©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transient Free Radicals in Iron/Oxygen Reconstitution of Mutant Protein R2 Y122F
POSSIBLE PARTICIPANTS IN ELECTRON TRANSFER CHAINS IN RIBONUCLEOTIDE REDUCTASE (*)

Margareta Sahlin (1), Günter Lassmann (3) (4), Stephan Pötsch (3), Britt-Marie Sjöberg (1), Astrid Gräslund (2)

From the (1) Department of Molecular Biology and the (2) Department of Biophysics, Stockholm University, Arrhenius Laboratories, S-106 91 Stockholm, Sweden, the (3) Max Delbrück Center of Molecular Medicine, Robert-Rössle-Strasse 10, D-13125 Berlin, Germany, and the (4) Max Volmer Institute of Biophysical and Physical Chemistry, Technical University of Berlin, D-10623 Berlin, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

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 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.

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 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.

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 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.


EXPERIMENTAL PROCEDURES

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 mMFe, 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.

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 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.


RESULTS

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).

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 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.

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 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.

Attempts to observe possible deuterium effects in the rapidly decaying room temperature singlet have so far not given any conclusive results.

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.

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 = 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.




DISCUSSION

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/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.

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/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.

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)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.

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.

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).

The transient broad light absorption, with 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).

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 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.

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 -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.

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 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.

The isotropic 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 CH 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°).

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 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).

In the cell respiration process, the O 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)).

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 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.

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 R1R2 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).

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.

  
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



FOOTNOTES

*
This study was supported by grants from the Tryggers Foundation (to M. S.); from Deutsche Forschungsgemeinschaft, Project No. 751/1-2 (to G. L. and S. P.); from the Swedish Cancer Society, the Swedish Research Council for Engineering Sciences, and the Swedish National Board for Technical Development (to B.-M. S.); and from The Swedish Natural Science Council, the Magn. Bergvall Foundation, and the Bank of Sweden Tercentenary Foundation (to A. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Rova, U., Goodtzova, K., Ingemarson, R., Behravan, G., Gräslund, A., and Thelander, L. (1995) Biochemistry, in press.

The abbreviations used are: EPR, electron paramagnetic resonance; T, tesla; W, watt(s).

M. Karlsson, personal communication.

F. MacMillan, M. Sahlin, F. Lendzian, R. Fiege, S. Pötsch, B.-M. Sjöberg, A. Gräslund, W. Lubitz, and G. Lassmann, submitted for publication.


ACKNOWLEDGEMENTS

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.


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