Structural examination of the transient 3-aminotyrosyl radical on the PCET pathway of E. coli ribonucleotide reductase by multifrequency EPR spectroscopy

Article abstract of DOI:10.1021/ja903879w

E. coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, a process that requires long-range radical transfer over 35 A from a tyrosyl radical (Y₁₂₂?) within the β2 subunit to a cysteine residue (C

Full text of DOI:10.1021/ja903879w

Published on Web 10/12/2009
Structural Examination of the Transient 3-Aminotyrosyl
Radical on the PCET Pathway of E. coli Ribonucleotide
Reductase by Multifrequency EPR Spectroscopy
Mohammad R. Seyedsayamdost,†,⊥ Tomislav Argirevic´,§ Ellen C. Minnihan,†
JoAnne Stubbe,*,†,‡ and Marina Bennati*,§
Department of Chemistry and Department of Biology, Massachusetts Institute of Technology, 77
Massachusetts AVenue, Cambridge, Massachusetts 02139-4307, and Max Planck Institute for
Biophysical Chemistry, Am Fassberg 11, 37077 Go¨ttingen, Germany
Received May 13, 2009; E-mail: Marina.Bennati@mpibpc.mpg.de; stubbe@mit.edu
Abstract: E. coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucle-
otides, a process that requires long-range radical transfer over 35 Å from a tyrosyl radical (Y122•) within the
ꢀ2 subunit to a cysteine residue (C439) within the R2 subunit. The radical transfer step is proposed to occur
by proton-coupled electron transfer via a specific pathway consisting of Y122 f W48 f Y356 in ꢀ2, across
the subunit interface to Y731 f Y730 f C439 in R2. Using the suppressor tRNA/aminoacyl-tRNA synthetase
(RS) methodology, 3-aminotyrosine has been incorporated into position 730 in R2. Incubation of this mutant
with ꢀ2, substrate, and allosteric effector resulted in loss of the Y122• and formation of a new radical,
previously proposed to be a 3-aminotyrosyl radical (NH2Y•). In the current study [15N]- and [14N]-NH2Y730
•
have been generated in H2O and D2O and characterized by continuous wave 9 GHz EPR and pulsed EPR
spectroscopies at 9, 94, and 180 GHz. The data give insight into the electronic and molecular structure of
NH2Y730•. The g tensor (gx ) 2.0052, gy ) 2.0042, gz ) 2.0022), the orientation of the ꢀ-protons, the
hybridization of the amine nitrogen, and the orientation of the amino protons relative to the plane of the
aromatic ring were determined. The hyperfine coupling constants and geometry of the NH2 moiety are
consistent with an intramolecular hydrogen bond within NH2Y730•. This analysis is an essential first step in
using the detailed structure of NH2Y730• to formulate a model for a PCET mechanism within R2 and for use
of NH2Y in other systems where transient Y•s participate in catalysis.
(Y122•) cofactor essential for initiating nucleotide reduction.11-13
A structure of the active RNR complex has not been solved.
Introduction
Ribonucleotide reductases (RNRs) provide the monomeric
precursors required for DNA replication and repair by catalyzing
the conversion of nucleotides to deoxynucleotides.1,2 The class
Ia RNRs are found in eukaryotes and prokaryotes with the RNR
from E. coli serving as a model. It consists of a 1:1 complex of
two homodimeric subunits: R2 and ꢀ2.3,4 R2 contains the active
site, including residue C439, which is oxidized to a transient thiyl
radical to initiate nucleotide reduction,5-8 and binding sites for
allosteric regulators that control the rate and specificity of
nucleotide reduction.9,10 ꢀ2 harbors a di-iron tyrosyl radical
However, on the basis of in silico docking of the individual
subunits, Uhlin and Eklund made the provocative proposal that
a pathway consisting of aromatic amino acids is involved in
transport of the radical from Y122• in ꢀ2 over 35 Å to C439 in
the active site of R2.14-17 We have expanded this model to
include the fate of the protons as well as the electrons, as
oxidation of Ys within the pathway requires proton-coupled
electron transfer (PCET).18,19 Within ꢀ2, the reaction is proposed
to occur by orthogonal PCET,20 while within R2 it is proposed
to involve collinear PCET (Figure 1).21,22
Recently we have developed new probes to examine the
pathway and the involvement of redox-active tyrosines (Y356
in ꢀ2 and Y730 and Y731 in R2) and to study the mechanism of
† Department of Chemistry, Massachusetts Institute of Technology.
‡ Department of Biology, Massachusetts Institute of Technology.
§ Max Planck Institute for Biophysical Chemistry.
⊥ Current address: Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, 240 Longwood Ave., C1-609,
Boston, MA 02115.
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A R T I C L E S
Seyedsayamdost et al.
Figure 1. Proposed radical initiation pathway in E. coli class Ia RNR. Radical transfer occurs by orthogonal PCET within ꢀ2, where long-distance ET
within the pathway is coupled to short-distance off-pathway PT, and by collinear PCET (or hydrogen atom transfer) within R2. Note that the position of Y356
is structurally unknown.19
this unprecedented radical propagation process.18,23 Incorpora-
tion of 3,4-dihydroxyphenylalanine (DOPA), which has a lower
reduction potential than Y (260 mV lower at pH 7), into residue
356 of ꢀ2 led to trapping of the DOPA radical (DOPA356•).24,25
Formation of DOPA356• occurred concomitantly with disap-
pearance of Y122• and was dependent on the presence of substrate
and allosteric effector. These studies support direct participation
of residue 356 in radical transfer. We have also inserted a series
of fluorotyrosine analogues (FnY, n ) 1-4),20,26-28 3-nitroty-
rosine,29 and 4-aminophenylalanine30 in order examine the
protonation state and the effect of the driving force for radical
transfer through residue 356.18
To investigate the roles of residues Y730/Y731 in radical
propagation, we recently used the suppressor tRNA/RS method,
pioneered by Schultz and co-workers,31,32 to replace site-
specifically each residue with 3-aminotyrosine (NH2Y).22 This
analogue is 190 mV easier to oxidize than Y (at pH 7), but the
pKa of the hydroxyl group is unchanged.33,34 Examination of
NH2Y-R2s in the presence of ꢀ2, substrate, and allosteric
effector demonstrated radical propagation across the subunit
interface in a kinetically competent fashion. Concomitant with
loss of the Y122•, a new radical species was detected, which we
proposed was a NH2Y• on the basis of UV-visible and X-band
EPR spectroscopies. Surprisingly, we also showed that these
NH2Y-R2s were capable of making deoxynucleotides, an
observation that has important implications for the mechanism
of radical propagation.22,35
Key to using NH2Y as a probe of PCET reactions in general
and the radical propagation step specifically in RNR is a detailed
characterization of the electronic properties of the putative
NH2Y• by EPR spectroscopy. Prior to our studies, no spectros-
copy of this unnatural amino acid radical had been reported.
However, studies on the o-aminophenol radical36-41 and the
Y•1,42-44 in many different proteins have been examined and
provide a foundation for expectations with NH2Y•.
9 GHz EPR studies on the o-aminophenol radical in different
solvent matrixes reveal two major coupling constants assigned
to the protons and nitrogen of the amino group (Table S1 and
Figure S1). The strong temperature dependence of the EPR
spectra in aprotic solvents indicated that the two protons may
be involved in an intramolecular dynamic process.41 An INDO
calculation suggested that of the two possible tautomeric
structures of the radical the H is bonded to the N, not the O,
and that the amino group is not planar, but at least partially
sp3-hydridized. These early calculations further suggested that
the •O---H-N hydrogen bond does not lie in the ring plane and
that an out-of-plane deformation caused by bending of the C-N
bond 5° relative to the plane of the ring stabilizes the radical.41
A more recent computational study about substituent effects on
the hybridization state of the nitrogen in anilines showed that
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Stubbe, J. J. Am. Chem. Soc. 2006, 128, 1562.
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Transient 3-Aminotyrosyl Radical
A R T I C L E S
pyramidalization of the amino group can occur, but the out-of-
plane bending takes place at the protons and not at the
nitrogen.45
Transient and stable Y•s have been characterized in detail in
many proteins. However, despite the extensive experimental
effort in this area, the variability of EPR line shapes associated
with different rotational conformations of the phenoxy ring and
different patterns of hyperfine interactions have made simulation
of Y• spectra challenging. High-field EPR and ENDOR spec-
troscopies and computational studies have revealed a number
of interesting generalizations about the structures of these
radicals. High-field EPR studies give rise to accurate g values
and to insight about the electrostatic environment and H-bonding
to the radical. The gx values for the Y• in class I RNRs range
from 2.0077 to 2.0094.46-52 A shift of gx to a lower value
indicates the possibility of a H-bond.53,54 Also, variability in
line shape and width of gx observed suggests there may be
variability of H-bonding within the Y• population.55-57 The EPR
spectrum is largely defined by the hyperfine interactions with
the ꢀ-protons of the methylene group of Y and its four ring
protons. The former are primarily responsible for the different
patterns in the hyperfine structure. Lessons learned from EPR
spectra of the o-aminophenol radical and for many Y•s in
different environments provide the foundation for understanding
the structure of the NH2Y• described herein.
Figure 2. Structure of NH2Y730•. (Top) Numbering scheme (inside
numbers), axis system, and the intramolecular H-bond represented by dashed
lines. (Bottom) NH2Y730• viewed along the C3-N and the phenol C2-C3
bond within the aromatic plane. Dihedral angles for the amino protons (left)
and the Cꢀ-protons (middle) of NH2Y730• with respect to the phenol plane,
which is indicated by dashed lines. (Right) Tetrahedral nature of the NH2
moiety and the 5° tilt of the C3-N bond away from the aromatic plane,
which is indicated by a bold line.
(99.8 atom % in D) was from VWR. ꢀ2 was purified as previously
described with a specific activity of 7200 nmol/min mg and 1.2
1
Y122•/dimer. H and 13C NMR spectra were recorded on a Varian
We now report the characterization of the new radical
generated with Y730NH2Y-R2 using multifrequency EPR spec-
troscopy. Our results demonstrate that this signal is associated
with the NH2Y• and provide insight into its conformation within
the active RNR complex (Figure 2). The results establish the
existence of an intramolecular H-bond between one of the amino
protons and the phenol oxygen of the NH2Y730•. This detailed
spectroscopic analysis is an essential step in identifying the
importance of H-bonding between residues in the radical
propagation pathway (Figure 1), which in turn is required for
assessing the proposed model for collinear PCET.
300 MHz NMR spectrometer at the MIT Department of Chemistry
1
Instrumentation Facility. Aqueous samples for H NMR and 13C
(1H-decoupled) NMR were acquired in D2O with 3-(trimethylsi-
lyl)propionic acid-d4 (TSP) as a standard. Absorption spectra were
recorded on an Agilent 8453 diode array or a Cary 3 UV-vis
spectrophotometer.
Synthesis of 3-[15N]-Nitrotyrosine, [15N]-NO2Y. [15N]-HNO3
(10 N) contained 98% 15N as confirmed by mass spectrometry.
Synthesis of [15N]-NO2Y was carried out as previously described
with minor modifications.34 L-Y (7.0 mmol) was added to a 25
mL pear-shaped flask, equipped with a stir bar, and dissolved in 5
mL of H2O at room temperature. The mixture was supplemented
with 1.55 mL of [15N]-HNO3 (11.6 mmol, 7.5 N) by dropwise
addition over 1 min and stirred for 30 min at room temperature to
fully dissolve L-Y. The mixture was then cooled by stirring for
15 min in an ice-water bath. Then 4 mL of chilled [15N]-HNO3
(30 mmol) was added to the mixture at 4 °C over 2 h. The solution
was then stirred for an additional 5 h at 4 °C and refrigerated
overnight, which caused precipitation of [15N]-NO2Y. The precipi-
tate was isolated and dried by vacuum filtration on a coarse Buchner
funnel, providing product in 80% yield (5.6 mmol). The product
was analyzed by 1H and 13C NMR, and by UV-vis spectroscopy.
1H NMR (300 MHz, D2O, 25 °C): δ 3.18 (dd, 1H, Cꢀ-H1, 7.4
Hz, 14.8 Hz), 3.30 (dd, 1H, Cꢀ-H2, 5.8 Hz, 14.8 Hz), 4.24 (dd, 1H,
CR-H, 5.8 Hz, 7.4 Hz), 7.14 (dd, 1H, arom. C-H, 1.1 Hz, 8.7 Hz),
7.54 (dd, 1H, arom. C-H, 2.2 Hz, 8.7 Hz), 8.01 (t, 1H, arom. C-H,
2.2 Hz). 13C NMR (300 MHz, D2O, 25 °C): δ 34.7 (s, Cꢀ), 54.2 (s,
CR), 120.4 (d, arom. C, 2.1 Hz), 126.1 (d, arom. C, 1.6 Hz), 126.8
(d, arom. C, 2.4 Hz), 134.2 (d, arom. C, 15.2 Hz), 138.2 (s, arom.
C), 153.1 (d, arom. C, 1.2 Hz), 172.1 (s, COOH). UV-vis (H2O/
HCl, pH 2): λmax ∼277 nm (ε ∼5500 M-1 cm-1), λmax ∼358 nm (ε
∼2600 M-1 cm-1); (H2O/NaOH, pH 12) λmax ∼286 nm (ε ∼4100
M-1 cm-1), λmax ∼431 nm (ε ∼4100 M-1 cm-1).
Materials and Methods
Materials. L-Tyrosine, [15N]-HNO3 (conc ∼10 N, 98% 15N
enrichment), palladium catalyst on carbon (10 wt % loading),
hydroxyurea (HU), Sephadex G-25, 3-nitrotyrosine (NO2Y), 3-ami-
notyrosine (NH2Y), cytidine-5′-diphosphate (CDP), and adenosine-
5′-triphosphate (ATP) were obtained from Sigma-Aldrich. D2O
(45) Alabugin, I. V.; Manoharan, M.; Buck, M.; Clark, R. J. THEOCHEM
2007, 813, 21.
(46) Gerfen, G. J.; Bellew, B. F.; Un, S.; Bollinger, J. M., Jr.; Stubbe, J.;
Griffin, R. G.; Singel, D. J. J. Am. Chem. Soc. 1993, 115, 6420.
(47) Allard, P.; Barra, A. L.; Andersson, K. K.; Schmidt, P. P.; Atta, M.;
Gra¨slund, A. J. Am. Chem. Soc. 1996, 118, 895.
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A. J. Biol. Chem. 1996, 271, 23615.
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Fontecave, M. J. Biol. Inorg. Chem. 1997, 2, 586.
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Hagen, W. R.; Hoffman, B. M.; Andersson, K. K.; Gra¨slund, A. J. Am.
Chem. Soc. 1998, 120, 5080.
(51) Liu, A.; Barra, A. L.; Rubin, H.; Lu, G.; Gra¨slund, A. J. Am. Chem.
Soc. 2000, 122, 1974.
Synthesis of 3-[15N]-Aminotyrosine, [15N]-NH2Y. [15N]-NO2Y
(5.2 mmol) was combined with 18 mL of H2O and 70 mL of MeOH
in a 250 mL round-bottom flask, equipped with a stir bar, at room
temperature. The solution was supplemented with 0.52 mmol (550
mg) of Pd/C catalyst. The mixture was evacuated gently and filled
with H2(g) several times and then stirred for 2 h under H2 using a
H2(g) balloon. After the incubation, the H2 was replaced with N2
and the catalyst was removed by filtration through a fine Buchner
funnel. The solvent was removed in Vacuo, providing product in
(52) Bar, G.; Bennati, M.; Nguyen, H. H.; Ge, J.; Stubbe, J. A.; Griffin,
R. G. J. Am. Chem. Soc. 2001, 123, 3569.
(53) Un, S.; Atta, M.; Fontecave, M.; Rutherford, A. W. J. Am. Chem.
Soc. 1995, 117, 10713.
(54) Un, S. Magn. Reson. Chem. 2005, 43, S229.
(55) Wilson, J. C.; Wu, G.; Tsai, A.-L.; Gerfen, G. J. J. Am. Chem. Soc.
2005, 127, 1618.
(56) Svistunenko, D. A.; Cooper, C. E. Biochem. J. 2004, 87, 582.
(57) Warncke, K.; Babcock, G. T.; McCracken, J. J. Phys. Chem. 1996,
100, 4654.
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A R T I C L E S
Seyedsayamdost et al.
quantitative yield. The product was exchanged into D2O, lyophilized
to dryness, and assessed by 1H and 13C NMR and UV-vis
spectroscopic methods. It was pure on the basis of NMR spec-
troscopy.
frequency, we checked the 9 GHz simulations using the EasySpin
(3.1.0) EPR simulation package and used the option to perform
matrix diagonalization.58 The global solutions obtained with
SimFonia were also tested in EasySpin using least-squares fitting
with the choice of the so-called “genetic” algorithm.
Pulsed EPR Spectroscopy at 9, 94, and 180 GHz. Nine and
94 GHz pulsed EPR spectra were recorded on Bruker Elexsys
spectrometers of the series E580 and E680 using a π/2-π spin
echo sequence with typical π/2 pulse lengths of 32 ns; 180 GHz
pulsed EPR spectra were recorded on a home-built spectrometer
previously reported with typical π/2 pulse lengths of 32 ns.59 Other
experimental details are given in the figure captions. The pulsed
EPR spectra were recorded at 70 K, where the Y122• is not
detectable, as it relaxes more rapidly than an NH2Y• due to its
vicinity to the di-iron cluster.60 At this temperature, the electron
spin echo (ESE) spectra at all three frequencies contain contributions
only from the NH2Y730• signal.
Simulation of Pulsed EPR Spectra. The EPR powder spectra
were analyzed using SimFonia software (Bruker). To determine
independently the optimal number of parameters, g values were
obtained from the 180 GHz EPR spectrum, and the majority of the
hyperfine couplings were fixed by iterative simulations of the 15N
and 14N spectra in D2O at two different frequencies (9 and 94 GHz).
Finally, the optimized parameters obtained from the 15N and 14N
simulations in D2O were used as input for the simulations of the
spectra in H2O. The latter spectra contained additionally the
contribution of the two exchangeable protons of the NH2 group.
The simulation of the amino proton contribution was complex
because of their large hyperfine anisotropy and the noncollinearity
of the g and A axes for these protons. The simulation thus required
the introduction of the three Euler angles between the g and A axes
as additional parameters. The definition of the three Euler rotations
around the three axes z, y′, and z′ is illustrated in Figure S3, and
the explicit rotation matrix is given in Bennati and Murphy.61
1H NMR (300 MHz, D2O, 25 °C): δ 3.12 (m, 2H, Cꢀ-Hs), 3.90
(t, 1H, CR-H, 6.3 Hz), 6.99 (m, 1H, arom. C-H), 7.19 (m, 2H,
arom. C-H). 13C NMR (300 MHz, D2O, 25 °C): δ 35.2 (s, Cꢀ),
55.5 (s, CR), 116.9 (s, arom. C), 118.2 (d, arom. C, 9.4 Hz), 124.8
(d, arom. C, 1.7 Hz), 126.9 (d, arom. C, 1.9 Hz), 131.4 (s, arom.
C), 149.5 (s, arom. C), 173.3 (s, COOH). UV-vis (H2O/HCl, pH
1): λmax ∼275 nm (ε ∼1900 M-1 cm-1); (H2O, pH 7) λmax ∼289
nm (ε ∼3200 M-1 cm-1); (H2O/NaOH, pH 12) λmax ∼303 nm (ε
∼4700 M-1 cm-1).
Growth, Expression, and Purification of [15N]-NH2Y730-r2.
All procedures were carried out as previously detailed and gave 6
mg of pure [15N]-NH2Y730-R2 per gram of wet cell paste.22
Activity Assays. The activity of [15N]-NH2Y730-R2 was deter-
mined by the spectrophotometric assay as described.20
Reaction of [15N]-Y730NH2Y-r2 with ꢀ2, CDP/ATP Monitored
by EPR Spectroscopy. [15N]-NH2Y730-R2 was prereduced as previ-
ously described.22 Prereduced [15N]-Y730NH2Y-R2 and ATP were
mixed with ꢀ2 and CDP in a final volume of 250 µL to yield final
concentrations of 24 µM [15N]-Y730NH2Y-R2/ꢀ2, 1 mM CDP, and
3 mM ATP. The reaction was quenched after 20 s by hand-freezing
in liquid N2 and its EPR spectrum recorded (see below).
Reaction of Y730NH2Y-r2 or [15N]-Y730NH2Y-r2 with ꢀ2, CDP/
ATP in Deuterated Buffer Monitored by EPR Spectroscopy. Assay
buffer (50 mM Hepes, 15 mM MgSO4, 1 mM EDTA) was prepared
in D2O and its pD adjusted to 8.0. Nucleotide stock solutions were
prepared in deuterated assay buffer. Y730NH2Y-R2, [15N]-Y730NH2Y-
R2, and ꢀ2 were exchanged into deuterated assay buffer by multiple
concentration/dilution cycles at 4 °C using a Centriprep concentra-
tion device and a YM-30 membrane until the solution consisted of
>99% deuterated buffer. The reaction in D2O was carried out as
described above.
Results
Preparation of High-Field EPR Samples. Prereduced Y730NH2Y-
R2 and ꢀ2 were combined in an equimolar ratio and concentrated
at 4 °C in a Minicon concentration device (YM-30 membrane) to
a final complex concentration of 70-100 µM (ε280 nm (R2+ꢀ2) ) 320
mM-1 cm-1). The concentrated sample was divided into 100 µL
aliquots, placed in 1.5 mL Eppendorf tubes, and flash-frozen in
liquid N2. High-field EPR samples were prepared by thawing each
aliquot on ice and adding CDP and ATP to final concentrations of
2 and 6 mM, respectively. Each reaction was allowed to proceed
for 30 s at room temperature, at which point 15 µL of glycerol
buffer (100 mM Hepes, 30 mM MgSO4, 2 mM EDTA, 50%
glycerol, pH 7.6) was added to yield a final glycerol concentration
of ∼6%. The reaction was then quenched by hand-freezing in liquid
N2, and the EPR spectra were recorded.
Continuous Wave (CW) X-Band EPR Spectroscopy. EPR
spectra were recorded at 77 K in the Department of Chemistry
Instrumentation Facility (MIT) on a Bruker ESP-300 X-band
spectrometer equipped with a quartz finger dewar filled with liquid
N2. EPR parameters were as follows: microwave frequency ) 9.34
GHz, power ) 30 µW, modulation amplitude ) 1.5 G, modulation
frequency ) 100 kHz, time constant ) 5.12 ms, scan time ) 41.9 s.
The composite spectra obtained were analyzed as described
previously.22
Simulation of CW X-Band EPR Spectra. The X-band EPR
powder spectra were analyzed using SimFonia software (Bruker).
The g values used were determined by 180 GHz EPR spectroscopy
and held fixed during the simulations. The spectrum of [15N]-
NH2Y730• in D2O was simulated first, followed by that of [14N]-
NH2Y730•. Then simulation of spectra in H2O was attempted. The
strength and number of hyperfine couplings from I ) 1/2 (1H, 15N)
and I ) 1 (2H, 14N) nuclei were varied systematically to yield the
best fit to the experimental data. Since SimFonia employs perturba-
tion theory and forbidden transitions might not be taken into account
accurately when the hyperfine couplings exceed the nuclear Zeeman
Isotopic Labeling to Identify the NH2Y•. To assess the involve-
ment of residues Y730 and Y731 in radical propagation catalyzed
by RNR, we recently reported efficient site-specific incorpora-
tion of NH2Y into R2 using the amber suppressor tRNA/RS
method. We demonstrated formation of a putative NH2Y• in
the presence of CDP/ATP based on UV-visible and EPR
spectroscopies and by power saturation studies.22 To further
support our assignment of the new EPR species as an NH2Y730
•
and to obtain additional information regarding its electronic
structure, we have performed isotopic replacement studies
combined with multifrequency EPR spectroscopic analysis.
Preparation of [15N]-NH2Y. [15N]-NH2Y was prepared from
[15N]-NO2Y according to the synthetic route in Scheme S1 in
80% yield with 98% [15N] isotopic enrichment. As expected,
1
the nuclear coupling from 15N to 13C was observed in the H-
decoupled 13C NMR and in 1H NMR spectra of [15N]-NH2Y.62
To further confirm the identity of the product, UV-vis spectra
were acquired as a function of pH. The λmax and extinction
coefficients for 3-ammoniumtyrosine, 3-aminotyrosine, and
3-aminotyrosinate were determined with authentic NH2Y (Scheme
S2), revealing values that are identical within error to our
synthetically prepared [15Ν]-ΝΗ2Y (Table S2).
(58) Stoll, S.; Schweiger, A. J. Magn. Reson. 2006, 178, 42.
(59) Hertel, M. M.; Denysenkov, V. P.; Bennati, M.; Prisner, T. F. Magn.
Reson. Chem. 2005, 43, S248.
(60) Lawrence, C. C.; Bennati, M.; Obias, H. V.; Bar, G.; Griffin, R. G.;
Stubbe, J. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 8979.
(61) Bennati, M.; Murphy, D. Electron Paramagnetic Resonance Spectra
in the Solid State. In Electron Paramagentic Resonance; Brustolon
M., Giamello, E., Eds.; Wiley & Sons: New York, 2009.
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Transient 3-Aminotyrosyl Radical
A R T I C L E S
different frequencies were acquired. We previously reported that
the pulsed EPR method in class I RNRs filters the signal of
any new radical in R2 from that of Y122• in ꢀ2, when the electron
spin echo (ESE) spectrum is recorded at temperatures of 70 K
or higher.60 The method is illustrated in Figure 4A with the
180 GHz pulsed EPR spectrum of NH2Y•. At 6 K, the spectrum
is a composite of Y122• and NH2Y•. However, at 70 K, the signal
associated with Y122•, adjacent to the diferric cluster, is not
observed and only the NH2Y• is apparent.
Pulsed EPR spectra were also recorded at the intermediate
frequency of 94 GHz and at 9 GHz for comparison. The
frequency dependence of the [14N]-NH2Y• ESE spectrum in H2O
at 70 K is shown in Figure 4B. The absorption line shape at 9
GHz is quite symmetric and almost without structure, in
agreement with the spectra observed in the CW mode (Figure
3). At 94 GHz, an asymmetry caused by the g tensor appears
and anisotropic hyperfine interactions are resolved.
Figure 3. Normalized EPR spectra of isotopically substituted NH2Y730•.
Spectra were acquired with Y730NH2Y-R2/ꢀ2 containing [14N]-NH2Y or
[15N]-NH2Y in H2O or D2O assay buffer in the presence of CDP/ATP. The
ticks on the deuterated traces indicate the positions of the quartet and triplet
peaks with [14N]- and [15N]-NH2Y730•, respectively. See text for details.
The pulsed 180 GHz EPR spectrum is displayed in absorption
mode and shows a typical EPR line shape dominated by g
anisotropy (Figure 4), making it possible to extract g values
from the experimental spectrum at the canonical points that
characterize singularities in the powder pattern. These points
are marked in Figure 4B (bottom trace). The g values were
obtained from analysis of spectra at 6 and 70 K using the
spectrum of Y122• as a g-factor reference and yield gx ) 2.00520,
gy ) 2.00420, gz ) 2.00220 with a precision of (0.0001. The
intrinsic EPR line width is on the order of 22 MHz, and only
very large hyperfine couplings can be resolved at 180 GHz.
Pulsed 94 GHz Spectra of [14N]-NH2Y730• in H2O and D2O
Assay Buffer. To resolve and disentangle the hyperfine couplings
associated with the NH2 protons, we recorded pulsed 94 GHz
spectra at 70 K for [14N]-NH2Y730• in H2O and D2O and
computed the point-by-point first derivative spectrum (Figure
5). Both spectra show highly resolved hyperfine interactions
and indicate the expected anisotropy of some of the nuclei
involved. The spectrum in D2O is dominated by two large
hyperfine couplings, one almost isotropic and a second largely
anisotropic. At the high-field edge (B|gz), both hyperfine
couplings are large with a similar size, giving rise to a quartet
with 1:2:2:1 intensity, as observed in the 9 GHz CW spectra.
In the center and at the low-field side (B|gy and B|gx,
respectively) of the 94 GHz spectrum only one large hyperfine
interaction is detected. A large anisotropic interaction is typical
for the 14N nucleus, which has major spin density in the pz orbital
(see below).63,64 Conversely, a large isotropic coupling is typical
for ꢀ-methylene protons that have a C-H bond oriented almost
parallel to the pz orbital of C1 in the aromatic ring (Figure 2),
as has been well documented for Y122• of E.coli RNR and other
Y•s.65-67 The spectrum in H2O shows a substantially different
line shape, which arises from hyperfine contributions of the NH2
Preparation of [15N]-NH2Y-r2. [15N]-NH2Y was incorporated
into R2 as described for [14N]-NH2Y. Purification by dATP
chromatography gave 6 mg of [15N]-Y730NH2Y-R2 with 98%
15N enrichment per gram of wet cell paste. The product was
>95% pure by SDS PAGE (Figure S2). Spectrophotometric
assays revealed a specific activity of 115 ( 12 nmol/min ·mg.
This activity is similar to that observed with Y730NH2Y-R2 (100
( 8 nmol/min ·mg) and will be discussed subsequently.22
CW X-Band EPR Spectroscopy of [14N]-NH2Y730• and [15N]-
NH2Y730•. To characterize the new radical, [14N]- or [15N]-
Y730NH2Y-R2 in H2O or D2O was mixed with ꢀ2, ATP, and
CDP. The reaction was quenched after 20 s and the 9 GHz EPR
spectrum recorded. CDP/ATP were chosen as substrate and
effector due to the amount of radical produced versus other
substrate/effector pairs (data not shown). The observed spectra
were composites of Y122• and NH2Y730• signals. The NH2Y•
signal was obtained by subtracting the Y122• contribution as
described previously.22 The resulting spectra are shown in Figure
3. The spectra of [14N]-NH2Y730• and [15N]-NH2Y730• in D2O
show a marked perturbation relative to each other and relative
to those in H2O, as expected for substitution of 15N with 14N
and substitution of amine protons for deuterons. The spectrum
of [14N]-NH2Y730• in D2O appears as a 1:2:2:1 quartet, consistent
with a doublet of triplets with similar hyperfine couplings. The
triplet hyperfine coupling arises from the 14N nucleus (I ) 1)
and the doublet from one of the ꢀ-methylene protons (I ) 1/2)
of NH2Y. The spectrum of [15N]-NH2Y730• in D2O has collapsed
to an apparent anisotropic triplet, demonstrating the effect of
15N incorporation (I ) 1/2). These isotope-dependent spectral
shifts validate our hypothesis that the new signal is associated
with the NH2Y probe.
(63) Huyett, J. E.; Doan, P. E.; Gurbiel, R.; Houseman, A. L. P.; Sivaraja,
M.; Goodin, D. B.; Hoffman, B. M. J. Am. Chem. Soc. 1995, 117,
9033.
Analysis of NH2Y730• by Pulsed High-Field EPR Spectrosco-
py. To obtain more detailed information on the anisotropy of
the hyperfine interactions and g values, pulsed EPR spectra at
(64) Bleifuss, G.; Kolberg, M.; Po¨tsch, S.; Hofbauer, W.; Bittl, R.; Lubitz,
W.; Gra¨slund, A.; Lassmann, G.; Lendzian, F. Biochemistry 2001, 40,
15362.
(62) Interestingly, in the case of [15N]-NO2Y, 1J, 2J, and 3J couplings were
observed in the 13C NMR spectrum at 15.2, 2.1-2.4, and 1.2-1.6
Hz, respectively. With [15N]-NH2Y, however, only the 1J and 2J
couplings are present in the 13C NMR spectrum, at 9.4 and 1.7-1.9
Hz, respectively. This is consistent with previous NMR studies on
(65) Hulsebosch, R. J.; van den Brink, J. S.; Nieuwenhuis, S. A. M.; Gast,
P.; Raap, J.; Lugtenburg, J.; Hoff, A. J. J. Am. Chem. Soc. 1997, 119,
8685.
(66) Bender, C. J.; Sahlin, M.; Babcock, G. T.; Barry, B. A.; Chandrashekar,
T. K.; Salowe, S. P.; Stubbe, J.; Lindstrom, B.; Petersson, L.;
Ehrenberg, A.; Sjo¨berg, B.-M. J. Am. Chem. Soc. 1989, 111, 8076.
(67) Hoganson, C. W.; Sahlin, M.; Sjo¨berg, B.-M.; Babcock, G. T. J. Am.
Chem. Soc. 1996, 118, 4672.
1
2
3
aniline, which have shown J, J, and J 15N-13C couplings on the
order of 13.9-14.8, 2-4, and 0-1.5 Hz, respectively, depending on
the solvent and concentration.
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J. AM. CHEM. SOC. VOL. 131, NO. 43, 2009 15733

A R T I C L E S
Seyedsayamdost et al.
Figure 4. Multifrequency EPR spectra of NH2Y730• in the presence of CDP/ATP in H2O assay buffer. (A) 180 GHz spectrum at 6 K consisting of Y122• and
NH2Y730• signals. Inset: 180 GHz spectrum at 70 K containing only the NH2Y730• signal. (B) Spectrum of NH2Y730• at 9, 94, and 180 GHz, demonstrating
the higher g resolution with increasing frequency. The spectrum at 180 GHz yields g values of gx ) 2.00520, gy ) 2.00420, and gz ) 2.00220. The error
in these measurements was (0.0001.
that accounted for a coupling at C5 with the parameters of Table
S1 did not show any further improvement. For the [15N]-
NH2Y730• simulation the parameters were identical to those of
[14N]-NH2Y730•, except that the N-nucleus hyperfine constants
were divided by 1.4, based on the difference in the gyromagnetic
ratios of 14N and 15N. The fact that a single substitution in the
15N simulation with 14N nuclear parameters generates an
excellent fit to the [14N]-NH2Y730•/D2O data provides further
strong support that the new signal is associated with a neutral
NH2Y730•.
The parameters obtained from the simulations of the 9 GHz
CW spectra in deuterated buffer were subsequently employed
to simulate the 94 GHz spectrum of [14N]-NH2Y730• in D2O.
The simulation immediately led to satisfactory results. Never-
Figure 5. 94 GHz pulsed derivative EPR spectra of NH2Y730• in H2O (black
theless, it could be improved with the introduction of a small
trace) and D2O (red trace). Experimental parameters: echo sequence: π/2
anisotropy in the hyperfine tensor of the Cꢀ-proton, as previously
) 32 ns, τ ) 248 ns (in H2O), τ ) 260 ns (in D2O), π ) 64 ns; trep ) 5 ms;
found for Y122• at 140 GHz,46 and by introducing a 5° tilt angle
shots/point ) 50; scans ) 700; T ) 70 K. The ticks on the deuterated trace
for the Az 14N-hyperfine tensor axis away from the gz axis toward
indicate the positions of the quartet. See text for details.
the gx-gy plane.41 The best solution at 94 GHz was then tested
protons. The complexity of this spectrum is such that the visible
features cannot be interpreted with simple splitting schemes.
for consistency with the 9 GHz spectra, and finally a global
solution was found, which is shown in Figure 6 with the
parameters in Table 1.
Spectral Simulations. Analysis of the spectra at different
frequencies required a simulation strategy in which the con-
straints posed by the spectra at the different frequencies were
determined independently, as far as possible, and then combined
to find a global solution. The simulation of the 9 GHz CW-
EPR spectra for [15N] and [14N]-NH2Y730• acquired in D2O was
attempted first. In this case, hyperfine coupling to the exchange-
able amine deuterons is very small, thus facilitating the
simulations by lowering the number of unknown hyperfine
coupling constants. Consequently, the hyperfine couplings to
two nuclei were included in the simulations, as seen in the
spectra of Figure 6. One large hyperfine interaction was assumed
to be isotropic, as is typical for ꢀ-methylene protons, whereas
the second one, which generates the triplet pattern and arises
from the 14N nucleus, was set anisotropic with the largest tensor
component at Az. In this initial set of simulations, for simplicity,
all hyperfine tensor axes were assumed collinear with the g
tensor axes. The g values determined by EPR spectroscopy at
180 GHz were held as fixed parameters. The best simulation
for the 9 GHz spectra could be obtained with the two assumed
hyperfine tensors, indicating the couplings to the three ring
protons cannot be resolved but contributed to a line width of
∼11 MHz (see Table 1). A simulation with an additional proton
The analysis of the spectra was next attempted in H2O.
Simulations first focused on the 94 GHz spectra, as the 9 GHz
simulations did not provide sufficient constraints. Two additional
couplings associated with the amino protons were added to the
parameters for simulation of the spectra in D2O. Assuming that
the hyperfine tensor axes of the amino protons were not collinear
with the g tensor axes, simulations were performed by varying
systematically all hyperfine tensor components as well as the
Euler angles between hyperfine and g tensor axes for each
nucleus. Our starting parameters were based on the studies on
the o-aminophenol radical (Table S1). As a starting point two
equivalent tensors for the amino protons with an isotropic
coupling constant of 15 MHz were employed (Table S1, entry
1), as were a set of defined Euler rotations that accounted for
the directions of the N-H bonds within the molecular plane.
After several iterative steps, however, it became clear that the
simulation required nonequivalent dipolar tensor components
for the amino protons and N-H bond orientations noncollinear
with the ring plane. The parameters were further optimized with
the simulations of the 9 GHz spectra, and the global solution is
displayed in Figure 7 with the parameters shown in Table 1. It
is important to note that the simulations do not provide the
9
15734 J. AM. CHEM. SOC. VOL. 131, NO. 43, 2009

Transient 3-Aminotyrosyl Radical
A R T I C L E S
Figure 6. Simulations (blue ) SimFonia; green ) EasySpin) of the first
derivative EPR spectra (red) of [14N]-NH2Y730• and [15N]-NH2Y730• in D2O
assay buffer. (A) [14N]-NH2Y730• at 9 GHz. (B) [15N]-NH2Y730• at 9 GHz.
(C) [14N]-NH2Y730• at 94 GHz. See Table 1 for parameters. The simulations
obtained with EasySpin and the parameters of Table 1 are in agreement
with those from SimFonia.
Figure 7. Simulations (blue ) SimFonia; green ) EasySpin) of the first
derivative EPR spectra (red) of [14N]-NH2Y730• and [15N]-NH2Y730• in H2O
assay buffer. (A) [14N]-NH2Y730• at 94 GHz. (B) [14N]-NH2Y730• at 9 GHz.
(C) [15N]-NH2Y730• at 9 GHz. See Table 1 for parameters. The simulations
with EasySpin appear slightly broader due to the effect of forbidden
transitions.
Table 1. Summary of Parameters (in MHz) Obtained from
Simulation of [14N]-NH2Y730• and [15N]-NH2Y730• EPR Spectra in
Protonated or Deuterated Buffera
Structure of NH2Y•. In order to rationalize the results obtained
in Table 1, it is first necessary to establish the relationship
between the obtained Euler angles (rotation of the A into the g
tensor) and the molecular axes (Figure S3). The detailed location
of the g and hyperfine tensors, the latter specifically for the
nuclei of the NH2 group, is not known a priori. However, we
found that our simulations were very sensitive, within <5°, to
the γ angle of the N-(H2) proton (rotation about H(2)-Az′
collinear with gz, Figure S3), which is 60° (see Table 1). With
the H(2)-Ax tensor axis along the N-(H2) bond and following
the Euler rotations, the angle γ defines the orientation, with
respect to gx, of the N-H(2) bond projection in the gx,y plane,
i.e., the aromatic plane (Figure S3). Assuming that the gx axis
nucleus
|Axx|
|Ayy|
|Azz|
R
ꢀ
γ
14N
2.4
3.4
1.6 30.5
2.2 42.7
60°
60°
|5°|
|5°|
15N
Cꢀ-H
NH2 (H2) 13.0
NH2 (H1) 6.7
30.8 28.0 30.5
4.4 27.6
8.0 18.0 0° to (-40°)
|126°| ( 10° -60° ( 5°
|50°|
a Hyperfine tensor principal values (Aii) and Euler angles (R, ꢀ, γ) are
indicated. The intrinsic EPR line width was 11-13 MHz at 9 and 94
GHz. In some cases, a range of values is indicated, for which the
simulation does not change, as discussed in the text.
absolute sign of the hyperfine interaction matrix, which requires
ENDOR studies.
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A R T I C L E S
Seyedsayamdost et al.
lies along the C-O bond, then the projection of the N-H(2)
bond in the gx,y plane forms an angle of about 120° with the
C-N bond, a fact that is reasonably consistent with a C-NH2
group forming a trigonal pyramid. We note that the error
associated with the uncertainty of the simulated γ angle leads
to some error in the location of gx and, in turn, of the g tensor,
used to deduce the location of the protons and the nitrogen
group, which is difficult to quantify. Nevertheless, the results
for the N-H(1) proton (Table 1) show that the simulation is not
sensitive to the R angle (rotation about H(1)-Az, Figure S3), as
long as it is between 0° and 40°. An R value larger than 0°
represents a distortion of the trigonal pyramidal structure of the
NH2 group, i.e., a tilt of the N-H(1) bond toward the oxygen
atom.
The Euler angle ꢀ for the two amino protons is related to the
dihedral of each N-H bond with respect to the plane of the
aromatic ring (rotation about Ay, Figure S3). The ꢀ values in
Table 1 lead to an out-of-plane angle between 30° for the
N-H(1) and 54° for the N-H(2) bond. Owing to some
uncertainty in the direction of gx (see above paragraph), the
substantial values of the ꢀ angles required in the simulations
still infer that the bond direction of the amino protons is
considerably out of the molecular ring plane. Similar to the γ
angle discussed above, a slight change of the ꢀ angle of N-H(2)
(for instance by 5°) readily lowers the quality of the simulation.
Altogether, the results in Table 1 show that the orientation of
N-H(2) within the amino group seems well established, whereas
the location of the N-H(1) is less defined. We also note that
the hyperfine tensors of these two protons are substantially
different. This is likely caused by an additional dipolar interac-
tion of N-H(1) with the oxygen of the C-O group. We
conclude that the differences in the hyperfine tensors of the
amino protons combined with an R value consistent with a tilt
of N-H(1) toward the oxygen strongly suggest the formation
of an intramolecular hydrogen bond. A structure of the 3-ami-
notyrosyl radical is illustrated in Figure 2.
The 94 GHz simulation is not sensitive to the sign of the
Euler angles ꢀ, in contrast to the 9 GHz simulations. A physical
explanation for this observation arises from the largely aniso-
tropic hyperfine couplings of the amino protons and their
noncollinearity with the g tensor. Accordingly, the Euler
rotations produce large off-diagonal hyperfine tensor elements
in the laboratory frame that contribute more strongly to the low-
frequency rather than to the high-frequency spectra.61 In Figure
7, the 9 GHz simulations appear slightly narrower than the
experimental data. We suggest that some dynamics of the NH2
group could lead to an interconversion of the N-H bond
directions (i.e., change in the sign of ꢀ) and might account for
broadening of the experimental spectra. However, a more
quantitative model is not possible due to the low-resolution of
the 9 GHz spectra and missing information about detailed
conformational restraints of NH2Y730• in the protein environ-
ment. To make this point, a 9 GHz EPR simulation of the [15N]-
NH2Y730• spectrum with different signs of the N-H proton ꢀ
angles is displayed in Figure S4.
29.8 MHz for one of the ꢀ-protons (Table 1), a value of AH ,
13 MHz for the second ꢀ-proton (the coupling is not visible in
the EPR spectra and must be smaller than the intrinsic line
width), and a B1 of 162 MHz,70 one calculates θ1 ) (16 ( 1)°
with θ2 ) (104 ( 1)° and a spin density of 0.2 at C1 (Figure
2). With knowledge of θ1 and θ2 we can extract the dihedral
angle between CR and Cꢀ of the protein chain and pz at C1, which
is (46 ( 1)°. The information about the dihedral angle allows
us to generate a structure of the radical in R2 as illustrated in
the discussion.71
Discussion
We have previously shown that site-specific insertion of
NH2Y into the PCET pathway in R2 results in production of a
new radical, concomitant with loss of the Y122•. The surprising
observation that this mutant can support deoxynucleotide
production suggests this new radical is actually an intermediate
on the PCET pathway.22 In the present work, by 15N substitution
of the amino group of Y and by acquiring data in H2O and
D2O, we provide direct evidence that this new species is
NH2Y730• and obtain insight into its molecular and electronic
structure.
Our experiments and analysis have allowed us to determine
the g values and the largest hyperfine tensors of NH2Y730
•
(Figure 2). The g values, gx ) 2.00520, gy ) 2.00420, gz )
2.00220, have been resolved using 180 GHz pulsed EPR
spectroscopy and are in the range of those previously reported
for phenoxy and ortho-substituted phenoxy radicals.72-77 Com-
parison of the g values of NH2Y730• with those for Y122• (gx )
2.0091, gy ) 2.0046, gz ) 2.0023 for Y122•)46 reveal that gy
and gz are similar and that the gx shift is significantly suppressed.
Two mechanisms have been suggested for gx shifts in phenoxy
radicals and their derivatives. The first is ortho substitution of
the phenoxy ring with a heteroatom such as O or S, and the
second is H-bonding to the phenoxy radical oxygen.54,72-75,77
High-frequency EPR studies on phenoxy radicals substituted
at the ortho position with a thio-ether group such as that found
in galactose oxidase have revealed a gx value of 2.007 with axial
g tensor symmetry.75,77 For o-hydroxyphenoxyl radicals coor-
dinated to a metal, the gx values varied between 2.0045 and
2.006,72 and in our recent studies with Y356DOPA-ꢀ2,24,78 a gx
value of 2.0056 with an axial g tensor was observed upon
formation of the DOPA• (M. Bennati and J. Stubbe, unpublished
results).
On the other hand, suppression of gx has been found
experimentally and validated by several theoretical studies to
(69) Heller, C.; McConnell, H. M. J. Chem. Phys. 1960, 32, 1535.
(70) Fessenden, R. W.; Schuler, R. H. J. Chem. Phys. 1963, 39, 2147.
(71) We note that the large hyperfine coupling of the amine nitrogen
suggests that it bears significant unpaired spin density consistent with
recent computational work. Additional studies are in progress, using
the structural features obtained herein as input, to delineate the
complete distribution of the spin density.
(72) Brezgunov, A. Y.; Dubinskii, A. A.; Poluektov, O. G.; Prokof ’ev,
A. I.; Chemerisov, S. D.; Lebedev, Y. S. J. Struct. Chem. 1992, 33,
69.
Finally, the largest hyperfine coupling occurs at the ꢀ-me-
thylene proton and is due to hyperconjugation with the C1 of
the aromatic ring (Table 1, Figure 2). Its strength is modulated
by θ, the dihedral angle between the Cꢀ-H bond and the pz
orbital at C1, and by the spin density at C1 according to AH ≈
(B1 × FC1 × cos2 θ).68,69 Using the isotropic coupling value of
(73) Engstrom, M.; Himo, F.; Agren, H. Chem. Phys. Lett. 2000, 319, 191.
(74) Kaupp, M.; Gress, T.; Reviakine, R.; Malkina, O. L.; Malkin, V. G.
J. Phys. Chem. B 2003, 107, 331.
(75) Gerfen, G. J.; Bellew, B. F.; Griffin, R. G. J. Phys. Chem. 1996, 100,
16739.
(76) Himo, F.; Eriksson, L. A.; Blomberg, M. R. A.; Siegbahn, P. E. M.
Int. J. Quantum Chem. 2000, 76, 714.
(77) Lee, Y. K.; Whittaker, M. M.; Whittaker, J. W. Biochemistry 2008,
47, 6637.
(68) McConnell, H. M. J. Chem. Phys. 1956, 24, 764.
(78) Seyedsayamdost, M. R.; Stubbe, J. J. Am. Chem. Soc. 2007, 129, 2226.
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15736 J. AM. CHEM. SOC. VOL. 131, NO. 43, 2009

Transient 3-Aminotyrosyl Radical
A R T I C L E S
be associated with intermolecular hydrogen bonding to the
phenoxy radical oxygen.54,79 Indeed, the gx value of Y•s in
various RNRs has been used as a diagnostic for the presence
of a H-bond. Owing to the complexity of the electronic structure
of the NH2Y730•, which contains a heteroatom at the ortho
position as well as an intramolecular H-bond (Figure 2), its low
gx value (2.0052) could be associated with either or both of
these mechanisms. Furthermore, the putative intermolecular
H-bonding network at NH2Y730• (Figure 1) may also contribute
to suppression of gx and is the subject of current ENDOR
studies. Nevertheless, the form of the rhombic g tensor is
characteristic for the NH2Y730• radical and is visible not only
at 180 but also at 94 GHz.80 With this information at hand, it
will be possible to distinguish an NH2Y• from other Y•s within
enzymes using high-frequency EPR, if NH2Y is inserted as a
probe to trap radical intermediates.
There are several important features of the electronic structure
of NH2Y730• that have resulted from our study. First, the
requirement for two amino protons in the simulations, along
with their inequivalent hyperfine interactions with the nitrogen,
is more consistent with an sp3- and not an sp2-hybridized
nitrogen. Second, our model suggests the presence of an
intramolecular H-bond between the N-H(1) and the oxygen. Our
observations are in remarkable agreement with earlier experi-
mental and theoretical studies by Loth et al. on the o-
aminophenoxyl radical.41 Their studies suggested an out-of-plane
angle of 30° (related to our ꢀ angle; see above and Figure S3)
for the amino protons in an sp3-hybridized nitrogen, a confor-
mation proposed to stabilize the internal H-bond with the
phenoxyl oxygen. Our simulations is consistent with a 5° tilt
angle of the C-N bond relative to the ring plane (above or
below). Loth et al. found a similar tilt angle stabilized the
aminophenol radical by g5 kcal/mol and increased the isotropic
hyperfine coupling constant of 14N to match their experimental
value of about 8.4 MHz. This hyperfine coupling is similar to
our isotropic value of about 11 MHz assuming all tensor
elements either positive or negative. On the other hand, the
hyperfine couplings of their amino protons (Table S1) are
different from those determined herein, although only average
coupling values, and not full hyperfine tensors, were reported.
Finally, recent DFT calculations predicted formation of an
H-bond upon oxidation of o-aminophenol,76 in agreement with
our results and those of Loth et al.81
di-tert-butyl-2-tert-butyliminosemiquinone radical. The structure
showed bond lengths for C-N and C-O of 1.34 and 1.26 Å,
respectively, values intermediate between the reduced and
oxidized forms of this molecule. They reported an EPR spectrum
at 9 GHz recorded at room temperature in DMSO; however,
no simulations of the spectrum were presented. Their protonated
imino structure in which the N is sp2-hybridized is inconsistent
with our EPR data (see Figure S5 for simulations using their
model). Interestingly these authors also reported that their
observed radical has an absorption feature at 734 nm with an
ε734 that varied between 700 and 900 M-1 cm-1 depending on
solvent. These studies provoked a re-examination of the visible
spectrum of our NH2Y• in R, where we detected a broad
absorption feature between 700 and 760 nm with an ε735 of 800
M-1 cm-1 (Figure S6).
NH2Y was chosen as a probe, as it is 190 mV easier to oxidize
than Y at pH 7 and can function as a radical trap.33 It was hoped
that trapping the NH2Y• in conjunction with high-field EPR and
ENDOR spectroscopies could provide evidence for or against
the proposed collinear PCET process to oxidize C439 in R2. The
strength of the probe design is also its weakness: the intramo-
lecular H-bonding within NH2Y• makes it a more efficient
radical trap, but potentially complicates spectral analysis, as it
is not present in Y•. A complete understanding of the electronic
structure is thus essential, and computational studies are in
progress to understand the unpaired spin density distribution.
However, based on our observation that the NH2Y• can function
as a catalytically competent radical in nucleotide reduction and
on thermodynamic arguments, it is likely that oxidation of C439
occurs through hydrogen atom abstraction.22
Our EPR analysis has provided constraints to model NH2Y
at residue 730 in R in order to obtain insight into its position
relative to Y731 and C439. The hyperfine coupling of the ꢀ
methylene proton of the NH2Y• allows calculation of the
dihedral angle between CR and Cꢀ of the protein chain and pz
at C1 (θ ) 46° ( 1°). It is similar to the one determined for
Y730 in the crystal structure of wt R2 (θ ) 36°).14 Thus,
replacement of Y730 with NH2Y730 does not appear to signifi-
cantly alter the orientation of this residue. Four orientations of
the amino group are possible. Two of these orientations cause
steric clashes with the protein. Recent structural characterization
of NO2Y-R2 and NH2Y-R2s (Uhlin, Yokoyama, Minnihan, and
Stubbe, unpublished results) suggest that ortho substitution
requires the ring to have the orientation shown in Figure 8, thus
eliminating a third possible orientation (Figure S7). In this
model, the amino protons of NH2Y730• cannot form any
hydrogen bonds except the internal one. The distances to Y731
and C439 are 3.3 and 3.5 Å, respectively. Thus, this modeling
suggests that the amino group does not affect the distances
between the essential amino acids in the proposed PCET
pathway, indicating that the NH2Y probe can be used to measure
the distance to the protons on Y731 (OH) and C439 (SH). The
coupling to these two protons might be visible in high-field
ENDOR experiments using D2O buffer, a protocol that permits
observation of exchangeable protons and their orientation. The
experiment will be aggravated by the concomitant observation
of the amino protons, which are also exchangeable. Therefore,
the independent determination of the tensors for the amino
Additional insight into the structure of the NH2Y• is provided
by a recent report of Carter et al.82 on the synthesis and structural
characterization of a neutral imino-semiquinone radical, 4,6-
(79) Bennati, M.; Stubbe, J.; Griffin, R. G. Appl. Magn. Reson. 2001, 21,
389.
(80) The g values of the NH2Y• radical incorporated at position Y731 in R2
are the same as those for NH2Y730• (M. Bennati and J. Stubbe,
unpublished results).
(81) Preliminary calculations on the isolated NH2Y• lead to a planar amino
group. While this is a reasonable result, it is at variance with the
experimental data obtained in this work. This strongly suggests a
significant impact of the protein environment. Cluster and QM/MM
calculations with more than 100 atoms in the quantum region are
planned to clarify this point.
(82) Carter, S. M.; Sia, A.; Shaw, M. J.; Heyduk, A. F. J. Am. Chem. Soc.
2008, 130, 5838.
(83) Babcock, G. T.; Espe, M.; Hoganson, C.; Lydakis-Simantiris, N.;
McCracken, J.; Shi, W. J.; Styring, S.; Tommos, C.; Warncke, K. Acta
Chem. Scand. 1997, 51, 533.
(86) Bhattacharjee, S.; Deterding, L. J.; Jiang, J.; Bonini, M. G.; Tomer,
K. B.; Ramirez, D. C.; Mason, R. P. J. Am. Chem. Soc. 2007, 129,
13493.
(84) Pujols-Ayala, I.; Barry, B. A. Biochim. Biophys. Acta 2004, 1655,
205.
(85) Gupta, A.; Mukherjee, A.; Matsui, K.; Roth, J. P. J. Am. Chem. Soc.
2008, 130, 11274.
(87) Rigby, S. E. J.; Hynson, R. M. G.; Ramsay, R. R.; Munro, A. W.;
Scrutton, N. S. J. Biol. Chem. 2005, 280, 4627.
9
J. AM. CHEM. SOC. VOL. 131, NO. 43, 2009 15737

A R T I C L E S
Seyedsayamdost et al.
the evolved tRNA/tRNA synthetase pair to incorporate a NH2Y
site specifically into any protein and our characterization of the
NH2Y• reported herein should make this a useful probe to study
PCET processes in general.
Acknowledgment. We thank Prof. T. Prisner (Univ. of Frank-
furt) for allowing us to record the 180 GHz spectra and Dr. J.
Fritscher for valuable discussions. We are indebted to Prof. Frank
Neese and Christoph Riplinger (Univ. of Bonn) for starting quantum
chemical calculations on NH2Y730• incorporated into R2 in support
of this work. We also thank one of the anonymous reviewers for
the constructive feedback. This work was supported by the NIH
grant 29595 to J.S., the DFG-IRTG 1422 (T.A. and M.B.), and the
Max Planck Society.
Supporting Information Available: Summary of EPR param-
eters previously reported for the o-aminophenol radical; UV
spectral properties of authentic [14N]-NH2Y and synthetic [15N]-
NH2Y determined in H2O at 25 °C; numbering scheme for the
spectral data on the o-aminophenol radical data in Table 1; SDS
PAGE analysis of purified [15N]-NH2Y730-R2; Euler rotations
for the N-(H1) and N-(H2) protons; simulations of the 9 GHz
CW-EPR spectrum of [15N]-NH2Y730• for the four possible
combinations of the ꢀ angle signs; simulations of the 9 and 94
GHz spectra using a planar NH2 arrangement; spectrum of the
730 nm feature associated with NH2Y•; models of the
Y731-NH2Y730-C439 pathway; synthetic scheme used for prepa-
ration of 15N-NH2Y; different protonation states and pKas of
NH2Y. This material is available free of charge via the Internet
at http://pubs.acs.org.
Figure 8. Model of the Y731-NH2Y730-C439 pathway in NH2Y730-R2.
NH2Y has been modeled into residue 730 of R2 using the coordinates from
ref 14. The pz orientation and Cꢀ- and amino proton dihedral angles are
based on results herein. The distance between the NH1 and the phenol O is
indicated. The distances between the essential residues around NH2Y730 are
illustrated as discussed in the text.
protons reported herein is a prerequisite for future ENDOR
studies with NH2Y. These ENDOR studies as well as studies
with C439S-R2 variants are currently underway.
Finally, understanding PCET within proteins is a major focus
of interest of many laboratories, and transient Y•s have been
identified in many of these processes.83-87 The availability of
JA903879W
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15738 J. AM. CHEM. SOC. VOL. 131, NO. 43, 2009