From the Department of Biophysics, Stockholm
University, Arrhenius Laboratories, S-106 91 Stockholm, Sweden, the
¶ Max-Volmer-Institut für
Biophysikalische Chemie und Biochemie, Technische Universität
Berlin, D-10623 Berlin, Germany, and the
Department of Medical Biochemistry and Biophysics,
Umeå University, S-901 87 Umeå, Sweden
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ABSTRACT |
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The ferrous iron/oxygen reconstitution reaction in
protein R2 of mouse and Escherichia coli ribonucleotide
reductase (RNR) leads to the formation of a stable protein-linked
tyrosyl radical and a µ-oxo-bridged diferric iron center, both
necessary for enzyme activity. We have studied the reconstitution
reaction in three protein R2 mutants Y177W, Y177F, and Y177C of mouse
RNR to investigate if other residues at the site of the radical forming
Tyr-177 can harbor free radicals. In Y177W we observed for the first
time the formation of a tryptophan radical in protein R2 of mouse RNR with a lifetime of several minutes at room temperature. We assign it to
an oxidized neutral tryptophan radical on Trp-177, based on selective
deuteration and EPR and electron nuclear double resonance spectroscopy
in H2O and D2O solution. The reconstitution
reaction at 22 °C in both Y177F and Y177C leads to the formation of
a so-called intermediate X which has previously been
assigned to an oxo (hydroxo)-bridged Fe(III)/Fe(IV) cluster.
Surprisingly, in both mutants that do not have successor radicals as
Trp· in Y177W, this cluster exists on a much longer time scale
(several seconds) at room temperature than has been reported for
X in E. coli Y122F or native mouse protein R2.
All three mouse R2 mutants were enzymatically inactive, indicating that
only a tyrosyl radical at position 177 has the capability to take part
in the reduction of substrates.
The reduction of ribonucleotides to their corresponding
deoxyribonucleotides is an essential reaction for all living cells and
provides precursors for DNA synthesis. This reaction is catalyzed by
the enzyme ribonucleotide reductase
(RNR).1 A number of RNRs of
different species have been discovered and classified into three major
classes (1). A common feature of all RNRs within these three classes is
that they use radical mechanisms to reduce ribonucleotides. RNR of
class Ia, found in mammals, plants, DNA viruses, and some procaryotes
like Escherichia coli, consists of two components, proteins
R1 and R2 (2, 3).
The three-dimensional structures of proteins R2 of E. coli
(4) and mouse (5) and of E. coli protein R1 (6) provide a
basis for understanding the enzymatic reaction. A main step in
ribonucleotide reduction, which takes place in the large subunit, protein R1, is the abstraction of the 3'-hydrogen from the ribose moiety of the substrate by a protein-linked radical (probably on
Cys-439) (7, 8). Since Tyr-122 in the small subunit, protein R2 of
E. coli (Tyr-177 in protein R2 of mouse RNR), is deeply
buried inside the protein structure, it cannot take part directly in
the substrate reduction in protein R1. A structure model of the enzyme
(based on the known R1 and R2 structures) shows that Tyr-122 is
connected to the substrate-binding site in protein R1 over a total
length of about 35 Å via a network of conserved hydrogen-bonded amino
acids, the so-called radical transfer pathway (2). It is believed that
coupled electron/proton (H· radical) transfer takes place via
this pathway during the enzymatic reaction (9). The enzyme loses
activity when this network is disturbed, e.g. by mutations
(10, 11).
The tyrosyl radical and its adjacent µ-oxo-bridged diferric iron
center is formed in vitro in the so-called reconstitution reaction, when ferrous iron reacts with apoprotein R2 and molecular oxygen. This reaction, which results in the reduction of molecular oxygen to water, requires four reducing equivalents. Three are provided
by the oxidation of the tyrosyl residue and the two ferrous irons. The
fourth may come from an external ferrous iron ion via the radical
transfer pathway. Schmidt et al. (12) showed recently that
the radical transfer pathway in mouse protein R2 is involved in the
generation of the tyrosyl radical/diferric iron site. In the E. coli protein R2 mutant Y122F, where the tyrosyl radical cannot be
formed, other aromatic amino acids in the vicinity of the diferric iron
center become oxidized, like tryptophans (13, 14) or a tyrosine (15).
These radicals did not yield enzyme activity but show the existence of
side pathways for the radical transfer in protein R2 when Tyr-122 is
not available.
The unique high stability of the protein-linked tyrosyl free radical on
Tyr-122 in protein R2 of E. coli RNR has been discussed since its discovery and spectroscopic assignment in 1972 (2, 16). As
shown by site-directed mutagenesis, the tyrosyl radical might be
protected by a cluster of hydrophobic amino acids, like in E. coli protein R2; changing only one hydrophobic amino acid for a
hydrophilic one leads to a drop in the lifetime of the radical by
several orders of magnitude (17).
In the present study we addressed the question whether another
redox-active amino acid (for example tryptophan) may take over the role
and enzymatic function of a tyrosine. In solution both tyrosine and
tryptophan have similar redox potentials, 0.94 and 1.05 V, respectively
(18). We have constructed protein R2 mutants from mouse and E. coli RNR,2 where the radical
carrying Tyr-177 (122 in E. coli) is replaced by other
oxidizable amino acids with side chains of different sizes, like
tryptophan, cysteine, and histidine or a non-oxidizable amino acid like
phenylalanine. The latter was constructed to look for side pathways or
suppressed radicals as previously observed in E. coli
protein R2 Y122F (13, 14). We have used EPR and ENDOR to study the
paramagnetic species formed in the Fe2+/oxygen
reconstitution reaction of the mouse protein R2 mutants Y177W, Y177C,
and Y177F. The results underline the uniqueness of the tyrosyl radical
in the catalytic reaction of the native enzyme.
Materials--
L-Tryptophan
(indole-d5, 98%) and 57Fe (95.2%)
were purchased from Larodan Fine Chemicals AB, Sweden; D2O,
99.97%, was obtained from Studsvik Energiteknik AB, Sweden. Tris
buffer in D2O was prepared by freeze-drying a solution of
50 mM Tris-HCl, 0.1 M KCl, pH 7.5, and
dissolving it in D2O. This procedure was repeated twice.
Plasmids--
The mouse R2 protein is produced in E. coli by expression from its cDNA using the plasmid pETM2 (19,
48). A new modified plasmid was constructed expressing the mouse R2
mutants Y177W, Y177F, and Y177C. By using the R2-expressing pETM2
plasmid as a template, a 246-base pair DNA fragment was amplified by
the polymerase chain reaction with the mutated primer. The following upstream primers were used: for Y177W, 5'-CAA ATT GCC ATG GAA AAC ATA
CAC TCT GAA ATG TGG AGT CTC CTTA-3'; for Y177F, 5'-CAA ATT
GCC ATG GAA AAC ATA CAC TCT GAA ATG TTC AGT CTC CTTA-3';
and for Y177C, 5'-CAA ATT GCC ATG GAA AAC ATA CAC TCT GAA ATG
TGC AGT CTC CTTA-3'. The downstream primer in each reaction
was 5'-GAG CCA GAA TAT CGA TGC AA-3'. The changing amino acid codon 177 is underlined. The second downstream primer contained the restriction cleavage site of MluI. The resulting PCR fragment was
cleaved with the restriction enzymes NcoI and
MluI and introduced into the plasmid pETM2 cleaved in the
same two unique restriction sites. The new constructions pETM2-Y177W,
pETM2-Y177F, and pETM2-Y177C were verified with dideoxyribonucleotide sequencing.
Expression and Purification of the Mutant R2 Proteins--
The
plasmid pETM2-Y177W (pETM2-Y177F or pETM2-Y177C) was transfected into
E. coli strain BL21(DE3)pLysS that contains an
IPTG-inducible chromosomal copy of the T7 RNA polymerase gene and a
plasmid with the T7 lysozyme gene (20). LB medium (5 liters) containing
carbenicillin 50 mg/ml and chloramphenicol 25 mg/ml was inoculated with
10 ml of overnight cultures, shaken (275 rpm) at 37 °C, and
supplemented with IPTG (0.5 mM) at
A590 = 0.7. After a further 4 h at
37 °C, the cultures were chilled, centrifuged at 2,500 × g for 20 min at 2 °C, and gently resuspended in 100 ml of
50 mM Tris-HCl, pH 7.6. The suspensions were frozen in
liquid nitrogen and stored at
For preparation of protein R2 Y177W with
indole-d5-Trp, the bacteria were grown in a
minimal medium (21) including the antibiotics described above. At the
time for induction with IPTG the medium was supplemented with a
solution of indole-d5-Trp to a final
concentration of 0.003% (w/v). The time between induction with IPTG
and harvest was prolonged to 6 h.
Frozen bacteria were gently thawed at 25 °C in a water bath, and the
viscous solution was cleared by centrifugation at 45,000 rpm for 1 h at 2 °C (Beckman L-90 ultracentrifuge, Ti70 rotor). The purification was performed as described earlier for the mouse R2
recombinant protein (19, 48). The R2 proteins were in the apo form
(metal-free) after purification. Protein purity was analyzed by
SDS-polyacrylamide gel electrophoresis. The protein R2 concentrations were measured by light absorbance and calculated using the molar extinction coefficient E280-310 = 124,000 M
In order to exchange the H2O to D2O buffer 200 µl of apoprotein was kept in 2 ml of 50 mM Tris-HCl, 0.1 M KCl in D2O, pH 7.5, at 5 °C. After 12 h incubation the diluted apoprotein was concentrated with a Centricon
10 (Amicon) (10-kDa exclusion size). When the volume was approximately
200 µl, an additional 2 ml of 50 mM Tris-HCl, 0.1 M KCl in D2O, pH 7.5, were added to the
apoprotein, and the solution was again concentrated to approximately
200 µl. The concentrating procedure was repeated twice.
Reconstitution Reaction of Apoprotein R2 with Fe2+
and Oxygen--
The reconstitution of the iron site in the mutant
proteins R2 in H2O or D2O buffer at room
temperature (22 °C) was carried out as follows: apoprotein R2 in
oxygen-saturated 50 mM Tris-HCl, 0.1 M KCl, pH
7.5, was mixed with an equal volume of an anaerobic (NH4)2Fe(SO4)24H2O
solution (six times molar excess) dissolved in 50 mM
Tris-HCl, 0.1 M KCl, pH 7.5. A rapid freeze quench
technique (System 1000 from Update Instruments) was used to obtain time points from 8 ms to 2 s. Both the aerobic apoprotein R2 and
anaerobic Fe2+ solutions were rapidly mixed and sprayed
into a cold isopentane bath (
The amount of bound iron in protein R2 was colorimetrically determined
after removing unspecifically bound iron by running the protein R2
through a Sephadex G-25 column (23, 24). The apoproteins contained
approximately 0.2 iron ions/protein R2 after purification. An acidic
57Fe stock solution was prepared as described in Sturgeon
et al. (25). The reconstitution of the protein with
57Fe was done as described above except that the protein
was dissolved in 200 mM HEPES, pH 7.5.
EPR and ENDOR Instrumentation--
X band EPR spectra were
recorded on a Bruker ESP 300E spectrometer using a standard rectangular
Bruker EPR cavity (ER4102T) equipped with an Oxford helium flow
cryostat or on a Bruker ESP 380E spectrometer using a Bruker dielectric
ring flex line resonator (ER 4118-X-MD-5W2) equipped with an Oxford
helium bath cryostat. Continuous wave (cw) ENDOR spectra were measured
on a Bruker ESP 300E spectrometer using a self-built ENDOR accessory,
which consists of a Rhode and Schwarz RF synthesizer (SMT02), an
ENI A200L solid state RF amplifier, and a self-built
high-Q TM110 ENDOR cavity (26). The cavity was
adapted to an Oxford helium flow cryostat (ESR 910). The measured
g values were calibrated using the known g value
standard Li/LiF, with g = 2.002293 ± 0.000002 (27). Spin concentrations were determined by comparison of double
integrals of EPR spectra with that of the tyrosyl radical in the mouse
R2 wild type Tyr-177 which had been previously calibrated with a 1 mM Cu2+/EDTA standard. Evaluation of the
microwave power saturation behavior of the radical was done by using
the plot described in Ref. 28. The microwave saturation and the kinetic
curves were fitted using the program Grafit 3.01.
Mass Spectrometry--
The correct incorporation of
isotope-labeled tryptophan into protein R2 was proved by electrospray
ionization or matrix-assisted laser desorption
ionization-time-of-flight mass spectrometry. Approximately 3 nmol of
apoprotein R2 was dissolved in 1 ml of 1% acetic acid and injected
into an liquid chromatography/mass spectrometry-coupled electrospray
single quadruple mass spectrometer (Applied Biosystems, Sciex,
Perkin-Elmer Corp., Canada). For determining the mass by
matrix-assisted laser desorption ionization-time-of-flight (Voyager;
Perspective Biosystems Inc.), approximately 1 pmol of apoprotein R2 was
crystallized in an excess of sinapinic acid as matrix. The following
molecular masses of mouse R2 proteins in Da were determined (calculated
polypeptide molecular mass in parentheses): native R2, 45,101.5 (45,099); Y177W R2 indole-h5-Trp, 45,129 (45,122.6); Y177W R2 indole-d5-Trp, 45155 (45,157.6).
Simulation of EPR Powder Spectra--
The EPR powder spectra
have been analyzed using a program for simulation and fitting of EPR
spectra with anisotropic g and hyperfine tensors based on
the work of Rieger (29). Resonant field positions were calculated to
second order for arbitrary orientations of the g and
hyperfine tensor principal axes (14).
Mouse Protein R2 Mutants--
All three mutated R2 proteins,
Y177W, Y177C, and Y177F, behaved like the native protein R2 during the
growth and purification procedure; the final yield was approximately
4-6 mg of pure protein R2/liter of bacterial culture. The light
absorption spectra of all three mutants after 6Fe2+/R2
reconstitution are shown in Fig. 1. The
absorbance of the mutants is significantly increased in the 300-400-nm
region after the reconstitution reaction. However, the two bands at 320 and 370 nm, which are characteristic for a µ-oxo-bridged diferric iron center like in the active form of the native mouse protein R2
dimer (Fig. 1e), are not resolved in the mutant proteins.
The Met form of native mouse protein R2 (19, 48) and other mouse R2
mutants (10) shows similar badly resolved light absorption spectra in
the iron absorption region as seen for the mutants in Fig. 1,
b-d. Upon reconstitution of Y177W, a broad transient light
absorption band in the range 500-600 nm with a maximum at 550 nm and a
half-width about 60 nm appeared initially (Fig. 1, inset).
This band is completely gone after 2 min reaction time, which
corresponds approximately with the decay of the transient EPR doublet
signal in Y177W R2 (Fig. 3, discussed below). In an earlier work (13)
the light absorption spectrum of a neutral tryptophan radical in
E. coli Y122F R2 was described as a broad band centered at
545 nm. From the similar characteristics of the transient absorption
bands centered at 550 nm in mouse Y177W R2 and E. coli R2
Y122F (13), we assigned the observed transient here to the tryptophan
radical.
Further characterization of the amount of bound iron in the protein R2
mutants by spectrophotometric analysis showed that all three mutants
were able to bind approximately 3.5-4 iron ions/protein R2. In
holoenzyme studies the interaction between native protein R1 and mutant
protein R2 was analyzed by BIAcore studies as described earlier (30).
The binding efficiency of Y177W, Y177C, and Y177F to protein R1 was
about the same as found for native protein R2 (data not shown).
The enzyme activity of the mutated R2 proteins was measured by the
reduction of [3H]CDP in the presence of an excess of pure
recombinant mouse R1 protein and ATP as a positive effector (22).
Neither of the mutated proteins showed any significant activity
compared with native R2 protein (Table
I). As demonstrated earlier and again clearly seen here for the native R2 protein, reactivation of apoR2 protein occurs continuously and with high efficiency during the assay
in the presence of iron-dithiothreitol (31). Therefore, we should have
been able to detect activity even from an R2 protein with a radical
less stable than the normal tyrosyl radical.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C.
1 cm
1 (19, 48). The proteins
were kept and investigated in 50 mM Tris-HCl, 0.1 M KCl, pH 7.5. The enzymatic activity of the native and
mutated R2 proteins was measured in the presence of pure recombinant mouse protein R1 using the [3H]CDP reduction as described
earlier (22).
120 °C) to quench the reaction. The
frozen spray was then tightly packed into an EPR tube (12). For
reaction times >2 s, the anaerobic ferrous iron solution was mixed
with the apoprotein R2 in an EPR tube using a gas-tight Hamilton
syringe. The reaction was stopped by immersing the EPR tube into cold
n-pentane (
120 °C). Radical decay was monitored by
measuring the amount of free radical versus the reaction
time of the sample warmed up to room temperature and refrozen again.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Light absorption spectra of non-reconstituted
apo form (a) and reconstituted mouse R2 proteins Y177F
(b), Y177C (c), Y177W
(d), and native (e) at 10 °C
(22 µM protein R2).
Inset, transient light absorption band upon reconstitution
of Y177W measured after 15 s (a), 2 min (b),
and 5 min (c) reaction time; spectrum recording time,
15 s.
Ribonucleotide reductase activity of native and mutated protein R2
Reconstitution of Protein R2 Y177W with 6Fe2+ and Oxygen
EPR Spectroscopy-- After addition of ferrous iron to aerobic apoY177W and a reaction time of 3 s at room temperature, an EPR spectrum was observed at 40 K with giso = 2.0029 (see Table II) and a dominating doublet hyperfine structure (hfs) (Fig. 2, top). The radical decays with a half-life time of 49 s at 22 °C (Fig. 3). Quantitation of this radical showed a maximum value of 0.34 unpaired spins/protein R2 dimer. The measured microwave power at half-saturation of the new radical at 30 K was at least 1 order of magnitude lower (P50 = 0.2 mW) than that of the tyrosyl radical at Tyr-177 in native protein R2 (P50 = 2-3 mW) at the same temperature, indicating a weaker magnetic interaction of the new radical with the diferric site in R2 Y177W.
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To identify the origin of the new radical in protein R2 Y177W we
exchanged all tryptophans in the protein with selectively deuterated
indole-d5-Trp. The incorporation of
indole-d5-Trp into protein R2 Y177W was checked
by mass spectroscopy (see "Experimental Procedures"). A comparison
with the calculated molecular mass of Y177W R2
indole-d5-Trp shows that the isotopically
labeled amino acid has been incorporated properly into the protein. A significant change of the hfs of the EPR spectrum was observed with the
mutant R2 Y177W containing ring-deuterated tryptophans, indole-d5-Trp (Fig. 2, bottom). The
small sub-splittings of each doublet component (Fig. 2, top)
into three lines disappeared in the
indole-d5-Trp labeled sample, proving that these
lines resulted from a hyperfine interaction with the ring -protons
of Trp. Furthermore, well resolved lines became visible at the outer
wings of the spectrum (Fig. 2, bottom). These lines are
assigned to the Az component of an anisotropic
hyperfine powder pattern from a 14N coupling. A similar
line shape was observed also for tryptophan radical Trp-111· in
mutant Y122F of E. coli R2 (14).
The dominating doublet splitting of 2.25 mT (Fig. 2, top),
which is not affected by indole-d5-Trp labeling
is assigned to a large isotropic hfc of a -proton. In the spectra
simulations, the best fit of the spectrum in Fig. 2, bottom,
was obtained by using a large isotropic proton hfc
(Aiso = 2.25 mT) and an axially symmetric
hyperfine tensor of 14N (Ax = 0.07 mT,
Ay = 0.07 mT, and Az = 0.94 mT,
see Table II). Due to the large Az tensor component
of the 14N hyperfine tensor, we expect a significant spin
density on the nitrogen of about 20%. An N-H proton coupled to the
indole nitrogen, as would be expected for a tryptophan cation radical,
would exhibit a hyperfine tensor component of about 0.5 mT (32). Such a
large coupling is, however, not observed in the EPR spectrum of Y177W indole-d5-Trp in H2O buffer (Fig. 2,
bottom). Therefore, we conclude that there is no NH proton
involved in the hfs. The observed tryptophan radical must therefore be
neutral with the nitrogen atom deprotonated.
ENDOR Spectroscopy-- cw-ENDOR experiments were performed at X band on Y177W with indole-h5-Trp or Y177W indole-d5-Trp in H2O or in D2O buffer. The ENDOR resonance condition is given to first order by Equation 1.
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(Eq. 1) |
The large hfcs of the -proton and the Az
component of the nitrogen were already determined by EPR. We attempted to observe ENDOR lines corresponding to the large
-proton coupling, which are expected at 17.6 and 45.4 MHz, but with no success. For such
large
-proton couplings large ENDOR line widths are expected, in
particular for methylene protons for which a variation of the side
chain geometry leads to a distribution of the observed hyperfine
couplings (see Equation 2). This effect and the low radical yield of
0.34 in Y177W reduce signal amplitudes for these couplings beyond detection.
The smaller hfcs, which are not resolved in EPR, were obtained from the
ENDOR spectra. Fig. 4A shows
the X band cw-ENDOR spectrum of Y177W with
indole-h5-Trp in H2O buffer measured
at 8 K. The spectrum is characterized by a strong matrix signal at the
proton Larmor frequency H (13.9 MHz) and several pairs of
lines that are symmetrically placed around 13.9 MHz and discussed
below. Another pair of rather broad lines, at 4.8 MHz and at 23 MHz, yielding a proton hfc corresponding to 0.65 mT, is absent in the ENDOR
spectrum of Y177W with indole-d5-Trp in
H2O buffer (Fig. 4B). We conclude that this
coupling represents one hyperfine tensor principal value of one (or
two) indole ring
-protons. Proton ENDOR signals corresponding to the
other hyperfine principal values were not observed. This is due to the
expected large hyperfine anisotropy of
-protons, which broadens the
ENDOR signals in frozen solution beyond detection.
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Corresponding hyperfine components are, however, observed for deuterons
at the ring -positions in Y177W with
indole-d5-Trp. Fig. 4B shows
additional lines between 2 and 4 MHz. These signals belong to
-deuterons of the indole ring of ring-deuterated tryptophan. Their
positions are shifted by a factor of 6.5 to lower frequencies, and
their ENDOR spectrum is compressed by the same factor in comparison with the
-protons in normal tryptophan due to the smaller magnetic moment of D as compared with H. The amplitudes of
the ENDOR signals are correspondingly increased. The lines are
symmetrically spaced around the deuterium Zeeman frequency,
D = 2.1 MHz. Only the high frequency part of the
deuterium-ENDOR spectrum appears at useful ENDOR frequencies (>1.5
MHz, Fig. 4B, see inset 2.1-5 MHz with expanded
frequency scale). Three couplings (A1 = 0.6 MHz,
A2 = 2.1 MHz, and A3 = 2.8 MHz) have been obtained. The second component is seen mostly as an
inflection on the third component (Fig. 4, B and
C). The couplings were assigned to the three principal values of one hyperfine tensor, since for
-deuterons, as for
-protons, a rhombic hyperfine tensor is expected. The values for the
corresponding
-proton are 3.9, 13.7, and 18.2 MHz or 0.14, 0.49, and
0.65 mT. The largest value agrees well with that obtained from the
proton-ENDOR spectrum of the protein with
indole-h5-Trp (see above, and Fig.
4A). This interpretation is supported by ENDOR simulations
(dotted traces in Fig. 4A, 15-31 MHz, and Fig. 4C, 2.1-5 MHz) using solely the tensor principal values of
the two
-protons and assuming an inequivalence for the two smaller components of both tensors within the error margins given in Table II.
The simulations show that for this case the largest component gives
rise to the most intense peak as is observed in the proton- and
deuterium-ENDOR spectra.
The three -deuteron and derived proton hyperfine tensor principal
values agree remarkably well with those obtained earlier for the
tryptophan neutral radical Trp-111· in E. coli
protein R2 Y122F (14), see Table II. In this earlier work the tensor
principal values were corroborated by EPR simulations and by
McConnel-Strathdee based dipolar tensor calculations accounting for all
carbon and nitrogen spin densities in the radical (14). The EPR
simulations performed for Trp-177· (Fig. 2, top)
showed, as in the earlier work (14), that two
-protons have to be
assigned to the observed three hyperfine tensor values and that their
hyperfine tensor axes of the largest value (0.65 mT) are rotated by
90° relative to each other in order to obtain the observed pattern in
the experimental spectrum (see Table II). Based on earlier density
functional calculations, these two proton hyperfine tensors were
assigned to ring protons H-5 and H-7 (14) (see Fig.
5 for numbering of tryptophan ring). Since the largest hyperfine tensor component of an
-proton is expected for the magnetic field oriented perpendicular to the C-H bond
and in the tryptophan ring plane, the largest component (0.65 mT) is
assigned to Ax (H-5) and Ay (H-7) and the smallest component (0.14 mT) to Ay (H-5) and Ax (H-7), whereas Az is assumed
to be similar (0.49(6) mT) for both
-protons (compare Fig. 4 and
Table II).
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The ENDOR spectra of Fig. 4, B and C, show two
lines at 11.8 and 16.0 MHz corresponding to a 4.2-MHz coupling which is
not affected by indole-D5-Trp nor by D2O
buffer. Since a proton weakly coupled to the nitrogen would exchange in
D2O buffer, we assign this small coupling of 0.15 mT to the
second methylene -proton. This coupling is not resolved in the EPR
spectrum in Fig. 2, bottom.
Exchangeable proton hfcs were not observed in the EPR spectra. However,
from the ENDOR difference spectrum Y177W
indole-d5-Trp in H2O
minus Y177W indole-d5-Trp in
D2O buffer (Fig. 4D), three small hyperfine
tensor values of 0.12, +0.18, and
0.06 mT were obtained and
assigned to one exchangeable proton. Previous ENDOR experiments on the
neutral tryptophan radical Trp-111· in E. coli Y122F
yielded very similar tensor components of an exchangeable proton of
0.12, +0.19, and
0.08 mT. Based on spin density and dipolar
hyperfine tensor calculations, they were assigned to a hydrogen-bonded
proton at the indole nitrogen (14). Since the tryptophan radical in
Y177W exhibits a very similar 14N hyperfine tensor as
Trp-111· in Y122F, we likewise assign the three values
0.12,
+0.18, and
0.06 mT to the three hyperfine tensor principal values of
a proton hydrogen-bonded to the nitrogen in the tryptophan indole ring (for signs, see Table II, legend and footnotes).
Reconstitution of Y177F and Y177C R2 with 6Fe2+ and Oxygen
EPR Spectroscopy--
When aerobic apoprotein R2 Y177F was mixed
with 6Fe2+ at 22 °C, an EPR singlet signal at
g = 2.00 with a peak-to-peak line width of 1.8 mT was
observed at 20 K. Fig. 6A
shows this signal when the reaction was quenched after 3 s. A very
similar EPR singlet signal, with regard to g value, line
width, and microwave saturation behavior, appeared also after
reconstitution reaction in protein R2 Y177C under the same conditions
(Table III). The EPR singlet in Y177F
appeared with a rate constant of about 0.5 s1 and decays
with a rate of 0.25 s
1. The EPR singlet in Y177C had
similar kinetics and appeared with a rate of 0.48 s
1 and
decayed with a rate of 0.23 s
1. Both EPR singlets had
completely disappeared after 20 s reaction time (Fig.
7). Upon reconstitution with apoprotein
Y177F and 57Fe, we observed a doublet splitting
(A = 2.6 mT) of the EPR signal due to the nuclear spin
of 1/2 for 57Fe (Fig. 6C). A similar transient
EPR signal has also been observed in the reconstitution of wild-type
and Y122F protein R2 of E. coli (34-36) and native mouse R2
(12) and has been assigned to an oxo (hydroxo)-bridged Fe(III)/Fe(IV)
cluster, intermediate X (25). For comparison, Fig. 6,
B and D, shows the corresponding spectra of
E. coli R2 Y122F after a freeze-quenched reaction with 56Fe and 57Fe, respectively. The dotted
trace is a simulation. Microwave saturation studies of the singlet
EPR spectrum in mouse Y177F R2 and of intermediate X in
E. coli Y122F R2 gave coinciding values of
P50, 0.02 mW at 5 K and 0.2 mW at 20 K (data not
shown). From the almost identical g values, line shapes,
microwave saturation behavior, and 57Fe hfcs, we assign
also the transient EPR signals in Y177F and Y177C to such a bridged
Fe(III)/Fe(IV) cluster, similar to intermediate X observed
earlier in E. coli protein R2.
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DISCUSSION |
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Among free radical enzymes (3), RNR from E. coli was the first enzyme in which a protein-linked tyrosyl free radical was identified. In a kinetic study of the formation of the tyrosyl radical, Bollinger et al. (36) have shown that an intermediate X, an Fe(III)/Fe(IV) cluster with spin delocalization, serves as precursor to the tyrosyl radical in the E. coli protein R2. When the tyrosyl radical cannot be formed due to a mutation like in E. coli protein R2 Y122F, intermediate X becomes longer lived and can oxidize other neighboring aromatic residues like tryptophans (13, 37). Two tryptophan radicals (located at Trp-111 and Trp-107) have previously been studied in detail and assigned to oxidized neutral radicals (13, 14).
An Oxidized Neutral Tryptophan Radical on Trp-177--
We have
observed a transient tryptophan radical in mouse protein R2 upon the
reconstitution of Y177W with ferrous iron and oxygen. Its light
absorption centered around 550 nm agrees with reported properties of
photochemically produced tryptophan radicals (38, 49). Its dominant EPR
doublet signal arises from the hyperfine splitting of one -methylene
proton, whereas two
-protons at the indole ring cause the small hfs
as seen in Fig. 2, top. In addition to the proton hfcs, the
full 14N hyperfine tensor was obtained with the help of EPR
simulations (see Fig. 2 and Table II). Finally, ENDOR difference
spectra of Y177W with indole-d5-Trp in
H2O and D2O revealed the hyperfine tensor of an
exchangeable proton assigned to a hydrogen-bonded proton at the
indole-nitrogen.
The tryptophan radical in Y177W can now be compared with the previously
reported oxidized neutral tryptophan radical at Trp-111 in E. coli R2 Y122F (13, 14). The tryptophan radical in Y177W exhibits
only one large -proton hfc. The hfc of the second
-proton is very
small and only resolved in the ENDOR spectrum. In the tryptophan
radical Trp-111· of Y122F, there are two large
-proton hfs.
This indicates a difference in the dihedral angles of the
-methylene
protons for the two tryptophan residues, whereas the spin densities in
the indole rings are very similar.
The spin density on C-3 in the tryptophan radical in Y177W can be
estimated from the observed hfcs of the -protons using the empirical
relationship (Equation 2)
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(Eq. 2) |
Another common feature of both tryptophan radicals in mouse R2 Y177W
and E. coli R2 Y122F is that they exhibit a rather small g anisotropy (gx gz
8 × 10
4, see Table II). This is typical for
radicals with high spin densities only on carbon and nitrogen atoms,
like tryptophan. For comparison, tyrosyl radicals with a high spin
density on an oxygen atom (larger spin-orbit interaction than nitrogen
or carbon) exhibit a larger g anisotropy (for native mouse
R2 gx
gz = 54 × 10
4 (42)). It should also be pointed out that in these
two mutant R2 proteins we have established that protein-linked neutral
tryptophan radicals exhibit a light absorption band centered around 550 nm (Fig. 1, inset, and see Ref. 13).
Which is the site of the observed oxidized neutral tryptophan radical
in protein R2 Y177W? Including Trp-177, there are 7 tryptophans per
polypeptide chain in mouse R2 Y177W. We expect the transient radical to
be located relatively close to the iron site (cf. Ref. 13),
and we expect the geometry of the -protons with respect to the
indole plane (dihedral angles
) as derived from the crystal
structure (5) to agree with the experimental observations. Tryptophans
103, 214, 210, 92, 246, and 117 have distances to their indole-nitrogen
from Fe2 (the iron ion most distant from Tyr-177) of 11, 15, 17.4, 21, 24.7, and 25.5 Å, respectively. Only tryptophans 92 and 246 have
dihedral angles of the
-methylene protons
1 =
27°,
2 = 93°, and
1 =
37°,
2 = 83° (5), which agree within ±10° with those
obtained for the tryptophan radical in Y177W (
30 and 90°). Trp-92
and Trp-246 are, however, 21 and 24.7 Å from the Fe2 atom, far away
from the iron site. Tryptophans 111 and 107 in E. coli,
which carry radicals in the mutant Y122F (13, 14), are much closer to
the iron site (5 and 7 Å). The closest tryptophan to the iron site in
native mouse R2 (Trp-103) is 11 Å from Fe2, but its dihedral angles
(
1 =
43° and
2 = 77°) do not agree
well with the experimental data. Therefore, we assign the observed
neutral tryptophan radical to residue Trp-177, which should be the
tryptophan closest to the iron site. Reconstitution with
57Fe did not change the EPR spectrum of the radical (data
not shown), indicating only weak interaction with the iron site like in
native enzymes with tyrosyl radicals (43) or the Trp-111·
radical in E. coli Y122F (13). The Trp-177 radical is not
enzymatically active (Table I).
The Long-lived Intermediate X in Protein R2 Mutants Y177F and Y177C-- The reconstitution of apoY177F and Y177C R2 with an excess of Fe2+ showed the formation of an S = 1/2 paramagnetic species. The X band EPR spectrum of 57Fe-labeled R2-Y177F protein could be successfully simulated using the g tensor and the two hyperfine tensors of 57Fe from the Q band studies of intermediate X (25) (Fig. 6D). We therefore assigned the intermediates in mouse R2 Y177F and Y177C to Fe(III)/Fe(IV) clusters, like intermediate X in E. coli R2, with similar structures as reported for intermediate X in E. coli R2 Y122F (25). However, the intermediates X in the two mouse mutants differ kinetically from that in E. coli Y122F in the rates of formation and decay, i.e. in their time window of appearance. Intermediate X in E. coli R2 Y122F became reduced after 2 s at 25 °C, whereas in the two mouse mutants intermediates X are still detectable after 10 s reaction time at 22 °C (Fig. 7).
In E. coli R2 Y122F intermediate X becomes reduced by the oxidation of the non-conserved tryptophans 111 and 107 (13). E. coli Trp-111 and Trp-107 correspond to Gln-167 and Phe-163, respectively, in mouse protein R2, which cannot be easily oxidized and may explain the missing successor radicals in Y177F. Besides Tyr-177 in native mouse protein R2, there are no other aromatic amino acids in the close vicinity of the iron site, which can reduce intermediate X. This may be the reason for its long lifetime.
Whereas phenylalanine in Y177F is not expected to be oxidized, cysteine in Y177C was expected to form a cysteine radical upon reconstitution. The crystal structure of the analogous mutant E. coli R2 Y122C shows a much larger distance of the -SH group to Fe1, the iron ion closest to Cys-122, (8.9 Å) than the OH group of tyrosine 122 in E. coli wild type R2 (5.3 Å) and a hole at the site of the missing phenyl ring.3 In neither of the R2 mutants E. coli Y122C and mouse Y177C did we detect any EPR signal at 77 K in the time window of 8 ms to 20 s (concomitant with the decay of X) up to 20 min reaction time at room temperature after the reconstitution. We have to conclude that either due to the larger distance between Cys-177 and Fe1 no cysteine radical has been formed or, if it was formed, it is short lived, or it has a low signal intensity caused by its large g anisotropy (44). Functionally related cysteine-based radicals have been detected in a class II RNR with strong magnetic interaction with a cobalt complex (45) and very recently in an E. coli protein R1 mutant with a lifetime of several seconds (46).
Concluding Remarks--
The present study shows that the
iron/oxygen reconstitution reaction in protein R2 is able to create
protein-linked free radicals in the close vicinity of the diferric iron
center in mouse protein R2. When the tyrosine 177 is mutated for
another redox-active amino acid with a suitable side chain and an
appropriate redox potential, like tryptophan, this residue can be
oxidized. Despite the formation of a transient tryptophan radical, the
loss of the enzymatic activity in all three mutants, Y177W, Y177F, and
Y177C, suggests strongly that the tyrosyl radical 177 cannot be
replaced by other amino acids. The long lifetime and high relative
yield of intermediate X in mouse Y177F and Y177C mutants
with no observable successor radicals may facilitate further studies of
the properties of this important intermediate in iron-oxygen chemistry
of diiron-oxygen proteins.
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ACKNOWLEDGEMENT |
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We thank Per Ingvar Olsson of the Department of Medical Chemistry and Biophysics, Umeå University, for providing the results of the mass spectrometric analysis.
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FOOTNOTES |
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* This work was supported by grants from the Deutscher Akademischer Austauschdienst (to S. P.), the Swedish Natural Science Research Council (to A. G.), the C. Trygger Foundation (to A. G.), Deutsche Forschungsgemeinschaft DFG:La 751-2/1 (to F. L. and G. L.), and Fonds der Chemischen Industrie (to G. L. and W. L.).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.
§ Present address: Institute for Physiological Chemistry and Biochemistry, University of Mainz, D-55099 Mainz, Germany.
** To whom correspondence should be addressed. Tel.: 46-8-162450; Fax: 46-8-155597; E-mail: astrid{at}biophys.su.se.
2 S. Pötsch, F. Lendzian, R. Ingemarson, A. Hörnberg, L. Thelander, W. Lubitz, G. Lassmann, and A. Gräslund, manuscript in preparation.
3 S. Pötsch and P. Nordlund, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
RNR, ribonucleotide
reductase;
cw, continuous wave;
EPR, electron paramagnetic resonance;
ENDOR, electron nuclear double resonance;
hfc, hyperfine coupling;
hfs, hyperfine splitting;
IPTG, isopropyl-1-thio--D-galactopyranoside;
mW, milliwatt;
mT, millitesla.
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REFERENCES |
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