From the
Ferrous iron/oxygen reconstitution of the mutant R2 apoprotein
Y122F leads to formation of a diferric center similar to that of the
wild-type R2 protein of Escherichia coli ribonucleotide
reductase. This reconstitution reaction requires two extra electrons,
supplied or transferred by the protein matrix of R2. We observed
several transient free radical species using stopped flow and freeze
quench EPR and stopped flow UV-visible spectroscopy. Three of the
radicals occur in the time window 0.1-2 s, i.e. concomitant with formation of the diferric site. They include a
strongly iron-coupled radical (singlet EPR signal) observed only at
Ribonucleotide reductases convert ribonucleotides to the
corresponding deoxyribonucleotides via a radical mechanism in an
allosterically regulated reaction (Reichard, 1993; Mao et al.,
1992; Sjöberg, 1994). The enzyme from Escherichia coli, a
model for the so called class I ribonucleotide reductases (Reichard,
1993), consists of two homodimeric proteins, R1 and R2. Protein R1
binds substrate and allosteric effector molecules. The active form of
protein R2 carries one diferric site/polypeptide chain and an adjacent
stable free radical on Tyr-122. The iron site consists of two high spin
ferric ions that are antiferromagnetically coupled by a µ-oxo
bridge. The tyrosyl radical is stable for days at 25 °C (Atkin
et al., 1973) and has been identified as an oxidized
deprotonated radical (Backes et al., 1989; Bender et
al., 1989). ApoR2 designates the form that lacks both iron center
and radical, and is obtained after chelation of the irons under mild
denaturing conditions (Atkin et al., 1973) or from
overproducing bacteria grown in iron-depleted medium (et al., 1993). The addition of Fe
From the crystal structure (Nordlund et
al., 1990) it is known that the tyrosyl radical and iron center
are embedded deep inside the protein, far from the substrate binding
site on protein R1. It is hence postulated that the involvement of the
tyrosyl radical in the substrate reaction requires long range electron
transfer. A network of hydrogen-bonded residues in R2, including
Tyr-122, two ligands to Fe1 (Asp-84, His-118), an intervening aspartic
acid (Asp-237), and a tryptophan (Trp-48) at the postulated interaction
surface with R1 has been proposed as a possible specific long range
electron transfer route (Nordlund et al.(1990), Nordlund and
Eklund(1993), cf. Beratan et al.(1992) and Franzen
et al.(1993)). Based on protein engineering studies, the
C-terminal residue Tyr-356, and possibly Glu-350, has also been
suggested as the most R1 proximal partner in the proposed electron
transfer chain (Climent et al., 1992; Sjöberg, 1994). In
mouse protein R2, mutations of the Asp and Trp residues, corresponding
to Asp-237 and Trp-48 mentioned above gave enzymatically inactive
mutant proteins, despite the fact that they contained iron and a stable
tyrosyl radical and bound normally to protein R1.
The R2 activation reaction (formation of diferric center
and tyrosyl radical) has been studied in extensive detail (Peterson
et al., 1980; Lynch et al., 1989; Sahlin et
al., 1990; Fontecave et al., 1990; Ochiai et
al., 1990; Bollinger et al., 1991a, 1994a, 1994b). It can
be formulated as
On-line formulae not verified for accuracy REACTION 1where P denotes the polypeptide chain and TyrOH denotes tyrosine 122.
The reduction of O
We
have studied the corresponding reaction (Reaction 1) in the mutant
protein R2 Y122F. Formation of the diferric center, in the reaction
between apoprotein, Fe
Rapid freeze quenching (about 0.3-s reaction at room temperature)
was achieved by using syringes driven by a Harvard 22 pump (Fa.
Kleinfeld Labortechnik, Hannover, FRG) and a homebuilt mixing chamber.
The reaction was stopped by spraying the mixture into isopentane at 170
K. The frozen spray was densely packed in EPR tubes and thereafter kept
at 77 K. Longer reaction times were achieved by mixing equal volumes of
anaerobic Fe
Quantitation of components
I and II showed that the concentration of the two components were
roughly equal at the 6-s reaction time and that each corresponded to
about 0.1 unpaired spins/R2. The amount of both components increased up
to four to five Fe
EPR spectra of the
quartet signal, component II, show that its linewidth increases
considerably up to 260 K leading to a 17-times decrease in amplitude.
These spectral changes are reversible, the spectra of the sample
recooled to 77 K and recorded at 77 K exhibits the same line shape
(quartet) and intensity as before the annealing (data not shown). These
results suggest that component II is magnetically interacting with the
iron site. In order to estimate whether component I or II is in fact
coupled with the iron center, apoR2 Y122F (with indole deuterated
tryptophan) was reconstituted with
Attempts to observe possible
deuterium effects in the rapidly decaying room temperature singlet have
so far not given any conclusive results.
Reconstructed
spectra between 350 and 450 nm are shown in Fig. 9, A and B for 4 and 2 Fe/R2, respectively. The transient
spectral component with a peak at 410 nm is clearly visible in the
three first reconstructed spectra from the Fe/R2 ratio of 2, whereas it
is hardly discernible in the reaction with 4 Fe/R2, also in agreement
with Bollinger et al. (1991a). In the present experiment with
2 Fe/R2 (Fig. 9B) the radical concentration at 0.3 s
corresponds to 0.1 radical/R2 (subtracting the 5-s spectrum from that
of 0.3-s), assuming the same absorbance index
In the generation of active ribonucleotide reductase protein
R2, oxidation of reduced R2 (ferrous form) by molecular oxygen leads to
formation of the diferric center and the oxidized tyrosyl radical at
Tyr-122 (see Reaction 1 above). Even though the reduction of molecular
oxygen to water requires four electrons, only three can be accounted
for by oxidation of the iron ions and tyrosine 122. Some transient
intermediates have recently been trapped in this fast reaction
(Bollinger et al., 1991a, 1994a, 1994b). Our approach to
identify possible further electron donors in this reaction has been to
modify and slow down the process by using the mutant R2 protein Y122F
and thereby removing one of the wild-type electron donors. Some studies
on reconstitution of apoR2 Y122F have previously been reported
(Bollinger et al., 1991, 1994a, 1994b; Ravi et al. 1994; Sahlin et al., 1994). Using stopped flow (0.1 s to
1 h) and rapid freeze (0.3 s to 20 min) EPR spectroscopy, as well as
stopped flow (0-10 s) and conventional (10 s to 20 min)
UV-visible spectroscopy, we here report on six transient EPR visible
species and two transient light absorption spectra. One of the
transient EPR species relates to a half-oxidized (semi-Met,
Fe
The room temperature singlet EPR spectrum is similar in line
shape to the 77 K singlet and occurs in the same time window (maximal
concentration at 0.3 s). Could the two species that give rise to the
singlet spectra at room temperature and 77 K be the same? We consider
this unlikely since the strongly metal-coupled free radical observable
at 77 K is not likely to have an observable EPR spectrum at room
temperature.
The stopped flow light absorption studies with single
and double mutant derivatives assigned the transient 410 nm species to
a tyrosyl radical at Tyr-356. The kinetic behavior of the Tyr-356
radical coincides approximately with the singlet EPR spectra observed
at room temperature and 77 K. However, the EPR spectra of the double
mutant protein Y122F/
In summary,
our data suggest that there are at least three intermediates (room
temperature and 77 K EPR singlets, Tyr-356 410-nm radical) occurring
before or concomitant with formation of the diferric center in R2
Y122F. At least one species is closely coupled with the iron site (77 K
singlet), whereas we expect the room temperature singlet EPR species
not to have strong paramagnetic metal interaction since it is
observable at room temperature. The Tyr-356 radical is at the surface
of R2 and more than 10 Å from the iron site. In comparison with
studies by Bollinger et al. (1991a, 1991b, 1994a, 1994b) on
the reconstitution of Y122F, we note both similarities and differences.
A short-lived tyrosyl radical and a strongly iron-coupled singlet
(called X or diferric radical
(Fe
The major differences between the results
of Bollinger et al. and ours occur in species observed after
formation of the diferric center (see below). Although we cannot at
present explain the origin of these differences, we note that the
reconstitution experiments have been performed differently in the two
research groups. We have mixed the apoprotein solution with an
anaerobic neutral ferrous mixture, whereas Bollinger et al. have worked with an aerobic acidic ferrous solution that is
neutralized by mixing with the buffered protein. Perhaps some of the
differences relate to transient local pH effects or to availability of
oxygen.
The transient broad
light absorption, with
The
line shape of component I at 77 K with the axial spectrum and g values 2.036 and 2.009 is very similar to that observed for the
tryptophan radical in cytochrome c peroxidase (CCP). However,
the latter signal was observable only at 4 K (Hoffman et al.,
1981; Sivaraja et al., 1989; Houseman et al., 1993).
The line shape of the cytochrome c peroxidase tryptophan
radical, which is quite unexpected for a planar
Another transient tryptophan radical was observed by
stopped flow EPR experiments. The doublet EPR signal implies that its
hyperfine structure is due to one major
A simple
explanation as to why the low temperature signals are not observed at
room temperature is that they are broadened beyond detection at 25
°C, as was experimentally verified for component II (quartet Trp).
The room temperature broadening of the EPR signals of components I and
II is probably related to paramagnetic metal interaction. This is
supported by their observed microwave saturation behavior
(), which shows that they have an even more rapid
relaxation than the tyrosyl radical at Tyr-122 in active protein R2
(Ehrenberg and Reichard, 1972). It is less straightforward to explain
why the room temperature doublet is not visible in the 77 K spectra of
25 s or longer incubation times. It might be saturated and broadened
even at low microwave powers so that it becomes masked by the other low
temperature components.
In summary, we observe at least two,
possibly three, different tryptophan radicals (the 77 K quartet or 545
nm band, the room temperature doublet, and perhaps the 77 K component
I) after formation of the diferric center. These radicals differ in
molecular geometry, in the extent of metal interaction and in the
kinetics of appearance. The geometry of the tryptophan radical residues
relative to the protein backbone may be estimated from the relative
magnitudes of the
The isotropic
Possible candidates to harbor these transient
radicals are Trp-48, Trp-107 and Trp-111, which are at distances of
4-8 Å from the iron center and have distinctly different
geometries in the three-dimensional structure of R2 (Nordlund et
al. 1990; Nordlund and Eklund, 1993). The geometry of Trp-111,
which is
In the cell respiration process, the O
The water splitting reaction in
photosystem II involves the channeling of electrons from the substrate
water, via a four-manganese cluster and a tyrosyl radical on the
oxidizing side (Andersson and Styring, 1991). Also in this case protons
are translocated during the reaction. One could speculate that a
tyrosyl radical may be a useful one-electron gate in a system otherwise
set up to transfer an even number of electrons. This seems certainly to
be the case in ribonucleotide reductase, where the catalytic reaction
is proposed to be initiated by long range one-electron transfer to the
tyrosyl radical (Stubbe and Ackles, 1980; Nordlund et al.,
1990).
Taken together, these results imply that the four-electron
reduction of O
The generally less than
stoichiometric amounts of radical in purified protein R2 may reflect
the considerable problem to keep a stable free radical inside a dynamic
protein. This also points to the obvious need for the living cell to be
able to regenerate the radical continously by redox reactions involving
iron and oxygen. The reason for the extremely slow rate of electron
transfer to the iron-oxygen reaction site in the present mutant study
can be rationalized in terms of the highly protected environment of
this site. In the resting state of the native protein R2, the tyrosyl
radical at Tyr-122 has to be effectively shielded from external
reductants. Protein R2 (and the R1
The present study revealed two or more
tryptophan radicals and most likely one tyrosine radical upon
reconstitution of protein E. coli R2 Y122F, i.e. residues of the kinds that have been suggested to function in long
range electron transfer. The tyrosyl radical on Tyr-356 is even
localized on a conserved residue that is supposed to have this function
(Climent et al., 1992; Sjöberg, 1994). A speculative
suggestion is that some species observed in the present study may be
functional in long range electron transfer also under physiological
conditions in wild-type R2 and may become long-lived enough to be
observed here, since the electron transfer processes are not effective
enough in the Y122F mutant protein.
We thank Agneta Slaby for invaluable technical
assistance, Christof Gessner of the Max-Volmer-Institute, Technical
University, Berlin for help with Q-band EPR measurements, and Per
Ingvar Olsson of the Department of Medical Chemistry and Biophysics,
Umeå University for performing the mass spectrometric analyses.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
77 K, a singlet EPR signal observed only at room temperature, and a
radical at Tyr-356 (light absorption at 410 nm), an invariant residue
proposed to be part of an electron transfer chain in catalysis. Three
additional transient radicals species are observed in the time window 6
s to 20 min. Two of these are conclusively identified, by specific
deuteration, as tryptophan radicals. Comparing side chain geometry and
distance to the iron center with EPR characteristics of the radicals,
we propose certain Trp residues in R2 as likely to harbor these
transient radicals.
to apoR2
under anaerobic conditions results in reduced R2 with a diferrous
center (Sahlin et al., 1989). The addition of oxygen to
reduced R2 leads to immediate generation of active R2, and it was
recently shown that the µ-oxygen of the diferric center is
initially derived from molecular oxygen (Ling et al., 1994).
Protein R2 has also been characterized in a radical-free state, metR2,
where the diferric site remains but the tyrosyl radical is reduced to a
normal tyrosine residue.
(
)
to water requires four electrons. Three
electrons are supplied via oxidation of the two ferrous ions and
formation of the oxidized tyrosyl radical. The fourth reducing
equivalent may be supplied by additional Fe
ions, at
least in vitro (Ochiai et al., 1990; Bollinger et
al., 1991a). Recent studies by Bollinger et al. (1991a,
1991b, 1994a, 1994b) have revealed two transient free radical species
that are formed in the activation reaction prior to formation of the
tyrosyl radical. One species is suggested to be a tryptophan free
radical and the other an iron-coupled radical of unknown origin.
and O
, hereafter
called reconstitution of R2 Y122F, requires supply of two additional
reducing equivalents besides the two ferrous ions in the iron site,
since the mutant protein cannot form the stable tyrosyl radical. In the
reconstitution of R2 Y122F, we have observed six transient paramagnetic
intermediates by stopped flow and freeze quench EPR
(
)
and two intermediates with UV-visible spectroscopy.
Reconstituted protein R2 Y122F has, after decay of the transient
species, most features common with the metR2. In an earlier report, two
of the free radical species were identified as being located on
tryptophans (Sahlin et al., 1994). In the present report, all
six EPR visible species have now been further characterized kinetically
and chemically. In a first attempt to assign the different
intermediates to specific residues in R2, we have studied the
time-resolved reconstitution reaction in the two double mutants
Y122F/Y356A and Y122F/
30C. The appearance in time of the different
intermediates relative to the formation of the diferric center may be
taken as an indication of which radicals may be part of the normal
electron transfer pathways during the Fe
/O
reaction and which are a result of the lack of the normal
electron donor, i.e. tyrosine 122. An attempt is made to
correlate the different radicals with electron routes in protein R2,
and plausible structures/residues are suggested for all observed
transients.
Materials
L-Tryptophan-indole-d (98% deuterium) from Cambridge Isotope Laboratories,
-d
-DL-tyrosine was prepared as
described elsewhere (Lindström et al., 1974; Achenbach
and König, 1972).
Fe foil (95.2% iron-57) was from U.
S. Services Inc.
Protein Purification
Wild-type protein R2 as well
as proteins Y122F, Y122F/30C, and Y122F/Y356A were purified as
described earlier (Sjöberg et al., 1986). Apo forms of
the different proteins were either produced by treating the protein
with a chelating agent (Atkin et al., 1973) or for Y122F by
purification from E. coli grown in iron-depleted medium
(et al., 1993). Overproduction of mutant proteins
was obtained in the E. coli strain MC1009 containing plasmids
pGP1-2 and MK5, the latter being a recombinant derivative of
pTZ18R containing the mutant nrdB gene
(
)
(Climent et al., 1992; Larsson and Sjöberg,
1986). Apoproteins with deuterated amino acids were prepared using the
low iron medium procedure.
Conditions for Reconstitution of apoR2 with Ferrous Ions
and Oxygen
All kinetic reactions were performed at room
temperature (25 °C). ApoR2 (50 or 100 µM) in
air-saturated 50 mM Tris, pH 7.6, was mixed with an equal
volume anaerobic solution of ferrous ammonium sulfate (0.05-1.0
mM) in 50 mM Tris, pH 7.6. Fe foil was
dissolved in 10 M HCl to give a stock solution of 120
mM in iron. Immediately prior to an experiment the stock
solution was diluted in anaerobic 200 mM Tris, pH 7.6, to 0.4
mM
Fe
, which gives a neutral
pH.
Stopped Flow EPR Spectroscopy
An EPR spectrometer
ESP 300E (Bruker) was coupled with a stopped-flow EPR accessory
specially designed for biological applications as described previously
(Lassmann et al., 1992). The volume of each reactant was 70
µl/shot. For the recording of EPR spectra of short living transient
species, a rapid field scan was started by an external trigger of the
stopped-flow apparatus immediately after stopping the flow. For the
longer living transient species, EPR spectra were recorded after
varying delay times. Since the EPR stopped flow equipment could not be
thermostatted, all reactions were performed at room temperature with
prewarmed solutions. Kinetic measurements were performed at a field
corresponding to the maximum of the EPR first derivative line of the
studied species. The kinetic scan was initiated by a trigger of the
closing valve of the stopped flow apparatus.
Freeze Quench EPR Spectroscopy
Low temperature EPR
spectra were recorded, with the ESP 300E spectrometer, at 77 K using a
cold finger Dewar or at 11 K using an Oxford-Instrument cryostat. An
active R2 with known radical concentration, originally calibrated
against a Cu-EDTA standard sample, was used for
quantitation of the radical species, comparing the double integrals.
solution and apoR2 protein
directly in the EPR tube and freezing the mixture after 6, 10, 25, 60,
600, or 1200 s incubation at 25 °C by immersing the EPR tube in
cold isopentane (170 K).
Stopped Flow UV-visible Spectroscopy
A DX.17MV
BioSequential stopped-flow ASVD spectrofluorimeter from Applied
Photophysics was used with 55 µl of each reactant/shot. Spectra
were compiled from kinetic traces at every fifth nanometer in the range
450-350 nm absorption. Kinetic spectra between 480-600 nm
were recorded with a speed of 480 nm/min in a Perkin Elmer 2
spectrophotometer.
Mass Spectrometry
In order to ascertain that the
deuterated amino acids had been properly incorporated into the
proteins, we determined the masses of the apoproteins of wild-type R2,
R2 Y122F, R2 [-d
-Tyr]Y122F,
and R2 [indole-d
-Trp]Y122F.
Approximately 3 nmol of protein R2 was dissolved in 1 ml of 0.1% acetic
acid and injected into an electrospray mass spectrometer from Perkin
Elmer (API
LC/MS system). The results presented in
clearly show that deuterated tyrosine or deuterated
tryptophan had been incorporated. The extremely good agreement between
the measured and calculated masses makes electrospray mass spectrometry
a potent method for confirming the correctness of incorporation of
isotope-labeled amino acids, as well as of single point mutations, in
this large protein.
Room Temperature EPR Spectroscopy of Tyrosyl Radical in
Wild-type Protein R2
With the present experimental setup for stopped flow EPR
studies, formation of the typical doublet EPR spectrum of the stable
tyrosyl radical at Tyr-122 in protein R2 can be observed at room
temperature (Fig. 1). The large line width (2 mT) at room
temperature is due to the interaction of the radical with the diferric
center (Gräslund et al., 1985; Sahlin et al.,
1987). The rate of formation of the tyrosyl radical could be followed
kinetically by EPR (Fig. 1, inset), and the rate
constant for this reaction was obtained as 2.5 s
(for a Fe/R2 ratio of 4). This rate constant is in reasonable
agreement with the corresponding value of
1 s
at 5 °C, reported by Bollinger et al. (1991a; 1994b)
using light absorption spectroscopy and limiting iron conditions (ca. 2
Fe/R2).
Figure 1:
EPR doublet spectrum at room
temperature of the tyrosyl radical at Tyr-122 in wild-type E.
coli. The spectrum was recorded immediately after 1:1 mixing of
aerobic apoprotein (50 µM) with anaerobic Fe (200 µM) in the stopped flow apparatus. Recording
conditions: microwave power, 40 mW; modulation, 0.5 mT; sweep time, 84
s; time constant, 0.3 s. Inset, kinetics of formation of the
EPR signal observed at the field position indicated by the upperarrow (microwave power, 40 mW; modulation, 2.8 mT; time
constant, 20 ms).
Low Temperature EPR Spectroscopy during Reconstitution of
Apoprotein R2 Y122F
EPR Singlet at 77 K
Freeze quenching after about
0.3 s of reconstitution of apoR2 Y122F with an Fe/R2 ratio of 4 gives
rise to an EPR singlet with g = 2.001 and a line width of about
2.6 mT (Fig. 2). The signal could not be saturated with available
microwave power (200 milliwatts) at 77 K, suggesting that the signal is
due to a free radical species that is very closely coupled to the iron
center. This transient signal, which is similar to that described by
Bollinger et al. (1991a, 1991b, 1994a, 1994b) for wild-type as
well as Y122F protein, is not present in samples that were freeze
quenched after 6 s or longer reaction times (see below).
Figure 2:
EPR singlet spectrum at 77 K from the
mutant E. coli R2 Y122F after freeze quenching of the reaction
at 25 °C of aerobic apoR2 (50 µM) with anaerobic
Fe (200 µM). Reaction time, 0.3 s.
Recording conditions: microwave power, 10 mW; modulation, 0.2 mT; sweep
time, 41 s; time constant, 0.6 s.
Composite EPR Spectra at 77 K
A very different and
complex EPR spectrum appears after a 6-s reconstitution of apoR2 Y122F
with an Fe/R2 ratio of 4 (Fig. 3A). With even longer
reaction times, the shape and intensity of the spectrum changes
(Fig. 3D), suggesting a composite spectrum with at least
two different paramagnetic species of different life times. The
presence of two different EPR species was confirmed by the microwave
saturation behavior of the different parts of the composite spectrum.
Extreme points 1 and 3 in Fig. 3E have P (as
defined in Sahlin et al., 1986) about 14 mW, whereas points 2
and 4 have P 160 mW at 77 K (data not shown). In a study of
reaction time dependence, peaks 1 and 3 showed a common decay with a
half-life of 7.5 min, whereas peaks 2 and 4 decayed faster (t
1.8 min). The longer living species (peaks 1 and 3) is
essentially the only visible EPR species at 77 K after 10-min reaction
time (Fig. 3D). This species, hereafter denoted
component I, exhibits a typical line shape of axial
g-anisotropy (g
= 2.036 and
g
= 2.009; Fig. 3D).
The shorter living species is denoted component II. Its line shape,
dominating at high microwave power (Fig. 3E), can be
obtained by partial subtraction of component I from the composite
spectrum. Component II may be described as a hyperfine quartet
spectrum, as will be presented in greater detail below. A Q-band EPR
spectrum exhibited the same quartet splitting for component II (data
not shown).
Figure 3:
EPR composite spectra at 77 K of the
intermediates after long reaction times at 25 °C of aerobic apoR2
Y122F (100 µM) with anaerobic Fe (400
µM). A, ApoR2 from cells grown in normal medium.
Reaction time, 6 s. Recording conditions: microwave power, 4 mW;
modulation, 0.3 mT; sweep time, 167 s; time constant, 2.6 s.
B, ApoR2 from cells grown in medium containing
indole-d
-tryptophan. Reaction time, 6 s. Recording
conditions: microwave power, 4 mW; modulation, 0.3 mT; sweep time, 167
s; time constant, 2.6 s. C, ApoR2 from cells grown in medium
containing
-d
-tyrosine. Reaction time,
25 s. Recording conditions: microwave power, 4 mW; modulation, 0.4 mT;
sweep time, 41 s; time constant, 1.3 s. D, ApoR2 from cells
grown in normal medium. Reaction time, 10 min. The g values
characterizing the axial spectrum are marked with arrows.
Recording conditions: microwave power, 80 mW; modulation, 0.4 mT; sweep
time, 41 s; time constant, 0.6 s. E, as in A, but
recorded with 80 mW and 0.3 mT modulation. Hyperfinelines of the quartet spectrum are indicated together with an arrow for its g value. F, partial subtraction of a
spectrum of an indole-d
-tryptophan sample reacted
for 10 min (similar to D) from spectrum (B). The
results show the hyperfine quartet spectrum.
The reconstitution reaction was also performed with
protein purified from cells grown in media containing either
deuterium-labeled tryptophan or tyrosine. Replacing a proton involved
in EPR hyperfine coupling with deuterium causes the hyperfine lines to
change from two to three and the distance between them to decrease
about 6-fold (Sjöberg et al., 1977). In practice this is
observed as a decrease in linewidth for small hyperfine couplings or a
collapse of the proton pattern if large hyperfine couplings are
affected (small and large are defined relative to the linewidth). The
transient 77 K EPR spectra obtained with specifically deuterated R2
Y122F are shown in Fig. 3, B and C. Deuteration
of the indole protons of tryptophan caused a significant change of the
EPR spectrum by yielding increased resolution of the quartet spectrum
of component II. There is no apparent change by deuterated tryptophan
in the axial spectrum of component I (Fig. 3B).
Incorporation of deuterated tyrosine gave no significant effects in the
composite EPR spectrum (Fig. 3C). This result clearly
links component II to a tryptophan residue. Partial subtraction of the
isolated component I from the composite spectrum with deuterated
tryptophan (Fig. 3B) gave the result shown in
Fig. 3F. The resulting spectrum, i.e. component
II associated with an indole-deuterated tryptophan residue, has a
g-value of 2.004 and is dominated by a 1:1:1:1 quartet from
the two -protons (A
= 2.8 mT,
A
= 1.3 mT).
added per R2. Higher Fe/R2 ratios
gave only a minor increase in the concentration of component I, whereas
that of component II continued to increase up to at least a Fe/R2 ratio
of 10 (data not shown). Preliminary experiments indicate that component
II is preferentially formed when high concentrations of apoR2 Y122F
(around 1 mM) are reacted with a Fe/R2 ratio of about 4. In
this case, oxygen may be a limiting factor.
Fe, in separate
experiments, for 10 s and 10 min. No significant changes were observed
in the composite spectra at 77 K (data not shown), indicating that the
coupling is weak in both components and similar in magnitude to
e.g. that between the diferric center and the tyrosyl radical
in active wild-type R2 (Sahlin et al., 1987).
Composite EPR Spectra at 11 K
The 6-s
reconstituted apoR2 Y122F sample (4 Fe/R2) was also studied at 11 K. As
shown in Fig. 4, the dominating EPR visible species has
components at g = 1.923 and 1.817 besides the signal at
g = 2.004 from component I and II. The g <
2 signals, characteristic for a mixed valent state of the iron center
(Davydov et al., 1994), exist up to 2 min reaction time, but
are lost after 10 min. It accounts for 0.02 mixed-valent centers/R2 at
6 s. The g = 4.3 signal (Fig. 4) is typical of an
isolated ferric iron in a rhombic environment and may be due to
unspecifically bound iron; about 0.08 Fe/R2,
corresponding to about 2% of the added iron.
Figure 4:
EPR spectra at 11 K of E. coli R2 Y122F after 6-s reaction time at 25 °C of aerobic apoR2
(100 µM) with anaerobic Fe (400
µM). Magnetic field scan 0-500 mT; microwave power,
10 mW; modulation, 1.0 mT. Some g values are indicated by
arrows.
Room Temperature EPR Spectroscopy during
Reconstitution of Apoprotein R2 Y122F
Singlet EPR Spectrum
Shortly after
reconstitution of apoR2 Y122F, a transient singlet-like EPR spectrum
can be observed with room temperature EPR spectroscopy (Fig. 5).
It appears with a rate constant of about 7 s and
decays with a rate of 0.35 s
. The singlet has
disappeared completely after approximately 8 s (Fig. 5,
inset). The line width is 1.6 mT, and the line shape shows
slightly asymmetric shoulders.
Figure 5:
EPR spectrum at room temperature of the
mutant E. coli R2 Y122F recorded immediately after 1:1 mixing
of aerobic apoR2 (100 µM) with anaerobic
Fe (400 µM) in the stopped flow
apparatus. A rapid scan was started by the stopped flow trigger.
Recording conditions: microwave power, 40 mW; modulation, 1.0 mT; scan
time, 1 s; time constant, 40 ms. Inset, kinetics of formation
and decay of the singlet EPR spectrum at the position marked by an
arrow, started by the stopped flow trigger. (Recording
conditions: microwave power, 40 mW; modulation, 3.0 mT; time constant,
20 ms.)
The relative amount of EPR singlet
formed depends strongly on the amount of Fe added. In
Fig. 6
, its appearance is visible as the steep increase and decay
of the signal amplitude during the first few seconds of the reaction.
The amount of singlet signal is maximal at a Fe/R2 ratio of 2,
decreases at a ratio of 4, and does not appear at a ratio of 10.
Figure 6:
Kinetics traces at room temperature of the
formation and decay the singlet EPR spectrum (see Fig. 5) and formation
of the doublet EPR spectrum (see Fig. 7) showing the dependence on
stoichiometry of added ferrous iron in the reconstitution of E.
coli Y122F apoR2. From top (a) to bottom (d) the
Fe:R2 ratios are 1:1, 2:1, 4:1, 10:1, respectively,.
Recording conditions: microwave power, 40 mW; modulation, 1.0 mT; time
constant, 1.3 s. The field position is the same as is marked by an
arrow in Fig. 5.
Doublet EPR Spectrum
After the complete
disappearance of the EPR singlet, a slow transient EPR spectrum with a
doublet line shape (Fig. 7A) grows in, and reaches its
maximum intensity approximately 2 min after mixing. The room
temperature doublet has a hyperfine splitting of 1.9 mT and a line
width of 1.1 mT. The rates of formation and decay of the doublet are
0.02 s and 0.005 s
, respectively
(Fig. 7A, inset). The kinetics of formation of
the room temperature doublet signal are clearly different from the
kinetics of components I and II observed at 77 K. Whereas high
concentrations of the two latter signals are visible after 6 s reaction
time, the room temperature doublet only starts to form after 10 s. The
relative intensity of the doublet (Fig. 6) increased gradually
with a Fe/R2 ratio from 1 to 4, whereas the addition of 10 Fe/R2
slightly decreased the yield of this species. The rate of formation of
the doublet was almost independent on the iron to R2 stoichiometry
(Fig. 6).
Figure 7:
EPR doublet spectra at room temperature of
the mutant E. coli R2 Y122F after mixing of aerobic apoR2 (100
µM) with anaerobic Fe (400
µM) in the stopped flow apparatus. Recording conditions:
microwave power, 40 mW; modulation, 0.5 mT; scan time, 84 s; time
constant: 1.3 s. 32 scans in the time interval 1-12 min have been
added and background has been subtracted. Inset, kinetics of
formation and decay of the EPR signal at room temperature at the field
position marked by the upper arrow triggered by the stopped flow
apparatus (microwave power, 40 mW; modulation, 1.0 mT; time constant,
1.3 s). A, ApoR2 from cells grown in normal medium.
B, ApoR2 from cells grown in medium containing
indole-d
-tryptophan. C, ApoR2 from cells
grown in medium containing
-d
-tyrosine.
Singlet and Doublet EPR Spectra in Specifically
Deuterated Proteins
In an attempt to probe the origins of the
room temperature singlet and doublet EPR species, we also reconstituted
apoR2 Y122F, containing either deuterium-labeled tryptophan or
tyrosine. Fig. 7, B and C show the results of
these experiments under conditions suitable for observing the room
temperature doublet spectrum. The results clearly show that
incorporation of deuterated tryptophan caused a significant change of
the doublet line shape (Fig. 7B), whereas deuterated
tyrosine had no significant effect (Fig. 7C). The EPR
line shape of the indole-d-tryptophan-labeled
protein (Fig. 7B) is a doublet with a decreased
linewidth and with poorly resolved shoulders. The decrease in line
width from 1.1 mT to about 0.6 mT, indicates that the indole ring
protons of tryptophan contribute to the unresolved line width of the
original doublet, whereas the doublet splitting must originate from one
of the
protons of tryptophan.
EPR Experiments during Reconstitution of Double
Mutant ApoR2 Proteins
To further trace the origin of the different transient EPR
species, two double mutants were used. Reconstitution of apoR2
Y122F/30C (a mutant that lacks the 30 C-terminal amino acid
residues) gave EPR spectra that were essentially the same as for the
single mutant Y122F for the two room temperature species, the
short-lived freeze-quench singlet, and the composite spectrum at 77 K.
The Y122F/Y356A double mutant was found to give essentially the same
results as the Y122F mutant regarding formation of the room temperature
singlet and the composite 77 K spectrum. These results suggest that
none of the five transient EPR active species discussed above is
located at Tyr-356 or any of the 30 C-terminal residues.
Stopped Flow UV-visible Absorption Spectroscopy during
Reconstitution of Mutant ApoR2 Proteins
Kinetics of Formation of the Iron Center in
Y122F
Fig. 8A shows the light absorption spectra
before and after reconstitution of apoR2 Y122F, with the typical iron
absorption bands at 325 and 370 nm clearly visible at the end of the
reaction. This indicates that a seemingly normal diferric iron center
is formed in the Y122F protein. To understand the relation between the
iron center formation and the appearance of the transient EPR signals
previously described, we studied the kinetics of the formation of
light-absorbing species in protein R2. The kinetics of the appearance
of the 370 nm absorbance is shown in Fig. 8B for 2 and 4
Fe/R2. The second-order rate constant of the 370 nm band depends on the
ratio Fe to protein R2 and is more rapid for 2 Fe/R2
than for 4 Fe/R2, in agreement with the result of Bollinger et al. (1991a). For both ratios of Fe/R2, however, the reaction is
essentially completed within 5 s at 25 °C (Fig. 8B).
Figure 8:
A, light absorbance spectra
before (lowertrace) and after (uppertrace) addition of 4 Fe/R2 to E.
coli apoR2 Y122F at 25 °C. Protein concentration was 11
µM. B, stopped flow kinetic traces at 370 nm
following the reconstitution of 50 µM apo-Y122F with 100
(trace1) and 200 (trace2)
µM Fe
. The spectra have been normalized
to an end point absorbance of 1.0. C, kinetic traces at 410 nm
following the reaction of 50 µM apo-Y122F with 100
(trace1) and 200 (trace2)
µM Fe
. The spectra have been normalized
to an absorbance of 1.0 at maximum
absorbance.
Kinetics of Formation of a Transient Tyrosyl
Radical
Fig. 8C shows the kinetics at 410 nm
(typical for a tyrosyl radical of the phenoxy type; Land et
al., 1961) for the reaction with 2 and 4 Fe/R2. The kinetic trace
for the reaction with 2 Fe/R2 showed that a short lived intermediate
was formed. The maximum amplitude was reached after 0.3 s, and the
intermediate had essentially decayed after 5 s.
= 3250 M
cm
as for the tyrosyl radical of wild-type R2 (Petersson et
al., 1980).
Figure 9:
Light absorpton spectra reconstructed from
stopped flow kinetic traces (as in Fig. 8, B and C)
after reconstitution of apoR2 Y122F (50 µM) at 25 °C
with (A) 4 Fe/R2, and (B) 2 Fe/R2. The spectra
correspond to 62-ms (a), 125-ms (b), 312-ms
(c), and 5.0-s (d) reaction times at 25 °C.
Traces were recorded at every 5th nm in the range 350-450 nm. A
reference scan of the buffer has been subtracted from each
reconstructed spectra.
To investigate if the transient 410-nm peak
emanates from the second conserved tyrosine 356 in protein R2, iron
reconstitution was performed with the two double mutants Y122F/30C
and Y122F/Y356A, which both lack Tyr-356. The kinetics for the
appearance of the 370-nm absorption of the iron site in these double
mutants are shown in Fig. 10A and found to be similar to
that of the Y122F single mutant. However, neither of these double
mutants gave rise to any intermediate at 410 nm upon reconstitution
with 2 Fe/R2 (Fig. 10B). These results conclusively show
that the transient 410 nm optical band in the single mutant Y122F is
dependent on the presence of Tyr-356 and most probably arises from a
tyrosyl radical located at Tyr-356. Light Absorption (at 480-600 nm) and Kinetics of Formation of
Longer-lived Transient Radicals-Photochemically produced
tryptophan radicals have been reported to have visible optical spectra
with absorbance indices around 2000 M
cm
in the wavelength range 500-600 nm
(Baugher and Grossweiner, 1977; Land and Prütz, 1979). In an
attempt to observe any of the EPR-detectable tryptophan radicals by
optical methods, we reacted Y122F with 4 Fe/R2, in a conventional
spectrophotometer. Repeated scanning of the range 480-600 nm
revealed a transient absorption in the 520-560 nm region, and an
isosbestic point around 505 nm (Fig. 11A). This weak
light absorption is superimposed on the end absorption from the
dinuclear iron center at longer wavelengths (see
Fig. 8A). Subtraction of the end point spectrum from the
intermediate spectra gave difference spectra with a broad band centered
at 545 nm (Fig. 11B). The transient species is rapidly
formed (
10 s) but has disappeared completely 13 min after iron
addition. The decay of the 545 nm absorbance band is on the same time
scale as that of the tryptophan-derived EPR quartet observed at 77 K.
Figure 10:
Light absorbance kinetic traces after
reconstitution of E. coli R2 double mutants in the reaction of
50 µM apoR2 with 100 µM Fe.
A, kinetic traces at 370 nm for Y122F (trace1), Y122F/Y356A (trace2), and
Y122F/
30C (trace3) are normalized to an end
absorbance of 1.0. B, kinetic traces at 410 nm for Y122F
(trace1), Y122F/Y356A (trace2),
and Y122F/
30C (trace3) are normalized to a
maximum absorbance of 1.0.
Figure 11:
Light absorption spectra at 10 °C in
the range 480-600 nm recorded upon reconstitution of apo Y122F
with Fe. The reaction was started by adding 200
µl anaerobic 800 µM Fe
to 400 µl
of 100 µM apo Y122F in a 4
10-mm cuvette. The scan
rate was 480 nm/min, and 30 consecutive spectra were registered.
A, spectra at 10 s (trace1), 1 min
(trace2), and 13 min (trace3)
after the addition of iron. B, difference spectra where the
13-min spectrum has been subtracted from the 10-s spectrum. Smoothing
has been performed on the difference
spectrum.
/Fe
) dinuclear iron center,
whereas the remaining five intermediates have characteristics of free
radical species. The formation and decay kinetics of the different
transient species is schematically presented in Fig. 12together
with the formation kinetics of the diferric center. Species occurring
prior to or concomitant with the diferric center in R2 Y122F may be
common with transient species occurring during the reactivation of the
wild-type R2, whereas species persisting and/or occurring after
formation of the diferric center in R2 Y122F may identify catalytically
important electron transfer pathways in wild-type R2. Below we will
consider possibilities to assign the transient species observed in the
reconstitution of apoR2 Y122F to molecular entities (or particular
residues) in the protein.
Figure 12:
Overview of time scale of formation and
decay and the suggested naure of of the transient free radicals formed
in the iron reconstitution reaction of E. coli R2 Y122F
apoprotein. The fulllines indicate continuous
registration of kinetic traces, or a deduction from spectra at later
time points showing that the transient is absent. Dottedlines for species observed the 77 K shows discrete
observations at the timepoints where the boxes are indicated.
Filledboxes indicate observed presence of species.
▾ in the baseline indicates observed absence
of species at that particular time point. The absence of a line
indicates that the time region has not yet been investigated. For each
species, the observed amplitudes are normalized to the same maximum
amplitude. The figure also shows the kinetics of the formation of the
stable diferric iron center. Note that the scale of the time axis is
broken at 10 s and 1 min.
Transient Radicals in R2 Y122F Occurring before or Concomitant
with Formation of the Diferric Site
The 77 K singlet EPR
spectrum observed at 0.3 s is similar to the transient radical observed
in wild-type protein R2 (species X in Bollinger et
al.(1991, 1994); see below). The inability to saturate our 77 K
singlet at 200 mW indicates that it is strongly coupled to the metal
site.
30C at room temperature and at 77 K cannot be
distinguished from those of the Y122F protein. Therefore none of the
EPR observable singlet signals could be due to a radical localized to
Tyr-356. One possible reason why this radical is not observed by EPR
may be its low concentration. Another, perhaps more important reason is
that Tyr-356 is located at the surface of R2 and at least 10 Å
from the iron center. Hence its relaxation properties should not be
much affected by the iron. Its EPR signal at 77 K may be saturated and
broadened beyond detection even at low microwave powers.
)
L) have also been observed in their
experiments. In addition, they report on a transient broad absorption
band at 560 nm formed under limiting iron conditions, which is
suggested to be a tryptophan cation radical on Trp-48 with a maximal
concentration at 0.3-0.4-s reaction time. It is interesting to
note that the room temperature singlet EPR spectrum observed by us is
also preferentially formed under limiting iron conditions and occurs in
a similar time window as the 560-nm species reported by Bollinger
et al. (1991a, 1994a, 1994b). Thus it seems quite likely that
the room temperature singlet EPR species observed here is in fact a
radical existing concomittantly with the
(Fe
)
L, as proposed in by
Bollinger et al. (1994b). Our deuterium-labeling experiments
have so far not been conclusive to determine whether this radical is
localized on tryptophan.
Transient Radicals Occurring after Formation of the
Diferric Site
Component II observed at 77 K has clearly been
shown to be a tryptophan radical by the isotope effect in the EPR
spectrum. The quartet nature of the EPR spectrum, which is not changed
by indole deuteration, corresponds to two large -proton hyperfine
splittings of 1.3 and 2.8 mT from the methylene group of tryptophan.
The quartet EPR species has been investigated in more detail by EPR and
electron nuclear double resonance studies at 20 K revealing a
relatively large anisotropic
N hyperfine coupling in
addition to the two proton hyperfine couplings. The interpretation of
the spectrum based on comparison with molecular orbital calculations
indicate that the tryptophan radical is a neutral deprotonated
radical.
(
)
A radical with similar features has
previously been observed in studies of radiation-induced free radicals
in tryptophan single crystals (Flossman and Westhof, 1978). The
transient spin polarized tryptophan radical in DNA photolyase, on the
other hand, was shown to have a negligible hyperfine coupling to
nitrogen, indicating that the radical in that case is a protonated
cation radical resulting from electron abstraction by photoexcited
FADH
(Kim et al., 1993).
at 545 nm (Fig. 11) is
indicative of a tryptophan radical and the time scale for this species
is similar to that of the EPR quartet observed at 77 K. Hence, this
light absorption could have the same origin as the EPR 77 K quartet,
i.e. a neutral tryptophan radical. Comparison with other
tryptophan radicals are in reasonable agreement with this proposal. The
width at half-height of the 545 signal is approximately 35 nm. This is
narrower than observed for a neutral tryptophan radical (
75 nm),
but a cation radical would be even broader (
130 nm) (Baugher and
Grossweiner, 1977). The absorbance maximum we observe is between those
observed for the neutral tryptophan radical (520 nm) or the cation
radical (570 nm) generated by pulse radiolysis (Baugher and
Grossweiner, 1977). Recently, another report based on light absorption
on transient tryptophan radicals in protein R2 has appeared. They
relate, however, to more short-lived species than the 545-nm species
observed by us. Lam et al.(1993) have observed transient
(
1 s) tryptophan radicals, with absorbance maximum at 510 nm, in
wild-type and Y122F R2, upon reaction with pulse radiolysis-generated
azide radicals. These were proposed to originate from tryptophan
residues close to the protein surface, distant from the iron center,
and to be involved in electron transfer processes between tyrosine and
tryptophan residues in the protein (Lam et al., 1993).
radical, was
found to be due to weak exchange interaction between a S = 1 oxyferryl (Fe=O)
moiety and
the tryptophan radical. Since the line shape was only partly affected
by hyperfine interactions, deuterium exchange experiments revealed the
tryptophan origin of the signal of the cytochrome c peroxidase
radical only after careful electron nuclear double resonance studies
(Sivaraja et al., 1989). One possible interpretation is that
also the present component I in R2 is a tryptophan radical, weakly
coupled to a metal site. An alternative explanation suggested by the
g values is a nonprotein-derived radical, e.g. a
peroxy radical.
-proton coupling, with a
large spin density on C-3 in the tryptophan ring. The second
-methylene coupling is less than 0.4 mT and hidden in the line
width. The striking spectroscopic differences of the room temperature
doublet and the 77 K quartet concerning
-methylene conformation,
kinetics of appearance and decay as well as the relaxation behavior
(distance to the iron) supports that the room temperature doublet
originates from a tryptophan radical at a site different from that
which gives rise to the tryptophan quartet spectrum.
proton hyperfine coupling constants. This
method was first used to determine the different geometry of the native
tyrosyl radical in the T4 bacteriophage protein R2 relative to the
E. coli one (Sahlin et al., 1982). Since the crystal
structure of protein R2 is known, the geometrical information can be
used to distinguish between possible tryptophan residues as candidates
for radical localization.
proton coupling
constant B can be described by the empirical relationship
B = B` + B"cos
(Stone and Maki, 1962), where B` and B" are constants
and
is the dihedral angle between the radical z orbital
axis (normal to the indole plane) and the projected
C
H
bond. Normal values of B`
and B" are 0 and 5 mT, respectively (Morton, 1964).
Introducing the spin density
at the C-3 carbon of tryptophan, the
following equations are obtained (Sahlin et al., 1982):
B
=
(B` +
B"cos
); B
=
(B` + B"cos
);
=
+ 120°, where
B
and B
are the
proton
hyperfine couplings and
and
are
the corresponding dihedral angles. The equation system may be solved
graphically, yielding two solutions, one of which can sometimes be
discarded. For the 77 K quartet, with B
=
2.8 mT and B
= 1.3 mT, we obtain an
approximate solution with
= 0.57,
= 13°, and
= 133°. For
the room temperature doublet, where only one
-proton hyperfine
coupling is resolved and the other one is hidden in the linewidth, a
rough estimation shows that one of the
-protons should have a
dihedral angle close to 90° (and the other one should be close to
-30°).
4 Å from Fe2, suggests that one methylene proton
would couple strongly with spin density at C-3 (
10° dihedral
angle to the normal of the indole plane) and that the other would
couple somewhat weaker (
130° dihedral angle). These structural
features are in agreement with the relatively strongly iron-coupled
quartet at 77 K (component II, ) with corresponding
experimentally determined dihedral angles of 13 and 133°. For
Trp-107,
8 Å from Fe1, the structure suggests that one
methylene proton is in the indole plane and the other has a dihedral
angle of
-30°. These structural features are compatible
with the room temperature doublet EPR spectrum, which has at most a
weak paramagnetic metal interaction since it is observable at room
temperature (). The three-dimensional structure of Trp-48,
about 8 Å from Fe1, indicates approximately similar dihedral
angles for both methylene protons (
68° and
-52°).
This should give rise to a relatively narrow (unresolved?) triplet EPR
spectrum with intensities approximately 1:2:1 and a hyperfine coupling
around 0.6 mT, assuming a spin density of 0.5 at C-3. The room
temperature singlet EPR spectrum actually fits this description quite
well, which adds further support to the assignment of this EPR spectrum
to Trp-48. As pointed out previously (Sahlin et al., 1994), if
component I is indeed a tryptophan radical, Trp-48 is a strong
candidate. Trp-48 in protein R2 is part of a triad of hydrogen bonded
amino acids linking it with one of the iron ions (Nordlund et
al., 1990). The situation is similar to that in cytochrome c peroxidase, where Trp-191 is part of a corresponding amino acid
triad linked to the heme iron (Finzel et al., 1984; Sivaraja
et al., 1989; Wang et al., 1990; Goodin and McRee,
1993), and Trp-191 is the site of the free radical assigned to the
axial EPR spectrum. If both the room temperature singlet and component
I at 77 K would arise from Trp-48, this implies that the properties of
these radicals are strongly dependent on the state of the iron site and
possibly its surrounding H-bonding network. The former appears during
the formation of the iron site with at most a weak interaction with a
paramagnetic site, whereas the latter exists long after its completion
and exhibits significant coupling to a paramagnetic metal ion.
Concluding Remarks
The present results demonstrate
the transient formation of a large number of EPR and/or optically
active paramagnetic species on different time scales when the E.
coli protein R2 mutant Y122F reacts with Fe and
O
. In analogy with the corresponding reaction in wild-type
R2, we consider the reconstitution to take place between a reduced
dinuclear iron center and oxygen (cf. Sahlin et
al.(1987) and Mann et al.(1991)). The oxygen becomes
reduced, with one atom forming the bridging oxygen in the diferric site
(Ling et al., 1994) and the other atom presumably forming
water. It is interesting to consider this reaction resulting in a
four-electron reduction of O
to water in comparison with
the corresponding four-electron reduction in cell respiration (Babcock
and Wikström, 1992), or the opposite four-electron oxidation
resulting in water splitting in photosystem II (Andersson and Styring,
1991).
reduction
site in the terminal oxidase is a bimetallic center composed of one
heme iron and one copper site. As pointed out in Babcock and
Wikström (1992), the iron is in a high spin state to overcome the
spin restriction on O
reactivity (O
is in a
triplet state), and the presence of the redox active copper ion in
addition to the heme iron avoids an unfavorable one-electron reduction.
Similarly, the fully reduced dinuclear iron center in protein R2 has
its iron ions in high spin form (Lynch et al., 1989), and is
thus set up for a favorable reaction with O
to form the
active protein. In contrast, the concomitant translocation of protons,
which dictates the kinetics of the terminal oxidase reaction, has not
so far been shown to have any correspondence in the reaction of protein
R2. However, preliminary results in the present study suggest that the
kinetics are significantly slowed down if the reactions are performed
in a deuterated solvent. There are also some indications that the
oxygen forming the bridge between the two iron ions may be hydrogen
bonded in protein R2 (Galli et al., 1994). In addition, recent
studies on the mutant R2 protein S211A indicated that proton
translocation may be required during reduction of the diferric center
in R2 (Regnström et al., 1994). The participation in the
R2 reaction of a ferryl intermediate in the O-O bond scission
would be analogous to the situation in the terminal oxidase, but this
is still a matter of controversy in the case of R2 (cf. Sahlin
et al.(1989), Ling et al.(1994), and Bollinger et
al. (1994a, 1994b)).
to water in wild-type protein R2 is as
strictly controlled as in the terminal oxidase reaction. The
participating electron donors must be precisely organized in space as
well as in redox properties for the reaction to yield the necessary
result: the oxygen-bridged iron center and the tyrosyl radical. This
leads to the possibility that the ferrous ion providing the fourth
electron in the wild-type reaction in vitro may be in a
specific site and directly or indirectly (via an electron transfer
pathway) in contact with the reaction site.
R2 complex) appears to be
prepared for the specific electron transfer along a very precise route,
which becomes operative only under functional conditions. A recent
preliminary report on the mouse ribonucleotide reductase system with
protein Y177F, corresponding to Y122F in E. coli, suggests
that transient radicals of a similar type as the ones presented here
could give a low (0.5%) but specific activity in the enzyme (Henriksen
et al., 1994).
Table:
Electrospray mass spectrometric analysis of R2
proteins containing protonated or deuterated amino acid residues
Table:
Overview of EPR
parameters of transient radicals obtained in iron reconstitution of
protein R2 Y122F
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.