EPR spectra
very similar to those of the enzyme-PCA-NO complex as
shown in Fig. SC, suggesting that most substrates bind in a
similar orientation in the active site. Substrates differed in
their affinities for the enzyme-NO complex. In the case
shown in Fig. 5C, the amount of 2,3,4-trihydroxybenzoate
added was insufficient to saturate the enzyme, so a small
amount of the g = 4.11, 3.95 species characteristic of
substrate-free enzyme-NO complex was observed. The order of substrate and NO addition was not found to be
critical. NO could be removed from the enzyme by evacuating the sample or exposing it to 02. After NO was
removed, full catalytic activity was observed, and NO could
again be bound to give the characteristic spectral features.
In a separate experiment, PCA was anaerobically added to
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3.7 OH
VOL. 175, 1993
TABLE 5. Mossbauer parameters of 57Fe-enriched 2,3-PCD'
(AE0, mm/s)
(8Fe, mm/s)
(I, mm/s)
2,3-PCD + 10 mM PCA
Typical Fe2" site (S = 2)d
Typical Fe3" site (S = 5/2)d
o.0k 5
Spectra measured at 4.2 K in zero applied field.
b Total iron concentration = 0.4 mM. Samples were prepared anaerobically.
c The AEQ value was temperature dependent. At 150 K, AEQ = 2.30 mm/s.
At 193 K, AEQ = 2.20 mm/s.
d From reference 20.
FIG. 6. Mossbauer spectra of 57Fe-enriched 2,3-PCD at 4.2 K.
(A) Uncomplexed enzyme as isolated (-500 ,uM). (B) Sample from
A after anaerobic addition of 10 mM PCA. The solid lines are
least-square fits of the data to one quadrupole doublet.
the enzyme and the EPR spectrum was recorded (data not
shown). The only change observed in the spectrum was a
small decrease in the signals atg = 8.14, 5.6, and 4.3, similar
to results seen in Fig. 4A versus B. No perturbation of the
Mn2+ signal nearg = 2 was observed, making it unlikely that
PCA binds to the Mn2+ (see reference 60).
Mdssbauer spectroscopy of enzyme and the enzyme-substrate complex. While the NO complex provides a rapid and
sensitive method to detect and quantitate Fe2+ in 2,3-PCD
and other enzymes, a direct technique that does not involve
binding of exogenous ligands to the metal is required to
validate the method. Mossbauer spectroscopy is such a
technique, but it requires substitution of "7Fe for 56Fe in the
active site. This was achieved by growth of the organism on
57Fe-enriched media. The zero-field Mossbauer spectrum of
2,3-PCD shown in Fig. 6A consists of a single doublet with
quadrupole splitting AE0 = 2.57 + 0.03 mm/s and isomer
shift 8 = 1.29 + 0.02 mm/s at 4.2 K. These parameters
unambiguously show that the iron site of the enzyme is in the
high-spin ferrous state (Table 5). The value of AEQ is
temperature dependent, suggesting that low-lying orbital
states become thermally accessible at the temperature of the
measurements; we found AEQ = 2.30 mm/s at 150 K and
substrate. The 4.2-K Mossbauer spectrum of the anaerobically prepared enzyme-PCA complex shown in Fig. 6B
consists of one quadrupole doublet with AEQ = 2.73 mm/s
and 8 = 1.19 mm/s. These parameters show that the iron site
remains high-spin ferrous upon binding of substrate. The
absorption lines of the enzyme-PCA complex are broader
(-0.48 mm/s) than those observed for the uncomplexed
enzyme (0.36 mm/s), suggesting some heterogeneity in the
ligand environment of the Fe2+ site, i.e., AE0 is distributed.
Iron and manganese content of 2,3-PCD and correlation
with subunit MW and enzymatic activity. While the quantity
of iron present clearly suggests that it is required for activity,
the possible role of Mn~
+in catalysis must also be considered. The metal-chelating reagents Chelex and Tiron were
used to remove any labile metals bound to the enzyme. Both
reagents bind Mn2 , while Tiron has a high affinity for Fe3+
and Chelex preferentially binds Fe2+. The EPR spectrum of
the enzyme after treatment with Tiron (Fig. 4C) shows that
much of the EPR-active Fe3+ and Mn2+ was removed by this
chelator. Samples of 2,3-PCD treated with Tiron and Chelex
were complexed with NO to produce the S = 3/2 EPR signal.
The height of the g = 4.11 peak was measured, and the ratio
of the enzymatic activity to the peak height for each sample
was determined (Table 6). Additionally, the ratios of total
iron and manganese (as measured by ICPES) to subunit
concentration and enzymatic activity were determined for
each sample. If a given metal is required for activity, the
ratio of the activity to the metal concentration should remain
constant. This was not observed for Mn2" or the total iron.
Only the amount of Fe2" that gives rise to the S = 3/2 signal
maintained a constant relationship to activity. This iron
could not be removed readily from the enzyme by these
chelators. This finding, in addition to the fact that only Fe2+
is present in a stoichiometric amount relative to the subunit
TABLE 6. Quantitation of metals bound to 2,3-PCD
2.20 mm/s at 193 K. (These values suggest that the
lowest orbital excited state is ca. 300 cm-1 above the ground
state). Studies in an applied magnetic field of 6.0 T elicited
magnetic patterns indicative of a site with a large and
positive zero-field splitting parameter, D > 10 cm-1. The
high-field spectra were, however, ill resolved, preventing a
detailed analysis. Identical zero-field spectra were observed
for aerobic and anaerobic samples, suggesting that the
enzyme does not form a complex with 02 in the absence of
Chelex treated
Tiron treated
iron/ Total iron/
subunit total activity
Total metal concentrations determined by ICPES.
Intensity of the S = 3/2 EPR signal of the NO complex of the enzyme
contained in the sample divided by the total units of activity. Values are
normalized to the Tiron-treated sample. It is argued in the text that the S = 3/2
signal is representative of the Fe2" in the active site of the enzyme.
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TABLE 7. Effect of metal chelators and oxidizing and reducing
agents on enzymatic activity
o-Phenanthroline (1 mM)
a,a-Dipyridyl (1 mM)
8-OH quinoline (8 mM)
EDTA (8 mM)
Nitrilotriacetic acid (8 mM)
H202 (1 mM)c
K3Fe(CN)6 (1 mM)
NaN3 (1 mM)
% Activity with
concentration, clearly shows that it is Fe2' rather than Mn2+
that is responsible for activity.
Effect of metal chelators and oxidizing and reducing agents
on enzymatic activity. As shown in Table 7, strong Fe2+
chelators, such as o-phenanthroline and oc,a-dipyridyl,
markedly inactivated 2,3-PCD at 1 mM concentrations. On
the other hand, the Fe3" chelator Tiron showed little inactivation even at 8 mM. Other less-specific metal chelators
such as EDTA and nitrilotriacetic acid inactivated the enzyme only at relatively high concentrations. A commonly
used Mn21 chelator, 8-hydroxyquinoline, showed moderate
inactivation, but this reagent will bind most divalent cations,
including Fe2 . Substrate was observed to protect against
inactivation only in the cases of N3-, o-phenanthroline, and
a,a-dipyridyl (data not shown). The basis for N3- inactivation is not known, although it may act as an iron ligand,
thereby inhibiting substrate binding as we have observed for
2,3-CTD (40).
The oxidizing agents H202 and K3Fe(CN)6 completely
inactivated 2,3-PCD. Full activity was restored if the samples were promptly reduced with sodium ascorbate. The
EPR spectrum of the nitrosyl complex of the H202-treated
sample showed nearly complete loss of the S = 3/2 signal.
This signal was restored after the sample was exposed to
ascorbate. All of these results are consistent with the proposed Fe2' requirement for enzyme activity and suggest that
the active site is accessible to chelators and oxidation
and/or reduction agents.
Through the use of altered growth conditions and the
identification of stabilizing reagents, we have purified 2,3PCD to homogeneity for the first time. The enzyme has been
characterized in terms of its physical, catalytic, and spectroscopic properties. The availability of the characterized enzyme permits comparisons with the other dioxygenases that
catalyze cleavage of the ring of aromatic substrates. In
particular, comparisons with 4,5-PCD and 3,4-PCD, which
utilize the same substrate as 2,3-PCD, may allow recognition
of features of particular relevance to the active site structure
or the molecular mechanism. Also, because 2,3-PCD is one
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Enzyme (4 FLM) was incubated anaerobically in 50 mM MOPS (pH 7.0) for
5 min at 40C with each addition. Values shown are the average of three trials
and have an error of approximately +10%.
b Ascorbate was added
at 1 mM and incubated for 5 min.
c Inactivation of 30 pIM enzyme by treatment with 5 mM H202 resulted in
comparable loss of the S = 3/2 EPR signal from the nitrosyl complex. Both the
activity and the S = 3/2 signal were restored after treatment of the sample with
5 mM ascorbate.
of the few dioxygenases purified from Bacillus species, it
may be possible to determine whether there are speciesspecific characteristics that distinguish these dioxygenases
from those of the better-studied genera such as Pseudomonas. Some of the characteristics of ring cleavage PCA and
catechol dioxygenases are brought together in Table 8 to
facilitate such comparisons in the following sections.
Structure. The holoenzyme MW, the subunit MW, and the
quaternary structure of 2,3-PCD from B. macerans are quite
similar to those of most other extradiol catecholic dioxygenases. All of the proximal extradiol-cleaving dioxygenases
have a single type of subunit with MWs of approximately
35,000. This group includes 2,3-CTD, which when presented
with PCA catalyzes its cleavage to the same product as
2,3-PCD (6, 48). In contrast, the distal extradiol-cleaving
dioxygenase 4,5-PCD is structurally quite distinct from
2,3-PCD, despite the fact that it utilizes the same substrate.
The 4,5-PCD has two nonidentical subunits ("212), the larger
of which is approximately the same size as the single subunit
of the proximal extradiol-cleaving dioxygenases (5). This
more complex structure for 4,5-PCD was controversial at the
time it was first reported. However, the recent report of two
structural genes for 4,5-PCD from Pseudomonas paucimobilis coding for subunits of the expected sizes shows that the
subunit structure is, in fact, different from those of the
proximal extradiol-cleaving enzymes (45).
In marked contrast to the more typical extradiol dioxygenase, each of the many intradiol 3,4-PCD dioxygenases that
have been characterized has two types of subunits. The
fundamental unit of structure is a1-Fe +, but many different
numbers of these protomer units are represented in the
family of 3,4-PCD enzymes (for a review, see reference 37).
Interestingly, the subunit structure of 1,2-CTD has recently
been shown to be variable (43). The active enzyme is always
composed of two subunits per iron, but any combination of
a and I subunits is permitted.
Ferrous iron site. The use in this study of EPR and
Mossbauer spectroscopic techniques to show unequivocally
that the active site metal of 2,3-PCD is Fe2+ validates
numerous previous studies of other extradiol dioxygenases.
In many of these studies (for example, see references 2, 47,
and 49), the assignment of the metal as Fe2+ was based on
either chelation studies, the observation of inactivation by
oxidants, or the requirement for Fe2+ in reconstitution
procedures. Unfortunately, each of these approaches is
subject to ambiguities; for example, reconstitution of apo3,4-PCD requires Fe2' despite the fact that the iron is Fe3+
in the active enzyme (27). The spectroscopic approach also
reveals differences in the active site structure that are more
subtle than simply the redox state of the iron. For example,
2,3-CTD appears to be like all of the other proximal extradiol
dioxygenases on the basis of structural criteria, but the
Mossbauer AEQ value is distinct (67). Moreover, the Mossbauer spectrum of 2,3-CTD does not change when the
substrate complex forms, unlike all other Fe2" dioxygenases
thus far investigated.
Nitrosyl complexes. The use of NO has proven to be a rapid
and convenient method to detect, quantitate, and distinguish
Fe2+ centers. It also offers information about the active site
structure. For example, 4,5-PCD and 2,3-CTD each have
more than one EPR-active species when complexed with NO
(5, 6). In the case of 4,5-PCD, the enzyme-NO complex is
homogeneous, but two species are observed when PCA
binds, suggesting either two binding orientations for the
substrate or two forms of the enzyme. Just the opposite is
observed for 2,3-CTD in which the enzyme-NO complex is
VOL. 175, 1993
TABLE 8. Comparison of the properties of intradiol and extradiol PCA and catechol dioxygenases
Structural characteristics
Enzyme and source
Extradiol dioxygenases
B. macerans (ATCC 35889)
plus PCA
P. testosteroni (ATCC 49249)
plus PCA
Pseudomonas anvilla (putida)
plus catechol
Intradiol dioxygenases
B. fuscum (ATCC 15993)
plus PCA
P. putida (aeruginosa) (ATCC
plus PCA
1,2-CTD; P. arvilla (putida)
(ATCC 23974)
(sv) EPRE/D Mossbauer
of NO
Subunit structure
143,000 Fe2" (a-Fe2")4
140,000 Fe2- (a-Fe2+)4
2.59 1.29 This work
2.73 1.21
a = 17,000, 13 = 33,000
2.23 1.28 5, 6
2.33 1.27
2.80 1.22
3.28 1.31 5, 6, 35, 47, 67
0.40 0.44 57, 70
0.40 0.44 50, 51, 59
315,000 Fe3+ (ap3-Fe3+)5C
587,000 Fe3` (a13-Fe3)12
103,000 Fe2
a = 22,500, 13 = 40,000
a = 22,300, P = 26,600
3.28 1.31
63,000 Fe3+ a-Fe3+, aa-Fe3+,
a = 30,000, 1 = 32,000
0.45C 33, 34, 43
0.50 3
a EPR spectra recorded near 4 K of the anaerobic nitrosyl complex of the enzyme.
b Mossbauer spectra recorded at 4.2 K in zero applied magnetic field.
c Preliminary crystallographic studies indicate a hexameric structure (21a).
d NO binds only to the reduced form of the enzyme (57).
e Value for the enzyme-substrate-NO complex.
f At least five species are present, presumably because of multiple substrate binding orientations.
g Many species were detected for the nitrosyl complex of the enzyme.
h Reference 55a.
heterogeneous, but addition of catechol gives a species with
a single sharp spectrum. The 2,3-PCD nitrosyl complexes
with and without PCA both appear as single species, suggesting that the active site is homogeneous and that the
substrate has a single binding orientation when a small
02-like molecule is bound to the iron. This unusual characteristic will facilitate more detailed spectroscopic studies
designed to characterize the intermediates in the reaction
cycle now in progress.
Turnover characteristics. The turnover number of 2,3-PCD
is comparable to those of 4,5-PCD and 2,3-CTD (5, 47).
the enzyme is not
Because of its relatively high Km for
saturated with this substrate under typical assay conditions.
It is generally thought that the Fe + enzymes have 2- to
10-fold higher turnover numbers than the Fe3" enzymes, but
this is not the case as shown by the high turnover number of
3,4-PCD from Brevibacterium fuscum (70) (Table 8). For
both 3,4-PCD and 2,3-PCD, the inherently high turnover
number at saturating 02is not observed under normal assay
conditions (or for that matter, in vivo). These results suggest
that the Fe2+ and Fe3+ dioxygenases can have comparable
catalytic capacities under favorable conditions and that
differences in the observed rate derive primarily from their
relative affinities for substrates or products.
Substrate specificity. It is shown here that 2,3-PCD has the
largest number of alternate substrates of any of the wellstudied intradiol and extradiol catecholic dioxygenases: 3,4PCD (25, 70), 4,5-PCD (5, 53), 1,2-CTD (34), and 2,3-CTD
(48). The substrate range of 2,3-PCD is most closely approx02
imated by 2,3-CTD and chlorocatechol dioxygenase (9), but
these enzymes do not readily accommodate bulky side
chains in the position equivalent to C-1 of PCA. The 2,3C1D also does not accept halogens in the position immediately adjacent to the vicinal hydroxyl groups. Thus, the
active site pocket of 2,3-PCD seems to be sufficiently open to
bind substrate analogs with significant alterations in structure from PCA. Moreover, the ring cleavage chemistry does
not appear to be as sensitive to inductive effects of the
substituents as proposed for other dioxygenase enzymes
(58). It seems likely that fundamentally the same molecular
mechanism of ring cleavage applies to a wide variety of PCA
analogs because the same site of ring cleavage is observed in
every case. From previous studies, we have suggested that
the site of ring cleavage depends strongly on the orientation
of the substrate relative to the iron and the activated oxygen
in the active site (4, 37). We have proposed that both the
substrate and the oxygen bind to specific sites in the iron
coordination field. Thus, 4,5-PCD and 2,3-PCD may use the
same fundamental mechanism, while controlling the site of
ring cleavage by altering the structure of the reactive complex. If proximity is the determining factor in the position of
ring cleavage for the extradiol dioxygenases, then the current results suggest that the binding orientation of the
substrate relative to the reactive oxygen species in the active
site is primarily controlled by the enzyme structure rather
than the functional groups of the substrates themselves. This
specificity might be achieved by establishing chelation of the
vicinal hydroxyl groups of the substrate to the iron as the
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(ATCC 23973)
primary binding determinant. Such chelate structures have
been observed for other extradiol dioxygenase-substrate
The observed turnover of catechol and the 3-substituted
catechols indicates that the presence of a C-1 carboxylate
group is not an absolute requirement for cleavage of catecholic compounds. Furthermore, a wide range of substitutions for the C-1 carboxylate is allowed, as evidenced by the
turnover of the 4-substituted catechols and esters of PCA.
However, the rate for the fastest substrate analog among any
of these groups is only 1/10 that of PCA, indicative of the
strong preference exhibited by the enzyme for the structure
of its natural substrate.
The role of Mn2". The results presented here show that
Mn is not required for catalysis. This conclusion is supported by the observation that 2,3-PCD is inactivated by
H202 and K3Fe(CN)6. It has been observed that these
oxidants selectively inhibit Fe2+-containing enzymes while
showing no effect against Mn2+-containing enzymes (38, 52,
60, 68). Moreover, the EPR spectrum due to Mn 2+ is not
altered by anaerobic addition of substrate to enzyme, while
large changes in the Fe2+ site are revealed by the changes
that occur in the EPR spectra of the enzyme-NO complexes
on addition of substrates. In a previous EPR study of an
Mn 2-containing extradiol dioxygenase from Bacillus species, anaerobic addition of substrate was clearly shown to
cause a perturbation of the Mn2+ spectrum (60), suggesting
that a perturbation would have been observable in the
present case had it occurred. Taken together, these results
suggest that the Mn2+ present in 2,3-PCD is likely to be
adventitiously bound to the surface of the enzyme. Studies
of homo-2,3-PCD have revealed that the enzyme from
Pseudomonas species utilized Fe2+ (2), while that from
Bacillus species (60) utilized Mn2+, raising the possibility
that Mn2+ is the metal of choice for Bacillus dioxygenases.
The present results also show that it is incorrect to generalize that dioxygenases from Bacillus species specifically
contain either iron or manganese, because enzymes containing both metals have now been characterized.
Order of substrate binding. The kinetic data presented
here are consistent with an ordered bi-uni mechanism in
which PCA binding must precede 02 binding. This order is
supported by the Mossbauer spectra, which show PCA
binding to be independent of 02 but provide no evidence for
an enzyme-02 complex in the absence of PCA. On the other
hand, we have also observed that the 02 analog NO can bind
either before or after PCA. Indeed, PCA and NO can bind
simultaneously, as shown by the EPR spectra of the complex. In the case of other Fe2" dioxygenase enzymes, we
have shown that the affinity for NO increases dramatically
when the organic substrate is added (6, 30). Preliminary
studies suggest that this will also be true for 2,3-PCD. Thus,
the binding of organic substrates and NO is positively
coupled for these enzymes. We have speculated that this
coupling may result from a decrease in the redox potential of
the iron due to the binding of a negatively charged catecholate or phenolate ligand to the iron (4, 30, 37). This would
allow the iron to form a stronger bond with NO. The same
phenomenon may apply to 02 binding, except that in the
absence of the organic substrate, the 02 affinity may be too
low to form a detectable amount of complex at physiological
concentrations of 02. The putative coupling of 02 and
organic substrate binding would assure that a substrate to
oxidize is always present when 02 binds and becomes
activated. The shift of electron density from the aromatic
substrate to the 02 via the iron may also be a fundamental
process in the oxygen activation mechanism (4, 30, 37).
Conclusion. The purification of 2,3-PCD completes the
isolation of all known types of enzymes of aerobic organisms
that cleave PCA. The resulting family of protocatechuate
dioxygenases represents all types of cleavage sites for catecholic dioxygenases as well as both of the mechanistically
distinct subclasses of these enzymes. The studies regorted
here strongly support the association of active site Fe + and
Fe2+ with intradiol and extradiol ring cleavage, respectively.
In contrast, the distinction between proximal and distal
extradiol ring cleavage is not related to the redox state of the
iron. Moreover, the spectroscopic studies reported here and
in previous studies failed to reveal a substantial difference in
the iron coordination sphere or the ability of the iron to
interact with substrates and small molecule ligands. It therefore seems likely that the mechanisms of proximal and distal
extradiol dioxygenase are fundamentally the same and that
the site of ring cleavage is determined by the influence of the
protein structure on the proximity of an activated oxygen
species to the site of attack on the aromatic ring of catecholic
This research was supported by National Institutes of Health
grants GM24689 to J.D.L., GM22701 to E.M., and ES-A1-02184 to
We thank Marcia A. Miller for measuring some of the data
contained in Table 4. We also thank Gerald Bratt for assistance in
use of the ultracentrifuge and Eric Eccleston for assistance in
performing the amino acid analysis and N-terminal amino acid
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