(Received for publication, January 6, 1997, and in revised form, February 12, 1997)
From the Reconstitution of the tyrosyl radical in
ribonucleotide reductase protein R2 requires oxidation of a diferrous
site by oxygen. The reaction involves one externally supplied electron
in addition to the three electrons provided by oxidation of the Tyr-122
side chain and formation of the µ-oxo-bridged diferric site.
Reconstitution of R2 protein Y122F, lacking the internal pathway
involving Tyr-122, earlier identified two radical intermediates at
Trp-107 and Trp-111 in the vicinity of the di-iron site, suggesting a
novel internal transfer pathway (Sahlin, M., Lassmann, G.,
Pötsch, S., Sjöberg, B.-M., and Gräslund, A. (1995)
J. Biol. Chem. 270, 12361-12372). Here, we report the
construction of the double mutant W107Y/Y122F and its three-dimensional
structure and demonstrate that the tyrosine Tyr-107 can harbor a
transient, neutral radical (Tyr-107·). The Tyr-107·
signal exhibits the hyperfine structure of a quintet with coupling constants of 1.3 mT for one Three classes of ribonucleotide reductases can be
distinguished on the basis of their composition and cofactor
requirements, but they have in common the generation of protein-linked
radicals during reduction of ribonucleotides to the corresponding
deoxyribonucleotides (1, 2). The class I ribonucleotide reductase of
Escherichia coli consists of two homodimeric proteins
denoted R1 and R2. The larger R1 protein contains the catalytic site
with redox-active cysteines, whereas the function of the smaller R2 is
to store a stable tyrosyl radical (Tyr-122· in E. coli) that is essential for catalysis (3). Tyr-122· is
formed by oxidation of an adjacent iron site and stabilized by the
resulting µ-oxo-bridged diferric center. Tyr-122 is located 10 Å from the surface and 5.3 Å from the closest iron (4).
Reduction of substrate by ribonucleotide reductase is proposed to
happen via a radical mechanism involving the formation of a thiyl
radical at cysteine 439 (E. coli numbering) at the substrate binding site in R1. This raises the question how a radical is transferred from Tyr-122 in R2 to Cys-439 in R1 and vice versa. By
means of separately solved crystal structures of R1 and R2 and
site-directed mutagenesis experiments, an electron transfer pathway, of
about 35 Å in length, has been suggested, leading from the assembled
cofactor in R2 to the active site in R1 (5). We prefer to call it a
radical transfer pathway (RTP),1
i.e. transfer of a hydrogen atom (radical), as the pathway
is hydrogen-bonded, and all radicals hitherto observed in protein R2
are of the oxidized, neutral form (6-9). The participating amino acids
are conserved among all class I ribonucleotide reductase species
described so far. The radical transfer in R2 is presumed to occur via
Trp-48, situated on the R2 protein surface, the hydrogen-bonded Asp-237
and His-118, a ligand of Fe-1 (see Fig. 1). In the mobile carboxyl-terminal part of R2, which interacts with R1, the invariant residues Tyr-356 and possibly Glu-350 might connect Trp-48 in R2
with Tyr-731 in R1, which is linked via Tyr-730 to Cys-439 in the
active site (3, 10).
ApoR2, lacking both iron center and tyrosyl radical, can be obtained
either by reducing the radical and chelating the iron or by growing the
overproducing bacteria in iron-depleted medium (11, 12). The iron
center and the tyrosyl radical assemble spontaneously in a
reconstitution process involving apoprotein R2, ferrous iron, and
molecular oxygen (Equation 1).
Department of Molecular Biology,
-methylene proton and 0.75 mT for each
of the 3 and 5 hydrogens of the phenyl ring. Rapid freeze quench
kinetics of EPR-visible intermediates reveal a preferred radical
transfer pathway via Trp-111, Glu-204, and Fe-2, followed by a proton
coupled electron transfer through the
-interaction of the aromatic
rings of Trp-(Tyr-)107 and Trp-111. The kinetic pattern observed in
W107Y/Y122F is considerably changed as compared with Y122F: the
Trp-111· EPR signal has vanished, and the Tyr-107· has
the same formation rate as does Trp-111· in Y122F. According to
the proposed consecutive reaction, Trp-111· becomes very short
lived and is no longer detectable because of the faster formation of
Tyr-107·. We conclude that the phenyl rings of Trp-111 and
Tyr-107 form a better stacking complex so that the proton-coupled
electron transfer is facilitated compared with the single mutant.
Comparison with the formation kinetics of the stable tyrosyl radical in
wild type R2 suggests that these protein-linked radicals are
substitutes for the missing Tyr-122. However, in contrast to
Tyr-122· these radicals lack a direct connection to the radical
transfer pathway utilized during catalysis.
Fig. 1.
A shows the structure of the wild type
R2 protein and B the structure of R2 mutant protein
W107Y/Y122F. Here only the residues in the vicinity of the di-iron site
that are believed to be involved in the reconstitution reaction are
depicted. At position 107 and 111 transient radicals are generated in
mutant R2 proteins lacking the essential Tyr-122. Iron ligand Glu-238
has been excluded for clarity. The water ligands of the iron center are
not shown. Hydrogen bonds to the ligands of the iron center are
indicated as dashed lines (see text). (Figure drawn with
Bobscript, a modified version of the program, Molscript (19) by R. Esnouf (personal communication), and Raster3D (43).)
[View Larger Version of this Image (28K GIF file)]
The reduction of oxygen to water requires four electrons. Two of
them are provided by the two Fe2+ forming the diferric
center and one by Tyr-122 forming the tyrosyl radical, and, at least
in vitro, the fourth electron can be supplied by external
reductants such as Fe2+ or ascorbate (13, 14). The
reconstitution reaction thus provides a possibility to trap and
characterize radical intermediates, and one
FeIII-FeIV intermediate and several other
intermediates have been observed prior to generation of the oxidized
di-iron site in wild type and mutant protein (9, 14-18). In addition,
recent kinetic investigations on wild type and mutant mouse R2 support
the use of the RTP for bringing in an external electron/H+
pair.2
(Eq. 1)
Mutation of residue Tyr-122 to the non-oxidizable residue phenylalanine
causes a deficit of one electron in the reconstitution reaction which
consequently must be supplied from elsewhere. Although it is possible
that one more electron could be delivered via the catalytically
essential RTP described above, it has been found that it may come at
least to some percentage from other oxidizable amino acids in the
vicinity of the iron center that are part of the hydrogen-bonded
network of the iron ligands. Transient radicals have been assigned to
the non-conserved tryptophans 107 and 111 based on their EPR and
electron nuclear double resonance characteristics and molecular
geometry (9, 15, 20). The conserved Trp-48 was proposed as a putative
third candidate based on comparison with tryptophan radical signals in
other proteins (9, 21, 22). Trp-107 is at 8 Å distance from Fe-2 and
hydrogen-bonded via a water molecule to the backbone of Fe-2 ligand
His-241 (Fig. 1). The transient neutral Trp-107· was observed as
a room temperature doublet EPR signal indicating relatively weak
interaction with the iron center (9, 20). Trp-111 is at 4 Å distance
from Fe-2 and hydrogen-bonded via N-1 to Fe-2 ligand Glu-204 (Fig. 1).
The transient Trp-111· was observed as a low temperature (77
K) EPR quartet with a weakly coupled proton and relatively strong
interaction with the iron center (9, 20).
In this report we have determined the three-dimensional structure of the double mutant W107Y/Y122F and compared transient radical kinetics in this double mutant with the single mutant Y122F during the reconstitution reaction. From the results we propose a pathway through which an electron/proton pair is delivered by the oxidizable amino acids surrounding the iron site.
[2H],
-Tyrosine was prepared as described
elsewhere (23, 24).
The oligonucleotide used for
mutagenesis (mismatches are underlined) W107Y
d(5-CTGGAAACCGTCGAAACC-3
) was synthesized and
purified by Scandinavian Gene Synthesis AB. The start plasmid to
construct the double mutant was pMK5. Plasmid pMK5 is a recombinant derivative of pTZ18R, containing the nrdB gene with a Y122F
mutation (25). Single-stranded sequencing primers, 6141 d(5
-CAGCTCTCTTTCTTC-3
), Pb1 d(5
-CGCTGCTGGATTCCATTCAGGG-3
), Pb2
d(5
-TGCGGAAGGGATCTCCAGC-3
), and Å1 d(5
-GCAGAACGCGAATTG-3
) were
synthesized and purified by Scandinavian Gene Synthesis AB, and
universe
40 d(5
-GTTTTCCCAGTCACGAC-3
) was purchased from United
States Biochemical Corp. The mutagenesis was performed using the
MutaGene Phagemid in vitro Mutagenesis kit from Bio-Rad,
based on the method described by Kunkel and coworkers (26, 27).
Transformants were screened by dideoxynucleotide chain termination
sequencing across the mutated site.
E. coli CJ236
(dut-1, ung-1, thi-1,
relA/pCJ105 Cmr) and E. coli MV1190
((lac-proAB), thi, supE,
(srl-recA)306::Tn 10/F
traD36, proAB, lacIqZ
M15),
obtained from Bio-Rad were used for mutagenesis and cloning. E. coli MC1009: (
(lacIPOZYA)X74, galE,
galK, strA,
(ara-leu)7697, araD139, recA, srl::Tn10)
was obtained from Pharmacia. Overproduction of mutant proteins from
pMK5 derivatives was obtained in MC1009 containing plasmids pGP1-2
obtained from Tabor and Richardson (28).
The mutant R2 proteins Y122F and
W107Y/Y122F were purified as described earlier (29). Apo forms of Y122F
and W107Y/Y122F were obtained by growing E. coli in
iron-depleted medium as described in Ref. 12 but modified slightly by
omitting the iron chelation procedure. Protein prepared in this way was
typically 90% pure and contained 0.2 eq of Fe per R2 as determined by
iron analysis with bathophenanthroline (30). The deuterated apoprotein
W107Y/Y122F was prepared by using depleted medium and adding the
labeled [2H],
-tyrosine at the time of heat
induction. Wild type apoR2 was obtained by chelation of the purified
protein with the agent Li-8-hydoxyquinolinesulfonate in the presence of
hydroxylamine (11). The specific activity of the R2 wild type protein
was 5000-7000 nmol min
1·mg
1, and the
mutant proteins were devoid of enzymatic activity.
Crystals of R2 mutant Y122F/W107Y were grown as
described previously (31). A crystal measuring approximately 1.0 × 0.3 × 0.3 mm was transferred from crystallization mother
liquor containing 20% PEG 4000, 0.2 M NaCl, 1 mM ethyl mercury thiosalicylate in 0.1 M MES
buffer, pH 6.0, to an almost identical solution containing 24% PEG
4000 and an additional 20% glycerol as cryoprotectant. After less than
1 min in this solution, the crystal was flash frozen in a stream of
liquid nitrogen at 170 °C and maintained at
160 °C for the
duration of the data collection.
The crystal diffracted to 1.95 Å on a rotating anode laboratory x-ray
source. Diffraction data were collected at two points, exploiting the
length of the crystal. The data set collected was 91% complete to 1.95 Å with Rmerge(I) = 0.092. In the highest resolution shell
(1.98-1.95 Å) the data were 73% complete, with Rmerge(I) = 0.354. The average I/(I) was 13.2, in the highest resolution shell
2.4. Reflection intensities were integrated using Denzo (32) and
reduced using Scalepack (32).
The starting model for refinement was the
structure of reduced R2 determined at 100 K (33). The occupancies of
the side chains of residues Tyr-122 and Trp-107 were set to zero before beginning refinement. The program TNT was used for all refinement (34).
A cross-validated R factor (35) was calculated on 5% of the data (2418 reflections). The initial model had Rmodel = 0.213 and
Rfree = 0.221 to 3.5 Å. Three cycles of rigid body
refinement at 3.5 Å followed by 28 cycles of restrained atomic
coordinate and B factor refinement at 1.95 Å gave Rmodel = 0.207 and Rfree = 0.275. Only minor adjustments of the
model at the di-iron center and refinement of the occupancies of
mercury atoms were carried out. Water molecules from the initial model
were retained: any refining with a B factor >60 Å2 was
removed, and new water molecules were placed at peaks above 4 in
difference electron density maps at plausible hydrogen-bonding positions. The program Quanta (Molecular Simulations Inc., Burlington, MA) was used for this and other model building operations.
After 15 cycles, clear difference and 2|Fo| |Fc| density was seen for a phenylalanine at
position 122 and a tyrosine at position 107 of both monomers. These
were refined as such from that point. The refined B factors of all four
iron atoms were higher than usual with respect to their surroundings, but more normal values could be obtained by setting their occupancy to
0.8. After 31 cycles, the reflections used for cross-validation were
returned to the refinement and a further 7 cycles gave
Rmodel = 0.208. The final model contains 5576 protein
atoms, 267 water molecules, 4 Fe atoms, and 14 partially occupied Hg
atoms and has good geometry: r.m.s. deviations from the ideal bond
lengths and angles of Engh and Huber (36) are 0.011 Å for bonds,
1.97° for angles, and 16.9° for torsion angles. All residues are in allowed regions of the Ramachandran plot.
All kinetic reactions
were performed at 25 °C by rapidly mixing equal volumes of an
aerobic solution of apoR2 in 50 mM Tris-HCl, pH 7.6, with
an anaerobic solution of
(NH4)2Fe(SO4)2
dissolved in 50 mM Tris-HCl, pH 7.6. To keep the iron to
protein ratio at 4:1 we generally used 80-100 µM apoR2
protein and 320-400 µM Fe2+. The protein
concentration was determined by absorbance at 280 nm
((280-310) = 120 mM
1·cm
1) using a Perkin-Elmer
Lambda 2 spectrophotometer. The iron content after reconstitution was
measured colorimetrically (30) after removing the unspecifically bound
iron by desalting the R2 protein on a G-25 column.
Rapid freeze quenching (System 1000, Update Instrument) was used to
obtain time points from 700 ms to 2 s. The solutions were mixed
rapidly and sprayed into a 120 °C isopentane bath to quench the reaction. The resulting crystals were packed in an EPR
tube.2 Longer reaction times (5 s to minutes) were achieved
by mixing the solutions directly in the EPR tube and freezing them in
isopentane at
120 °C.
For kinetics at room temperature (20 °C, not thermostated) the EPR spectrometer was coupled to a stopped flow accessory as described previously (37). The transient species was detected by starting a rapid scan after stopping the flow. The field was adjusted at the maximum of the first derivative line of a transient to measure the kinetics. The dead time is about 10 ms and a base line recorded under exactly the same conditions was subtracted.
EPR spectra at 9 GHz were recorded on a Bruker ESP 300E spectrometer at 8 K equipped with an Oxford Instrument liquid helium cryostat or at 77 K using a cold finger Dewar. Spin quantification was obtained with a Cu2+-EDTA sample (1 mM Cu2+, 10 mM EDTA) and as secondary standard an active wild type E. coli R2 protein (1.5 mM radical) by comparing the double integrals and signal intensities. Microwave saturation data were analyzed as described in Ref. 38 using GraFit 3.01® assuming inhomogeneous broadening, i.e. b = 1. The parameter P1/2 describes the microwave power at half-saturation. Simulation of the X-band EPR spectrum of Tyr-107· was made as described before (7) using iteration minimization to fit the spectrum (39).
Stopped-flow reconstitution experiments were done with a BioSequential stopped-flow ASVD spectrofluorimeter from Applied Photophysics. The obtained kinetic traces were fitted with the evaluation software DX.18MV using the double exponential equation.
Evaluation of KineticsThe kinetics of Tyr-107· and
Trp-111· in W107Y/Y122F and Y122F were obtained by partial
subtraction of the EPR spectra of the long lived axial spectrum
(component I in Ref. 9), as well as of the early formed singlet
spectrum from species X ((14), 200-700 ms, not shown in this work).
The transient radicals, which exhibit quintet (W107Y/Y122F) or quartet
(Y122F) EPR spectra at low temperature, respectively, were detectable
at 500 ms. They showed formation and decay phases indicating that
they are part of a consecutive reaction. The rate constants of the data
obtained by slow freeze and stopped flow EPR experiments were evaluated with Equation 2 for an intermediate of a consecutive reaction using
GraFit 3.01® including a "lag time."
![]() |
(Eq. 2) |
F(t) is the time-dependent ratio of radical to R2 dimer. [Trp·]/[R2] is the maximal yield of the intermediate; k1 is rate of formation; k2 is the rate of decay, and dt the lag time.
The structure of W107Y/Y122F is generally
well resolved at high resolution (1.95 Å) with good geometry (Fig.
1B). The electron density around residues 122 and 107 of both monomers (Fig. 2) establishes
unambiguously that the mutations to phenylalanine and tyrosine have
occurred. Tyrosine 107 has almost the same geometrical arrangement as
tryptophan 107 in the wild type protein. The dihedral angles,
H, of the
-methylene protons as defined by the
orientation to the pz axis of tyrosine 107 (perpendicular to the ring plane) are 40 and 80°. Two µ-oxo-bridged
di-iron sites are observed. However, the iron atoms do not appear to be
fully occupied, and a rough estimate of their occupancy based on
crystallographic B factors is 0.8. Perhaps for this reason the density
is poor for two of the coordinating side chains in both monomers,
namely Glu-238 and Glu-204. However, Glu-204 clearly has monodentate coordination, and since analysis of the radical transfer pathway presented herein does not depend on the coordination mode of Glu-238, we leave further analysis of the iron center for a future study.
Formation of the µ-Oxo-bridged Diferric Center in W107Y/Y122F
Formation of the di-iron site in W107Y/Y122F was
studied using light absorption. The features apparent at 325 and 370 nm
indicate that the µ-oxo-bridged diferric center in W107Y/Y122F is
properly formed (Fig. 3), and the extinction
coefficients are similar to those of Y122F (25). The stability of the
iron center is not affected by the additional mutation. The iron
content was of the same order of magnitude (~3 Fe/R2) for
W107Y/Y122F, for the single mutant Y122F, and for the wild type protein
implying at most 75% di-iron sites. In both mutants the iron center is
assembled spontaneously within 5 s. The observed kinetics at 370 nm are biphasic. From the inset of Fig. 3 we calculated for
W107Y/Y122F a rate constant of 2.1 ± 0.2 s1 for the
first phase, and 8.7 ± 0.2 s
1 was determined for
Y122F.
Stopped Flow and Freeze Quench EPR Studies of Transient Radicals
In Y122F we earlier assigned a transient room
temperature doublet EPR signal, visible in the time regime 10 s to
minutes, to Trp-107· (Fig. 4, Table
I) (9). In the double mutant W107Y/Y122F this room
temperature doublet is absent, confirming that this transient is indeed
likely to be generated at Trp-107 in Y122F. Instead a new radical
species in the time window 500 ms to 20 s is formed and decays
(Fig. 4, Table I). The hyperfine splitting of this room temperature
signal in W107Y/Y122F can be resolved in low temperature freeze quench
studies that show a quintet spectrum with estimated hyperfine couplings
from one proton of 1.3 ± 0.1 mT and from two additional protons
of 0.75 ± 0.1 mT each (Fig. 5A). In
Y122F a quartet signal appears in this time range which was assigned to
Trp-111 (Fig. 5C; adapted from Ref. 9). In Table
II the characteristics of the different EPR signals
found in W107Y/Y122F and Y122F are listed. The microwave power at
half-saturation (P1/2) of the quintet in W107Y/Y122F
is almost 2 orders of magnitude lower than that determined for the
quartet in Y122F, indicating a weaker magnetic interaction (38, 40) of
the new radical species with the iron center. Evaluation of the
intensity of the EPR signal showed that it is linearly dependent on the
temperature (8-190 K) as expected from the Curie-Weiss law. The total
spectral width defined by the spacing of the outermost peak to
outermost trough was not broadened by increasing temperature. However,
after annealing for 15 min at 260 K (13 °C) followed by cooling to 8 K, the yield of the transient radical was only recovered to 60%,
suggesting that this radical is not stable at higher temperatures.
|
|
In both mutants the axial EPR signal (Fig. 5C; component I
in Ref. 9) tentatively assigned to a Trp-48-derived radical is present,
even though it is considerably delayed in W107Y/Y122F and accumulates
to only 10% of the yield found in Y122F. This radical species is long
lived (20 min). The weak magnetic interaction with the iron center
would be consistent with the large distance of 8 Å between Trp-48 and
the di-iron site. A similar axial line shape was reported for a
tryptophan radical species weakly coupled to the heme iron in
cytochrome c peroxidase (21, 22) and for a peroxyl radical
observed in myoglobin treated with hydrogen peroxide (41). This
supports the hypothesis that the axial signal in Y122F or W107Y/Y122F
is a tryptophan-derived radical, probably an oxygen adduct of Trp-48 or
perhaps other surface-located tryptophans.
To assign the EPR quintet to a
tyrosyl radical the reconstitution reaction was performed with R2
protein from cells grown in media containing [2H],
-tyrosine. Replacing a proton involved in EPR hyperfine coupling
with deuterium causes the number of hyperfine lines to change from two
to three and their spacing to decrease about 6-fold (42). In practice
this is usually observed as a collapse of the line width of the EPR
signal. The EPR spectrum obtained with specifically deuterated R2
W107Y/Y122F is shown in Fig. 5B. The presence of deuterated
tyrosine in the mutant resulted in a significant narrowing of the
hyperfine splitting and yielded a singlet alike EPR spectrum.
We performed a computer simulation of the EPR spectrum of the new
tyrosyl radical (Fig. 5A). After iterations leading to a best fit to the experimental spectrum, we obtained the following parameters (hyperfine coupling constants in mT): 1.28,
0.1,
0.89
for H3,
0.87,
0.37,
1.02 for H5, and 1.69, 1.23, and 0.94 for one
of the
-methylene protons, whereas the hyperfine coupling of the
other one is not resolved outside the line width. The isotropic
hyperfine couplings are therefore 1.29 mT for the
-methylene proton,
and 0.76 and 0.75 mT for H3 and H5, respectively, in reasonable
agreement with the values estimated directly from the EPR spectrum. The
gz value was fixed to 2.0020 and the two other components
of the g tensor were obtained to g = 2.0075 and 2.0050. These EPR parameters are compatible with a tyrosyl radical of the oxidized, neutral form.3 According
to the theoretical calculations of Himo et al.3,
the simulated and experimental coupling constant with an isotropic value of about 1.3 mT for one
-proton corresponds to a dihedral angle of 40° relative to the pz axis, and the
invisibility of the second agrees with the dihedral angle of 80°.
This geometry matches very closely the values found for Tyr-107 in the
crystal structure of W107Y/Y122F determined here. Detection of the
transient EPR signal at room temperature as well as at low temperature
(see annealing experiment) is consistent with a relatively weak metal interaction and the observed distance of 8 Å between Tyr-107 and the
iron center. These considerations strongly indicate that the new
tyrosine at position 107 harbors the transient radical.
The transient Tyr-107· observed at low
temperature in rapid freeze quench samples of W107Y/Y122F showed rate
constants of formation (k1) and decay
(k2) of 1.4 and 0.03 s1,
respectively, and a lag time of 0.7 s. These rate constants of
Tyr-107· are confirmed by stopped flow EPR at room temperature
(Table I). The difference in k1 obtained by the
two methods, although still in the same range, may be caused by
systematic errors. In the low temperature measurements an EPR signal of
an early EPR singlet intermediate, possibly equivalent to species X (9, 14), is superimposed on the Tyr-107· quintet. The amount of
Tyr-107· was estimated by subtraction of the quintet spectrum
and inspection of the form of the residual, which should show a perfect
singlet. Small amounts of the quintet are difficult to determine by
this method and therefore the earliest values of Tyr-107· may be
judged too small. Thus, the lag time (dt) and the
k1 of Equation 2 will be increased and the
values obtained may be regarded as upper limits. The amount of
deuterated Tyr-107· was easier to calculate by this method and
gave the same results. The kinetic trace measured with EPR stopped flow
at room temperature showed different start and end values. Thus the lag
time of Tyr-107· could not be calculated accurately, so that
this formation rate can be regarded as a lower limit. Nevertheless, the
decay constants, for which these problems are not significant, are the
same by both methods. Taken together, the kinetic data and the results of the annealing experiment make it most likely that the transient radical species observed at 293 and 8 K are the same. Because the
kinetics of Trp-111· in Y122F and formation of the stable
Tyr-122· in wild type R2 were measured by rapid freeze
quenching, we considered only the kinetic data obtained by this
method.
The decay rate of 0.03 s1 for Tyr-107· is faster
than that for Trp-107· (0.001 s
1), suggesting that
a tyrosyl radical is less stable than a tryptophan radical in this
environment. Interestingly, the observed formation and decay rate
constants for the Tyr-107· transient in the double mutant are
almost identical to those found for the putative Trp-111· in
Y122F (Fig. 6, A and B, Table I).
We therefore assume a consecutive reaction forming first
Trp-111· and then Trp-(Tyr)107· in both mutant proteins.
In the W107Y/Y122F double mutant the formation of Trp-111· is
the rate-limiting step and formation of Tyr-107· is fast,
whereas in Y122F the formation of Trp-111· is 50-fold faster
than of Trp-107·. The overall yield (given in Table I) of
Tyr-107· and Trp-111· is about the same, with 0.07
0.08 unpaired spins/R2 obtained by fitting the kinetics to Equation 2.
The reconstitution reaction of R2 apoprotein with ferrous iron/O2 yields the µ-oxo-bridged diferric center and a stable tyrosyl radical at Tyr-122. Certain aspects of this reaction may serve as a model for delivery of hydrogen radicals to the iron site in R2 that may occur during catalytic reaction. The fact that transient protein-linked free radicals are trapped concomitant with and subsequent to the assembly of the diferric center in Y122F (9, 15-18) indicates that if Tyr-122 is missing other side chains can be oxidized to contribute an electron. Earlier EPR and electron nuclear double resonance studies revealed that neutral radicals can be generated at tryptophan residues in the near vicinity of the diferric center (9, 20). If we assume that radical transfer in proteins takes place via hydrogen bonds, tryptophans 48, 111, and 107 are the only candidates, as all three are hydrogen-bonded to ligands of the di-iron site (Fig. 1).
In the present work we investigated the W107Y/Y122F mutant R2 protein. In this double mutant a new transient tyrosyl radical is formed. It exhibits an EPR hyperfine quintet signal where the hyperfine splitting is consistent with the dihedral angles determined for Tyr-107 in the crystal structure of W107Y/Y122F. Microwave power saturation behavior and EPR visibility at room temperature indicate that the new radical species has very weak magnetic interaction with the diferric center, consistent with the distance of 8 Å from Tyr-107 to the diferric site. The Trp-107· transient in Y122F observed as an EPR doublet at room temperature disappeared as expected in the double mutant W107Y/Y122F. Together, these observations demonstrate that Tyr-107 harbors the new transient radical and thereby conclusively identify the earlier assignments of the transient tryptophan radicals in Y122F R2 protein (9, 15, 20).
The Tyr-107· EPR spectrum has a striking similarity with those
of the Tyr·D of photosystem II and the tyrosyl
radical of R2 from Salmonella typhimurium (7, 44). These
three EPR signals have in common that only one -methylene proton
shows a detectable hyperfine splitting. However, the isotropic
hyperfine splitting of 1.29 mT in Tyr-107· is somewhat larger
compared with 0.83 mT in Tyr·D of photosystem II and
0.91 mT Tyr· in R2 from S. typhimurium. The
anisotropy of the hyperfine structure of these signals is caused by
coupling with the 3 and 5 hydrogens of the phenyl ring and has similar
coupling constants.
Our kinetic studies on the reconstitution of the Y122F and W107Y/Y122F
mutant R2 proteins suggest that a hydrogen radical transfer occurs
along a hydrogen-bonded pathway, combined with an electron transfer,
utilizing the -interaction of aromatic rings. This last step is most
likely proton-coupled since again the observed transient radical is
neutral. Fig. 1 shows the crystal structure of W107Y/Y122F in
comparison with the structure of wild type R2. Trp-107 is
hydrogen-bonded via a water molecule to the backbone of the Fe-2 ligand
His-241, whereas Trp-111 is directly connected via a hydrogen bond to
the Fe-2 ligand Glu-204. Thus, the residues Trp-107 and Trp-111 in wild
type R2 and in Y122F are connected to Fe-2 in a similar fashion as are
Trp-48, Asp-237, and His-118 to Fe-1 (Fig. 1) and may therefore be
regarded as short distance "model RTP" of the catalytically
important RTP in wild type R2.
If we first consider Y122F, two possibilities exist for the delivery of
the "missing" electron from tryptophans Trp-107 and Trp-111 (Fig.
6A). The electron might be delivered in parallel from
Trp-111 and from Trp-107 using their specific hydrogen bonding described above. It then follows that the kinetics and yields of both
intermediates, Trp-111· and Trp-107·, may be quite
different. Another possibility is that Trp-111· is a direct
precursor of Trp-107·, because Trp-111 has the most direct
connection to the iron center considering the number of the covalent
and hydrogen bonds involved (Fig. 6A). The two tryptophan
planes are stacking in a "head to tail" manner with respect to
their indole rings, i.e. the pyrrole ring from one indole
stacks on the phenyl ring of the other and vice versa. The distance of
4-5 Å suggests electrostatic interactions between them. We
observed that generation of Trp-111· is followed by formation of
Trp-107·. The determined decay constant of 0.05 s
1
for Trp-111· and formation constant of 0.027 s
1
for Trp-107· and the accumulated yield of each of about 0.08 radical/R2 strongly suggest that Trp-111· is the precursor of
Trp-107·. We suggest that the radical is transferred utilizing
the "stacking complex" configuration of their aromatic rings as
illustrated by k2 in Fig. 6A and that
the route over the backbone of the ligand His-241 to the diferric
center is less efficient.
With the mutation Trp Tyr at position 107, a switch takes place
from the appearance of Trp-111· followed by Trp-107· to
the immediate observation of Tyr-107·. Surprisingly, the
transient Trp-111· signal is absent in the double mutant
W107Y/Y122F, and the formation and decay constants of Tyr-107·
are identical to those of Trp-111· measured in Y122F (Fig.
6B). The structure of the double mutant shows that Tyr-107
is still bound to His-241 via a water molecule and that the aromatic
rings of the Tyr-107 and Trp-111 now form a stacking complex with the
phenyl rings on the top of each other, the phenyl ring of Tyr-107 is
slightly tilted (Fig. 1). Assuming that this pathway (as was also
proposed for Y122F above) is valid for W107Y/Y122F, the transient
Trp-111· will no longer be detectable if Tyr-107· is
formed much faster than Trp-107· in Y122F. In the double mutant
the formation of Trp-111· becomes the rate-determining step, and
we measure the unchanged rate k1 for the
formation of Trp-111· as the overall rate constant of the
proposed consecutive reaction Trp-111
Tyr-107. The alteration of
the kinetics in W107Y/Y122F also underlines that the RTP
Fe-2-E204-W111-W107 is the preferred one in the Y122F single mutant
(Fig. 6A).
Our results indicate that the Fe-2-Glu-204-Trp-111-Trp-107 pathway
utilizes a radical transfer, combined with a proton-coupled electron
transfer via the -interaction of the indole rings. Why is the
Tyr-107 radical in the double mutant protein more rapidly formed than
the corresponding tryptophan radical in Y122F? According to earlier
studies on electron transfer proteins, the presence of aromatic
residues facilitates electron transfer in general (45). The kinetics of
the Tyr-107· formation in W107Y/Y122F may reflect that phenyl
rings arranged on top of each other allow a more efficient electron
transfer than does the head-to-tail configuration of the tryptophans in Y122F, where the phenyl rings do not overlap. An additional explanation might be found in slightly different redox potentials of tyrosine and
tryptophan. According to experiments with single amino acids in aqueous
(H2O) and hydrophobic (CH3CN) solutions,
tyrosine is easier to oxidize in a hydrophilic environment than
tryptophan, whereas tryptophan is easier to oxidize in a hydrophobic
environment (46). Probably the environment of the tryptophans 111 and
107 is predominantly hydrophilic because these residues are not
shielded in a hydrophobic pocket like Tyr-122. Therefore Tyr-107
becomes a better candidate for oxidation than Trp-111. The more rapid decay of the tyrosyl radical is not explained by this argument. Based
on the redox potentials an even slower decay of a tyrosyl radical would
be expected. The rapid decay is therefore most likely due to structural
differences, e.g. increased accessibility to solvent.
The radical transients formed during reconstitution in the R2 mutants
Y122F and W107Y/Y122F are substitutes for the missing stable tyrosyl
radical of wild type R2, and the rate constants for formation of
Tyr-107· and Trp-111· in the mutants (1.7 s1) and Tyr-122· in wild type R2 (1.4 s
1) are remarkably similar under our conditions. This
raises the question as to whether a tyrosine at position 107 would
compete with the Tyr-122 for radical formation. A situation, where
competition occurs, has been reported earlier with the mutant R2
protein F208Y. Here the introduction of a tyrosine in the near vicinity
of the iron center resulted in the hydroxylation of the Tyr-208 phenyl ring during reconstitution instead of formation of Tyr-122·, and
the formed catechol subsequently becomes an iron ligand (47). Our
results suggest that competition might be possible in a
Tyr-122-containing protein if Trp-107 or -111 is replaced by tyrosine.
Note that Trp-111 is H-bonded via a carboxylate to Fe-2 in almost the
same fashion as tyrosine 122 is linked to Fe-1. Analyzing the sequence
homologies among the class I R2 proteins with respect to positions 107 and 111 (E. coli numbering), one finds combinations like
tryptophan-tryptophan (e.g. E. coli), tyrosine
107-glutamine 111 (e.g. herpes simplex), or phenylalanine 107-glutamine 111 (e.g. mouse). Accordingly, in the Y177F
mutant R2 protein from mouse (corresponding to Y122F) no transient
tryptophan radicals can be observed.4 The
combination tryptophan (tyrosine)-tyrosine is not found among any class
I R2 protein so far sequenced. One might conclude that nature avoids
these combinations to have no competition for the stable radical
formation at the catalytically essential tyrosine site.
In class I ribonucleotide reductase a hydrogen-bonded chain of conserved amino acids is believed to transfer a radical from Tyr-122· in R2 to the active site in R1 during catalysis. Site-directed mutagenesis studies have revealed the importance of intact hydrogen bonding, but kinetic evidence is still lacking for a RTP during catalysis. The geometric arrangement of Trp-111 and Tyr-107 in R2 is reminiscent of the stacking configuration of the tyrosine residues 730 and 731 in the proposed RTP in protein R1. Both these tyrosine side chains in R1 are essential for catalysis: exchange of either of them to phenylalanine (Y730F or Y731F) results in an inactive enzyme, even though the positioning of the phenyl rings is unchanged (48). The present results in the mutant R2 protein, yet catalytically inactive, might model the RTP processes occurring between the R1 and R2 proteins during catalysis. Our kinetic and EPR results suggest that during reconstitution of Y122F and W107Y/Y122F a hydrogen radical is abstracted from Tyr(Trp)-107 via Trp-111 and Glu-204 by the iron site. This consecutive reaction of radical abstraction might resemble a consecutive radical transport from the active site in R1 to Tyr-122· in R2. In addition it presents the first kinetic evidence for the transfer of a radical (electron/proton) to the iron site.