From the Department of Biochemistry and the
§ Centre for Magnetic Resonance, The University of
Queensland, St. Lucia, Queensland 4072, Australia and the
¶ Department of Chemistry, Monash University, Clayton, Victoria
3168, Australia
Received for publication, October 26, 2000, and in revised form, January 30, 2001
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ABSTRACT |
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A purple acid phosphatase from sweet potato is
the first reported example of a protein containing an enzymatically
active binuclear Fe-Mn center. Multifield saturation magnetization data over a temperature range of 2 to 200 K indicates that this center is
strongly antiferromagnetically coupled. Metal ion analysis shows an
excess of iron over manganese. Low temperature EPR spectra reveal only resonances characteristic of high spin Fe(III) centers (Fe(III)-apo and Fe(III)-Zn(II)) and adventitious Cu(II) centers. There
were no resonances from either Mn(II) or binuclear Fe-Mn centers.
Together with a comparison of spectral properties and sequence
homologies between known purple acid phosphatases, the enzymatic and
spectroscopic data strongly indicate the presence of catalytic
Fe(III)-Mn(II) centers in the active site of the sweet potato enzyme.
Because of the strong antiferromagnetism it is likely that the metal
ions in the sweet potato enzyme are linked via a µ-oxo bridge, in
contrast to other known purple acid phosphatases in which a µ-hydroxo
bridge is present. Differences in metal ion composition and bridging
may affect substrate specificities leading to the biological
function of different purple acid phosphatases.
Purple acid phosphatases comprise a family of binuclear
metal-containing enzymes, the members of which have been identified in
plants, animals, and fungi. The animal enzymes contain an
antiferromagnetically coupled binuclear iron center. The active form of
the enzyme (Fe(III)-Fe(II)) exhibits a distinctive EPR signal with
principal g values of 1.93, 1.75, and 1.50 (1), whereas the inactive
(Fe(III)-Fe(III)) form is EPR-silent at X-band microwave frequencies
(2, 3). Plant purple acid phosphatases appear to be more diverse. Red kidney bean purple acid phosphatase, the best characterized plant enzyme, contains an Fe(III)-Zn(II) center (4). Replacement of Zn(II) by
Fe(II) yields an enzyme with full activity and with spectral properties
very similar to those of the animal enzymes (5, 6).
The sequences of three cDNAs encoding different isoforms of sweet
potato purple acid phosphatase have recently been reported (7, 8). Two
of these isoforms have been purified. One form was shown by Durmus
et al. (7) to contain one g-atom of iron and 0.77 g-atom of zinc/subunit. Comparison of its spectroscopic properties with
those of the red kidney bean enzyme indicated that the metal ions are
present as a binuclear Fe(III)-Zn(II) center (7). A different form
exhibiting 66% sequence identity to the Fe(III)-Zn(II) enzyme has
recently been characterized in our laboratory. It contains one g-atom
of iron and 0.6-0.8 g-atom of manganese/subunit (8). We now report
enzymatic, magnetic susceptibility and EPR studies on this enzyme
demonstrating that the enzyme contains a catalytically active
Fe(III)-Mn(II) center that is strongly antiferromagnetically coupled,
providing the first reported evidence for such a center in a protein.
Purification and Enzyme Assay--
The enzyme was purified by a
combination of juice extraction, acetone and ammonium sulfate
fractionation, DEAE-cellulose chromatography at pH 7.0, and gel
filtration on a Sephadex G-150 Superfine column at pH 4.90 as described
elsewhere (8). The enzyme was purple when concentrated and exhibited
essentially the same visible absorption spectrum ( Metal Ion Analysis--
Metal ion content was determined by
inductively coupled plasma mass spectrometry using a PerkinElmer
SCIEX-ELAN 5000 spectrometer. Samples and standards were prepared in
0.1% HNO3. Separate standard curves were routinely
prepared for iron, zinc, copper, and manganese. Samples were measured
in quadruplicate. Metal ion analysis showed that the sample of enzyme
used for magnetic susceptibility and EPR measurements contained
1.035 ± 0.108 iron, 0.582 ± 0.044 manganese, 0.183 ± 0.019 zinc, and 0.11 ± 0.028 copper/subunit of 55 kDa.
Oxidation and Reduction--
Pig purple acid phosphatase was
prepared as described previously (9). Protein concentrations for the
oxidation and reduction experiments were 300 µM for the
pig enzyme and 300 µM (subunit concentration) for the
sweet potato enzyme. Oxidation was carried out by incubating the
enzymes in 0.1 M acetate buffer, pH 4.90, containing 30 mM H2O2 at 4 °C for 45 min
followed by removal of the peroxide by gel filtration. Reduction of
oxidized pig purple acid phosphatase and untreated sweet potato enzyme
was carried out by incubation in pH 4.90 buffer containing 140 mM Partial Removal of Metal Ions--
Sweet potato purple acid
phosphatase (40 µM) in 0.1 M acetate buffer
(pH 4.9) was mixed with pyridine-2,6-dicarboxylate (2 mM)
at 20 °C (5). An aliquot of freshly prepared sodium dithionite solution (10 mM) was added. After a short incubation
(10-60 s) the mixture was separated on a Sephadex G-25 column, which
was equilibrated with 0.1 M acetate buffer (pH 4.9).
Multifield Saturation Magnetization--
Multifield saturation
magnetization (10, 11) data were collected with a Quantum Design MPMS
SQUID magnetometer and corrected for the magnetization of the
deuterated acetate buffer (pD 4.9, 0.1 M), the effects of
mismatches in residual ferromagnetic impurities in the quartz sample
holder (volume = 0.146 ml), background diamagnetism of the sample,
and temperature-independent paramagnetism (11). Fitting of the data
employed the program WMAG (11) and an S = 5/2
spin Hamiltonian. To reduce the number of variables, the g and E/D
values were held at 2.0 and 0.1925, respectively, as deduced from the
EPR spectrum. Reasonably good fits, as judged by the "goodness of
fit" parameter in the program, were obtained for |D| values
between 0.8 and 1.2 cm EPR Spectroscopy--
X-band (9-10 GHz, TE102
rectangular cavity) EPR spectra were measured on Bruker EPR
spectrometers (ESP300E and Elexsys E500). Low temperatures (1.5-2.5 K)
were obtained with a flow-through Oxford instruments ESR910 cryostat in
conjunction with an Oxford instruments ITC-4 variable temperature
controller. Spectrometer tuning, signal averaging, and subsequent data
manipulation were performed with either Bruker esp300e (v3.02) software
for the ESP300E spectrometer or Xepr® (v2.0) for the
Elexsys spectrometer. Instrument settings were as follows: microwave
power, 20 mW; modulation frequency, 100 kHz, and the modulation
amplitude was always less than one-tenth of the linewidth at
half-height. The microwave frequency and magnetic field were calibrated
using an EIP 548B microwave frequency counter and a Bruker ER035
M gaussmeter, respectively.
Computer simulation of the EPR spectra was performed using version 1.0 of XSophe© (13-15) running on a SGI R5K O2 work
station. Matrix diagonalization was employed within Sophe (13) for the
calculation of the eigenvalues/eigenvectors, which were used to
determine the resonant field positions and transition probabilities.
The field-swept EPR spectral intensity was normalized with the factor
(d Several independent preparations of sweet potato purple acid
phosphatase, including the one used in the present study (see "Experimental Procedures"), contained essentially stoichiometric amounts of iron, 0.6-0.8 g-atoms of manganese, and smaller amounts of
zinc (<0.05-0.18 g-atoms) and copper (0.08-0.11 g-atoms)/subunit of
molecular mass 55 kDa (8). In purple acid phosphatases from red kidney
bean, soybean, and one isoform from sweet potato, where the active form
of the enzyme contains an Fe(III)-Zn(II) center, the content of the
divalent metal ion is also substoichiometric, ranging from 0.6 to 0.8 g-atom/subunit (4, 7, 8). In all three enzymes the iron content is
stoichiometric, reflecting tighter binding of the trivalent metal ion.
For the red kidney bean and soybean purple acid phosphatases, it could
be shown that the specific activity correlates with the content of the
divalent ion, Zn (4, 8). Furthermore, smaller amounts of manganese, which are present in preparations of the soybean enzyme, are readily removed by dialysis against 5 mM EDTA without effecting
a change in specific activity. A similar treatment with EDTA of
the sweet potato enzyme characterized in our laboratory does not remove manganese, suggesting that the Fe-Mn binuclear cluster in sweet potato
purple acid phosphatase is responsible for the catalytic activity. To
substantiate this conclusion, the manganese and zinc contents
were correlated with the enzyme's specific activity.
First, prolonged storage of sweet potato purple acid phosphatase at
4 °C results in a loss of manganese with a proportional decrease in
specific activity. For example, when enzyme containing 1.0 g-atom of iron and 0.63 g-atom of manganese and having a specific activity of 730 units/mg was stored for one year at 4 °C, the manganese content decreased to 0.47 g-atom/subunit and the specific activity decreased to 500 units/mg. The zinc content remained invariant. Upon incubation with Mn(II) the manganese content increased to 0.71 g-atom/subunit and the specific activity increased to 800 units/mg (again, the zinc content did not change). We also investigated
the possibility of varying the metal ion composition. For mammalian and
red kidney bean purple acid phosphatases, a protocol has been
established for the generation of metal-free enzyme (5, 16). Although
the same treatment does not completely remove the metal ions from sweet
potato purple acid phosphatase, short incubations (10-60 s) with the
reductant sodium dithionite facilitated the generation of purple acid
phosphatase samples with varying manganese content but stoichiometric
iron content (1.02 ± 0.06 g-atom/subunit); the amount of zinc
present in the samples was below the detection limit (
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
max = 560 nm,
max = 3207 M
1 cm
1)
as the red kidney bean enzyme (
max = 560 nm,
max = 3360 M
1
cm
1) (4). Enzyme assays were performed at
25 °C using p-nitrophenyl phosphate (5 mM) as
substrate in 0.1 M acetate buffer, pH 4.90. The specific
activity of the sample used for magnetic susceptibility and EPR
measurements was 675 units/mg. The protein subunit concentration was
0.556 mM (based on a subunit weight of 55 kDa and an
A1%1 cm at 280 nm of 27.03 (8)).
-mercaptoethanol at 25 °C for 150 min followed by
gel filtration. The specific activities of the samples were determined
using the assay described above.
1.1
/dB). Spectral comparisons of the simulated and
experimental spectra were performed with Xepr® running on
the SGI work station.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
0.04
g-atom/subunit). The specific activity of these preparations was found
to be proportional to the manganese content; this is illustrated in a
plot of manganese content versus specific activity for
purified, manganese-depleted and reconstituted (see below) enzyme (Fig.
1). Clearly, the specific activity
increases proportionally with the increase in manganese content in the
range between 0.35 and 0.81 g-atom manganese/subunit (correlation
coefficient = 0.9952). The calculated specific activity for the
enzyme with a full complement of iron and manganese is 1260 units/mg,
corresponding to a turnover number for p-nitrophenyl phosphate of 1154 s
1.
View larger version (16K):
[in a new window]
Fig. 1.
Correlation between the manganese content and
the specific activity of purified, manganese-depleted and reconstituted
sweet potato purple acid phosphatase.
Second, dialysis of the manganese-depleted enzyme (iron remains stoichiometric) in the presence of MnCl2 was found to reconstitute enzymatic activity to a value proportional to its manganese content. In contrast, dialysis of the manganese-depleted enzyme against ZnCl2, (NH4)2Fe(SO4)2, FeCl3, MnCl3, or CuSO4 failed to reactivate the enzyme.
Third, we investigated the possibility that zinc, present as binuclear
Fe-Zn centers in sweet potato enzyme preparations, may be responsible
for the catalytic activity. The content of zinc ions varies from one
preparation to another and is likely influenced by the condition of the
soil. Several preparations, which vary in zinc content from below
detection limits (0.04 g-atom) to 0.18 g-atom/subunit (with
stoichiometric iron content and ~0.6 g-atom of manganese/subunit)
exhibit no variation in specific activity (8). Dithionite reduction of
purified enzyme (1 g-atom of iron, 0.58 g-atom of manganese, 0.15 g-atom of zinc, and a specific activity of 675 units/mg) and subsequent
dialysis against MnCl2 produced an increase in specific
activity (765 units/mg), which correlated with the manganese content
(0.68 g-atom/subunit) but not with the zinc (
0.04 g-atom) content.
The iron content after dialysis was found to be 0.96 g-atom/subunit.
Clearly, the small proportion of binuclear Fe-Zn centers present in the
preparations of sweet potato purple acid phosphatase are not
responsible for the observed catalytic activity.
Finally, to determine whether Fe(III)-Fe(III) centers are present in
sweet potato purple acid phosphatase, we subjected the enzyme to
reduction with -mercaptoethanol under conditions that restored
~90% of the activity of the oxidized pig enzyme. This treatment
increased the specific activity of the sweet potato enzyme by only 3%.
Similarly, treatment of this enzyme with hydrogen peroxide under
conditions that rapidly oxidized and inactivated the Fe(III)-Fe(II) pig
enzyme (16) had no effect on the specific activity of the sweet potato
enzyme. In the EPR spectrum of the reduced sweet potato purple acid
phosphatase (see Supplemental Material, Fig. S1) only very weak
resonances attributable to a binuclear Fe(III)-Fe(II) center (<2%)
were observed indicating that at best only a small proportion of sweet
potato purple acid phosphatase contains Fe(III)-Fe(III) and (in view of
the metal analysis) Mn-Mn centers.
In summary, the above observations clearly indicate that the
catalytically active form of sweet potato purple acid phosphatase contains binuclear Fe-Mn clusters. Iron in sweet potato purple acid
phosphatase is expected to be most likely in the Fe(III) state because
of the similarity of the visible absorption spectra (and the high
degree of sequence homology) of the sweet potato and red kidney bean
(4) enzymes, which suggests that the band observed at 560 nm in the
sweet potato enzyme arises from a tyrosine Fe(III) charge
transfer transition. The properties of this novel Fe-Mn center were
further investigated using magnetic susceptibility and EPR spectroscopy.
Multifield saturation magnetization of the type used by Day and
co-workers (10, 11) on the pig allantoic fluid purple acid phosphatase
was employed to measure the magnetic susceptibility of the enzyme at
four magnetic fields (0.2, 1.375, 2.75, and 5 tesla) over a temperature
range of 2 to 200 K (Fig. 2). The
magnetization values over this temperature range are very weak and show
typical Curie behavior (Fig. 2), characteristic of an isolated spin
system. The isofield best-fit plots of the experimental
susceptibilities (Fig. 2A) employing an S = 5/2 spin Hamiltonian produced reasonably good fits and yielded a
concentration of 0.082 ± 0.005 mM S = 5/2 centers. This concentration corresponds to only ~12% of
the total concentration of Fe(III) and Mn(II) centers present in the sample.2 For a weakly
antiferromagnetically coupled Fe(III)-Fe(II) center (e.g.
pig purple acid phosphatase) with 2J = 20
cm
1 (H =
2JS1·S2),
the susceptibility
= (M/H) versus T plot
would be expected to show a broad maximum centered at 82 K (Fig.
2B), and this is clearly not observed. Consequently, the weak susceptibility arises from a diamagnetic protein in which the two
metal ions (Fe(III), Mn(II)) at the binuclear active site are strongly
antiferromagnetically coupled (
2J
140 cm
1), producing an S = 0 ground state.
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A low temperature EPR spectrum of sweet potato purple acid phosphatase
(Fig. 3) consists of resonances arising
from high spin Fe(III) (g' (effective g value) = 3.8 9)
and Cu(II) (g|| 2.293, g
2.066, A|| 163.00 × 10
4
cm
1, A
8.10 × 10
4 cm
1).
The absence of signals that might reasonably be attributed to either
magnetically isolated manganese or a binuclear metal center is
consistent with the susceptibility data, indicating a strongly
antiferromagnetically coupled center. Computer simulation of the
resonances arising from high spin Fe(III) (Fig. 3) were performed using
a second-order fine structure spin Hamiltonian (H = g
B·S + S·D·S) and a distribution
of D and E/D values. The distribution of E/D was characterized with
five parameters using two Gaussian peaks, although in principle more
complicated distributions with more parameters could be used. A good
fit was obtained with one Gaussian peak centered at 0.1925 and a
half-width of 0.04 and another 50 times smaller at 0.3333 with a
half-width of 0.005. The resulting simulations are shown in Fig. 3,
b and c. The presence of these two Gaussian
distributions suggests the existence of two components with 1) E/D = 0.1925 and 2) E/D = 1/3. However, only a very small
component with E/D = 1/3 is included in the simulation to account
for the g = 4.3 feature; the spectrum could be reproduced by
extending the tail of the broad distribution to E/D = 1/3.
Therefore, it is more likely that there is a single species present
with a large distribution of E/D.
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Spin quantitation of the concentration of high spin Fe(III) centers
giving rise to the EPR spectrum shown in Fig. 3 is experimentally and
theoretically difficult to determine
accurately.3 The best way to
estimate the spin concentration is to compare the EPR spectra from the
red kidney bean and sweet potato enzymes, which suggests that the
proportion of sweet potato purple acid phosphatase containing high spin
Fe(III) centers (Fe(III)-Zn and Fe(III)-apo) is very small, certainly
less than 20%. This is in agreement with the estimate (~12%) from
magnetic susceptibility measurements. A comparison of the spin
Hamiltonian parameters for the sweet potato enzyme with those for red
kidney bean purple acid phosphatase (D ~ 1
cm
1, E/D = 0.13) reveals that the
Fe(III) site (i) is more rhombic in the sweet potato enzyme and (ii)
arises from either the one-iron apoenzyme or Fe-Zn centers.
In summary, the collective enzymatic and spectroscopic data presented herein indicate the presence of a strongly antiferromagnetically coupled binuclear Fe(III)-Mn(II) center in sweet potato purple acid phosphatase, which is responsible for the enzyme's biological activity.
Model systems containing µ-OH-µRCO2 or µ-RhO-
µ-RCO2 bridging combinations in Fe(III)-Mn(II)
complexes display weak antiferromagnetism with 2J 20 cm
1 (19, 21,
22).4 As a consequence
of the much higher coupling constant, µ-oxo Fe(III)-O-Mn(II)
bridge(s) are likely to be present rather than a µ-hydroxo
bridge, which is observed in the Fe(III)-Zn(II) and the Fe(III)-Fe(II)
centers of the native and iron-enriched red kidney bean purple acid
phosphatase (23). µ-Oxo bridges are known in Fe(III) and in Mn(III)
compounds but not, to date, in Mn(II) complexes. The seven amino acid
residues, which are known to coordinate to the two metal ions in the
binuclear centers in pig and red kidney bean purple acid phosphatases,
are invariant in the sweet potato enzyme (8). We have recently reported
the crystallization of and preliminary x-ray diffraction data on the latter enzyme (24). In a partially refined model of its crystal structure, it is obvious that these seven invariant amino acid residues
bind to the metal
ions.5 Furthermore,
the Fe(III)-Mn(II) internuclear distance at 2.7 Å resolution is
significantly shorter (2.9 Å) than in the µ-hydroxo bridged enzymes
from pig (3.31 Å; Ref. 26) and red kidney bean (3.26 Å; Ref. 27).
This observation is consistent with the presence of a µ-oxo bridge
rather than a µ-hydroxo bridge in sweet potato purple acid
phosphatase. Studies of additional model complexes with Fe(III)-Mn(II)
centers and additional refinement of the crystal structure of the sweet
potato enzyme with and without inhibitors are in progress in this
laboratory to clarify the interactions in the active site that may
contribute to the stabilization of the oxo bridge.
The presence of a µ-oxo bridge in the sweet potato enzyme described here may be of great significance for future mechanistic studies on binuclear metallohydrolases. Among all characterized metallohydrolases similarities in metal ion coordination are found, and a common mechanistic theme appears to be present in which the hydrolytic reaction is initiated by an activated solvent nucleophile (28). The identity of the attacking nucleophile remains uncertain. The three-dimensional structure of red kidney bean purple acid phosphatase demonstrates that a terminal Fe(III)-coordinated hydroxide is a likely candidate (27). More recently, metal replacement studies on bovine purple acid phosphatase proposed an alternative mechanism in which a hydroxo ligand of the trivalent metal ion acts as a general base to deprotonate a water molecule in the second coordination sphere, which then attacks the phosphate group of the substrate (29). Analysis of ternary fluoride-enzyme-phosphate complexes in both pig and bovine purple acid phosphatases (30, 31) has led to the suggestion that in the enzyme-substrate complex the phosphate group of the substrate and a hydroxide ion bridge the two metal ions in the active site. It is argued that because the nucleophilicity of this bridging hydroxide is too low for an attack on the phosphate group, the hydroxide is shifted toward the divalent metal ion upon substrate binding. This movement may increase this solvent molecule's nucleophilicity sufficiently for a nucleophilic attack on the phosphorus atom of the substrate (30). However, the oxo bridge in sweet potato purple acid phosphatase is expected to be a significantly stronger nucleophile; hence it cannot be ruled out at present that a direct attack from the bridging oxo moiety initiates the reaction in this enzyme. The differences in metal ion content and bridging ligands in purple acid phosphatases purified from animals (Fe(III)-µ-OH-Fe(II)), red kidney beans (Fe(III)-µ-OH-Zn(II)), and sweet potatos (Fe(III)-X-Zn(II) or Fe(III)-µ-O-Mn(II) in different isoforms) may affect the one-electron reduction potentials and substrate specificities, leading to different biological functions in vivo.
The discovery of three cDNAs encoding different isoforms of purple
acid phosphatase in sweet potato may provide the explanation for some
of the variations in metal content and EPR spectra observed by
different groups in early work on the enzyme. The enzyme isolated by
Hefler and Averill (32) was reported to contain two g-atoms of
iron/dimer (max = 545 nm,
max = 3080 M
1
cm
1/Fe). The native enzyme showed no evidence
for a g' = 1.70 signal characteristic of the antiferromagnetically
coupled Fe(III)-Fe(II) center of the mammalian purple acid
phosphatases, but the authors noted a complex signal in the g' = 4-6
range. The suggestion (32) that the enzyme may contain isolated
mononuclear iron centers is now most unlikely in view of the extensive
sequence homology between the three sweet potato isoforms and the red
kidney bean purple acid phosphatase (7, 8). Durmus et al.
(7), following a purification similar to that of Hefler and Averill
(32), showed that their enzyme contains an Fe(III)-Zn(II) center with
spectral properties similar to the red kidney bean enzyme. Furthermore, the positions of the resonances attributable to high spin Fe(III) in
their enzyme (pH 4.0) are very similar to those seen in the present
study (cf. Fig. 3a and Durmus et al.
(7)) indicating, as expected from the sequence homology, that the two
forms have similar iron coordination spheres. The sweet potato purple
acid phosphatase isolated by Sugiura et al. (25) may
be different from both the Fe-Mn and the Fe-Zn enzyme. Metal ion
analysis of this enzyme indicated the presence of one manganese and
negligible iron per 110-kDa dimer. The enzyme exhibited a
max of 515 nm and
max = 1230 M
1 cm
1
based on a monomer molecular mass of 55 kDa, values that are significantly different from those of the Fe-Zn and Fe-Mn enzymes. Their enzyme was EPR-silent, but upon denaturation in strong acid (0.8 M HCl, 3 min, 100 °C) resonances centered around g ~2,
typical of Mn(II), were observed. The authors concluded that the native enzyme contained a single Mn(III) coordinated to a tyrosine phenolate. It is feasible that the Mn(II) seen by EPR spectroscopy arose as a
result of disproportionation of Mn(III) to Mn(II) and Mn(IV). Although
the exact nature of the metal center of the purple acid phosphatase
purified by the Japanese group (20, 25) remains unclear, it is
possible that it may represent an isoform encoded by the third
cDNA, which we and Durmus et al. have independently cloned and sequenced but not yet characterized. If so, the metal center
is almost certainly a binuclear complex. Possible candidates, despite
the reported metal ion analyses, appear to be strongly antiferromagnetically coupled Fe(III)-Mn(II) or Mn(III)-Mn(III), both
of which would be EPR-silent and could generate a Mn(II) signal upon denaturation.
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FOOTNOTES |
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* This work was supported by grants from the Australian Research Council (to J. D. J., S. H., G. R. H., and K. S. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Fig. S1.
To whom correspondence should be addressed. Tel.:
+61-7-33654611; Fax: +61-7-33654699; E-mail:
hamilton@biosci.uq.edu.au.
Published, JBC Papers in Press, February 1, 2001, DOI 10.1074/jbc.M009778200
1 Slightly better fits were obtained using these g, D, and E values and an S = 2 Hamiltonian (e.g. Fe(II) or Mn(III)), but this does not agree with the EPR data.
2 The excess of iron over manganese, present as 1-iron (Fe(III)) apoenzyme and/or Fe(III)-Zn(II) centers would then be expected to comprise the S = 5/2 signal. This excess (0.20 ± 0.04 mM) is, however, considerably higher than 0.082 ± 0.005 mM. We know from recent work on well characterized S = 5/2 Fe(III) rubredoxin samples (17) that the errors between S = 5/2 concentration from metal ion analyses and magnetization fits is at most ±15%. Fitting attempts that included a combination of Fe(III), S = 5/2, and Cu(II), S = 1/2 centers yielded reasonable fits but with unreasonably low g values, and the concentrations of Fe(III) were still less than that expected if all of the iron were present in monomeric centers.
3
Spin quantitation experiments on high spin
Fe(III) centers require an accurate knowledge of the transition
probability (normally determined from the simulated spectrum (18)) as
this will affect the relative intensities of the individual resonances
in the spectrum. Unfortunately, the transition probability cannot be
determined uniquely from the computer simulation of the experimental
spectrum as the axial zero field-splitting parameter D (0.8 to
1.2
cm
1) is greater than the microwave quantum.
In addition, the distributions of D and E giving rise to the observed
line widths cannot be uniquely defined because D and E are unknown.
4 C.-J. McKenzie, B. Moubaraki, K. S. and Murray, unpublished data on Fe(III)Mn(II) model complexes.
5 G. Schenk and L. W. Guddat, unpublished results.
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