©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Residues Important for Radical Stability in Ribonucleotide Reductase from Escherichia coli(*)

(Received for publication, July 20, 1994; and in revised form, November 30, 1994)

Mats Ormö (1) Karin Regnström (1) Zhigang Wang (2) Lawrence Que Jr. (2) Margareta Sahlin (1) Britt-Marie Sjöberg (1)(§)

From the  (1)Department of Molecular Biology, Stockholm University, S-106 91 Stockholm, Sweden and the (2)Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The R2 protein of ribonucleotide reductase contains at the side chain of tyrosine 122 a stable free radical, which is essential for enzyme catalysis. The tyrosyl radical is buried in the protein matrix close to a dinuclear iron center and a cluster of three hydrophobic residues (Phe-208, Phe-212, and Ile-234) conserved throughout the R2 family. A key step in the generation of the tyrosyl radical is the activation of molecular oxygen at the iron center. It has been suggested that the hydrophobic cluster provides an inert binding pocket for molecular oxygen bound to the iron center and that it may play a role in directing the oxidative power of a highly reactive intermediate toward tyrosine 122. We have tested these hypotheses by constructing the following mutant R2 proteins: F208Y, F212Y, F212W, and I234N. The resulting mutant proteins all have the ability to form a tyrosine radical, which indicates that binding of molecular oxygen can occur. In the case of F208Y, the yield of tyrosyl radical is substantially lower than in the wild-type case. A competing reaction resulting in hydroxylation of Tyr-208 implies that the phenylalanine at position 208 may influence the choice of target for electron abstraction. The most prominent result is that all mutant proteins show impaired radical half-life; in three of the four mutants, the half-lives are several orders of magnitude shorter than that of the wild-type radical. This suggests that the major role of the hydrophobic pocket is to stabilize the tyrosyl radical. This hypothesis is corroborated by comparative studies of the environment of other naturally occurring tyrosyl radicals.


INTRODUCTION

The use of highly reactive entities, i.e. free radicals, in biological reactions puts high demands on the systems harboring these potentially harmful species. Yet, radical chemistry is used in a number of important catalytic steps. One such reaction is the production of building blocks for DNA synthesis. Even though at least three different classes of ribonucleotide reductases are known, all involve free radical chemistry (Stubbe, 1990; Reichard, 1993; Sjöberg, 1994). In aerobically growing Escherichia coli, this essential reaction is carried out by an enzyme (EC 1.17.4.1) consisting of two homodimeric proteins, whose three-dimensional structures have been solved to high resolution (Nordlund and Eklund, 1993; Uhlin and Eklund, 1994). The large protein, denoted R1, has a molecular weight of 171,477 and contains the active sites and regulatory functions. The small protein R2, with a molecular weight of 86,771, harbors the stable free radical in the amino acid side chain of tyrosine 122 (Larsson and Sjöberg, 1986). Indirect evidence suggests that the tyrosyl radical of R2 initiates the reduction of ribonucleotides by formation of a substrate radical in the active site of R1 (Sjöberg et al., 1983; Stubbe and Ackles, 1980). Since tyrosine 122 is buried in the R2 protein matrix, an electron transfer pathway through the R2 protein and across the R1-R2 interface is suggested (Nordlund and Eklund, 1993; Uhlin and Eklund, 1994; Sjöberg, 1994).

The tyrosyl radical in R2 is generated by abstraction of an electron from Tyr-122 during oxidation of two ferrous ions bound in the vicinity of the tyrosine. This reaction, which is driven by molecular oxygen, also generates the oxidized dinuclear iron center of R2. The two ferric ions are bridged by an oxide, derived from molecular oxygen (Ling et al., 1994), and a glutamate, and they are terminally liganded by two histidines, one aspartic acid, two glutamic acids, and two water molecules (Nordlund et al., 1990). The tyrosyl radical interacts magnetically with the iron center (Sahlin et al., 1987), which together with the unique environment of Tyr-122 may cooperate to give the radical its remarkable stability (a half-life of several days at 25 °C) (Atkin et al., 1973).

The environment of the radical-carrying tyrosine 122 in R2 is neither extremely hydrophobic nor particularly tightly packed (Nordlund et al., 1990). From sequence alignments, (^1)one can deduce that this region is highly conserved among 21 full-length R2 sequences that range from bacterial viruses to mammalian species. Three of the invariant residues, phenylalanine 208, phenylalanine 212, and isoleucine 234, form a hydrophobic pocket close to tyrosine 122. In ribonucleotide reductase from E. coli, these residues are accompanied by the non-conserved isoleucines 125, 230, and 231, leucine 77, and phenylalanine 216 (see Fig. 1). Of these residues, only isoleucine 234 is in van der Waals contact with Tyr-122. The occurrence of a hydrophobic pocket, constituted from conserved residues, between the iron center and tyrosine 122 and the involvement of molecular oxygen in the formation of the radical have led to speculations on the function of this structural element. It has been suggested that the pocket could serve as a binding pocket for oxygen and direct the radical generating reaction toward Tyr-122 (Nordlund et al., 1990, Nordlund and Eklund, 1993).


Figure 1: Residues constituting the surroundings for the radical-carrying tyrosine 122 in protein R2. The drawings were made with the program Molscript (Kraulis, 1991).



To investigate the role of these invariant hydrophobic residues in the R2 protein, site-directed mutagenesis was applied to introduce side chains with other chemical properties into the pocket. The mutations were chosen along three different lines: (i) changing of phenylalanine 208 and 212 to tyrosine would introduce a polar residue without changing the aromatic character of the area, (ii) replacement of phenylalanine 212 with tryptophan would lead to voluminal blocking of the pocket while still keeping the aromatic character of the residue, and (iii) introduction of an asparagine instead of an isoleucine in position 234 would change the electrostatic properties of the pocket to a more polar situation and at the same time decrease the van der Waals volume of the side chain. The importance of this hydrophobic region for normal protein function became apparent in the mutant R2 protein where phenylalanine 208 was changed to tyrosine. Instead of radical generation at tyrosine 122, this protein performs a self-hydroxylation at tyrosine 208 to form a dopa residue that is liganded to Fe1. This leads to largely changed spectroscopic and biophysical properties. The characteristics of the dopa residue and the iron center in protein R2 F208Y have been previously described (Ormöet al., 1992; Åberg et al., 1993a; Ling et al., 1994).

In this study, the different mutant proteins are characterized with respect to two functions: the ability to form a tyrosine radical during oxidative reactivation and the ability to stabilize the formed radical. The results presented show that, although the hydrophobic pocket was drastically disturbed, all mutant proteins (F208Y, F212Y, F212W, and I234N) were able to generate a tyrosyl radical. The differences with wild-type protein lie mainly in the stability of the radical, which, in all but one case, is dramatically lowered. These results suggest that the function of the conserved part of the hydrophobic pocket is not primarily to create an inert binding pocket for molecular oxygen, but rather to constitute a structure that can stabilize the radical. Support for such a conclusion is also found in comparisons with other tyrosine radical-containing proteins of known three-dimensional structure.


MATERIALS AND METHODS

Mutagenesis

The mutants F212W and I234N were made by oligonucleotide-directed mutagenesis using the method ``polymerase chain reaction overlap extension'' according to Higuchi et al.(1988). The primers for F212W were d(5`-CGTCAGCTGGGCTTGTTC-3`) and d(5`-GAACAAGCCCAGCTGACG-3`), and the primers for Ile-234 were d(5`-TTCGCCTGAACGCCCGC-3`) and d(5`-GCGGGCGTTCAGGCGAA-3`) with the amino acid-changing codons underlined. The mutant F212Y was constructed according to Kunkel et al.(1985, 1987) using the primer d(5`-CTACGTCAGCTACGCTTGTTC-3`) and the Mutagene phagemid in vitro mutagenesis kit (version 2) purchased from Bio-Rad. Construction of the mutant F208Y has been described earlier (Ormöet al., 1992).

Strains and Plasmids

The mutant proteins R2 F212W and R2 F212Y were overexpressed in E. coli MC1009 (Delta(lacIPOZYA)X74, galE, galK, strA, Delta(ara-leu)7697, araD139, recA, srl::Tn10) carrying a pTZ18R derivative, pTB2 (Climent and Sjöberg, 1992), with the nrdB gene under control of a T7 promoter. The T7 polymerase was introduced through a second plasmid, pGP1-2, where the gene is under control of the heat-sensitive repressor cI857 (Tabor and Richardson, 1985). The mutant protein R2 I234N was overexpressed by the pTB2 construct in E. coli BL21(DE3)/pLysS ((FompT rBmB), DE3 int::(lacI lacUV5 lacZ::T7RNApol)), which has the T7 polymerase gene under control of an isopropyl-1-thio-beta-D-galactopyranoside-inducible promoter and the activity of T7 RNA polymerase controlled by lysozyme (Studier et al., 1990).

Overexpression and Purification

The cells were fermented in 5 liters of SLBH medium (Green et al., 1974) in a New Brunswick Microferm fermentor except for cells overexpressing R2 I234N, which were fermented in a thermostatted shaker in five 5-liter flasks containing 1 liter of medium each. The cells were disintegrated with an X-press (AB Biox, Sweden). The mutant proteins were purified according to standard procedures as described earlier (Sjöberg et al., 1986). Iron-free R2 F212W protein was obtained by removing iron with the strong chelator Li-8-hydroxyquinoline-5-sulfonate in the presence of 1 M imidazole (Atkin et al., 1973). Iron-free R2 F208Y was obtained by fermenting the cells in iron-depleted medium as previously described (Åberg et al., 1993a).

Protein Stability Measurements

Denaturation by guanidine hydrochloride (Life Technologies, Inc.) was measured at equilibrium after 2 h of incubation at 20 °C. Circular dichroism spectra were collected on a J-720 spectropolarimeter (JASCO, Japan). The scanning speed was 20 nm/min, and the spectra were averaged from two subsequent scans. Protein concentration was 0.5 mg/ml, and the path length of the cell was 1 mm. The apparent fraction unfolded protein was measured as ellipticity at 222 nm relative to that of the corresponding native protein. All stability measurements were made on fully iron-reconstituted R2 proteins or, when needed, after addition of ferrous ions up to a final concentration of 4 Fe per R2 dimer. Stability measurements on apoR2 was in the absence of iron.

Assays

The iron content of the purified proteins was determined by the method of Atkin et al.(1973) as modified according to Sahlin et al.(1990). Occupation of the iron centers in the reconstituted proteins was determined as light absorbance at 370 nm ( 8700 M cm for radical-containing samples and 7200 M cm for samples lacking radical) (Petersson et al., 1980). Ribonucleotide reductase activity was measured spectrophotometrically as oxidation of NADPH followed at 340 nm (Thelander et al., 1978). The assay conditions were 0.5 mM CDP, 1.5 mM ATP, 11 mM magnesium acetate, 0.4 mM NADPH, 33 mM Hepes, pH 7.6, 13 µM thioredoxin, 0.5 µM thioredoxin reductase, 0.41 µM R1 protein, and 60 nM R2 protein. Molar absorption indices () used were for protein R2 120,000 M cm and for protein R1 180,000 M cm. The R1-R2 interaction was determined as described in Climent et al.(1991) with 0.158 µM R1 and 0.02-2 µM R2.

Measurements of Radical Content and Stability

Spectra of R2 F212Y were collected on a Hewlett Packard 8452 A diode array spectrophotometer. Stopped flow experiments were carried out with an Applied Photophysics rapid kinetic accessory (model RX 1000, Applied Photophysics, United Kingdom). Data were collected at 410 nm on a 2 spectrophotometer (Perkin-Elmer Corp.) at 22 °C. One syringe contained 24 µM R2 protein in 50 mM Tris-HCl, pH 7.6, and the other 96 µM Fe(NH(4))(2)(SO(4))(2) in 50 mM Tris-HCl, pH 7.6. The buffer in which the iron salt was dissolved was pretreated with argon bubbling to remove oxygen. Immediately prior to an experiment, the system was flushed with argon-bubbled buffer. Maximum radical content and radical decay were measured as the difference between maximal absorbance at 410 nm after iron addition to apoprotein and the absorbance after total decay of the radical (Delta 3250 M cm) (Petersson et al., 1980). The amount of radical in protein R2 F208Y was determined as the change in absorbance at 410 nm relative to a base line between 405 and 415 nm. Maximal radical content of R2 I234N was determined on reactivated protein (see below), and total radical decay was achieved by incubation with 3.5 mM hydroxyurea at 25 °C.

Reduction of Protein R2

To generate maximal amount of radical in protein R2 I234N, the protein was first reduced by addition of three molecular equivalents of dithionite to a solution containing 50 mM Tris-HCl, pH 7.6, 20% glycerol, 15 µM protein R2, and 2.5 µM benzyl viologen. Before exposure to oxygen needed for the radical generation, two equivalents of Fe were added as a (NH(4))(2)Fe(SO(4))(2) solution. All solutions were pretreated with argon bubbling to remove oxygen.

EPR Measurements

EPR spectra were recorded using a ESP 300E Bruker spectrometer, equipped with an Oxford Instrument cryostat.


RESULTS

Overall Stability of the Proteins

All of the mutant proteins, R2 F208Y, R2 F212W, R2 F212Y, and R2 I234N, were purified to homogeneity according to the standard procedures developed for the wild-type R2 protein. No difference to the wild-type CD spectrum was found for any of the mutant proteins. However, differences in iron and radical content were observed (see below), and all mutant proteins were reconstituted as described under ``Materials and Methods'' prior to further analysis. During such experiments to produce iron-free apoprotein, R2 I234N was found to be very sensitive to denaturation by imidazole, resulting in protein precipitation.

To investigate the overall effect on protein stability by the mutations introduced, guanidine hydrochloride (GdnHCl) (^2)denaturation experiments were performed and compared with those of active and apo-forms of the wild-type R2. Since R2 contains more than 70% alpha-helix, the relative amount of secondary structure could be determined by measuring ellipticity at 222 nm. The denaturation curve for the wild-type protein shows a gradual denaturation between 1 and 5 M GdnHCl (Fig. 2A). The curve is biphasic with a plateau in the range between 2 and 3.5 M GdnHCl, where half of the ellipticity at 222 nm is lost. The iron-free wild-type apoprotein is similar to active R2 above 2 M GdnHCl but differs from active R2 below 2 M GdnHCl, where it shows a more rapid loss of secondary structure (Fig. 2B). Precipitation was observed in the apoR2 sample between 1 and 2 M GdnHCl, which could explain in part the dramatic drop of the ellipticity at 222 nm. The apparent gain of structure between 1.5 and 2 M GdnHCl would then be a consequence of the increased solubility of apoR2 at higher GdnHCl concentrations and not a true gain of structure.


Figure 2: Guanidine hydrochloride denaturation of wild-type protein R2 and mutant proteins. The apparent fraction unfolded protein was determined as ellipticity at 222 nm.



The mutant proteins fall into two groups when it comes to their ability to withstand denaturation by GdnHCl (see Fig. 2, A and B). The proteins R2 F208Y and R2 F212Y both behave similarly to wild-type R2 with R2 F208Y having a less pronounced biphasic behavior. The other two, F212W and I234N, both have an increased sensitivity to GdnHCl at low concentrations. R2 F212W has a very fast initial denaturation and a long plateau, which corresponds to the plateau of the wild-type protein. The denaturation of R2 I234N was characterized by precipitation at low concentrations of GdnHCl, resulting in a curve similar to that of wild-type apoprotein.

Effects of the Mutations on the Iron Center

The mutant proteins were purified with different occupancies of their iron centers. The two mutant proteins R2 F208Y and R2 I234N were purified with normal or close to normal iron content. Mutations in position 212, however, resulted in large effects on the iron center. Protein R2 F212Y was purified essentially as apoprotein and R2 F212W with 50% iron content relative to wild-type protein. Measurements of iron content based on extinction coefficients at 370 nm after reconstitution with ferrous iron showed that the mutant proteins R2 F212Y and R2 F212W were capable of generating fully occupied and oxidized iron centers. As shown in Table 1, none of the mutations affected the initial ability of the R2 protein to form a diiron center.



Radical Generation in Mutant R2 Proteins

The mutant proteins were purified with little (R2 I234N) or no (R2 F208Y, R2 F212Y, and R2 F212W) radical content. To study the radical generating ability, different techniques had to be used to reconstitute the mutant proteins.

During iron center oxidation, protein R2 F208Y irreversibly forms a dopa at position 208 and a strong bidentate iron catecholate complex. Therefore, radical generation experiments with R2 F208Y required apoprotein that had never been exposed to ferrous iron. In vitro addition of Fe to such nascent apoprotein resulted in formation of a radical at tyrosine 122 as a parallel reaction to the dopa generation (Åberg et al., 1993a). In the present study, 0.18 tyrosyl radical per R2 protein was formed after 85 s of incubation of apoR2 F208Y with ferrous ascorbate.

For the reconstitution of mutants in position 212, R2 F212Y was obtained essentially as apoprotein after purification and could be used directly, whereas R2 F212W contained bound iron, which had to be extracted prior to reconstitution. Both R2 F212W and R2 F212Y formed a tyrosyl radical upon addition of Fe. The initial radical content was 0.65 and 0.60 radical per R2 protein, respectively.

The mutant protein R2 I234N contained 0.35 radical per R2 protein after purification. Since R2 I234N denatured irreversibly even during mild iron-chelating conditions, reduction of the iron center in situ by dithionite and benzyl viologen followed by oxidation was used as reconstitution procedure (see Fig. 3). After oxidation, protein R2 I234N contained 1.0 radical per R2 protein, which corresponds to the amount found in wild-type protein (Petersson et al., 1980; Elgren et al., 1991). Altogether, these results show that all mutant proteins described here can generate a tyrosyl radical, and, except for the mutant protein R2 F208Y, the amounts of tyrosyl radical were comparable with that of wild type (Table 1).


Figure 3: Reduction and reactivation of protein R2 I234N. Reduction was made anaerobically with dithionite and benzyl viologen as the mediator. Protein concentration was 15 µM. A, protein absorbance spectrum before reduction; B, reduced protein 35 min after addition of three equivalents of dithionite; C, reactivated protein R2 Ile-234; D, 30 min after subjection to 3.5 mM hydroxyurea. Inset, difference spectra between C and D showing the tyrosyl radical-attributed absorbance.



Structure and Saturation Behavior of the Radical Signals

EPR was used to further analyze the transient radicals formed in the different mutant proteins. Fig. 4shows that the EPR spectra for R2 proteins F208Y, F212Y, and F212W have a hyperfine splitting almost identical to that observed for E. coli wild-type R2, indicating that the aromatic ring of the tyrosyl radical has the same orientation relative to the beta-protons as in wild-type R2. R2 I234N, on the other hand, has a different hyperfine pattern, indicating a twist around the bond between the beta-protons and the aromatic ring relative to wild-type R2. The saturation behavior of the radicals was studied at 9, 11, 28, and 50 K. The evaluated P at 28 K, assuming inhomogeneous broadening, are presented in Table 1and show that R2 F208Y and F212W behave approximately as wild-type R2. R2 I234N is also similar to wild-type R2 with a P three times as high, whereas R2 F212Y exhibits a drastically different relaxation behavior with an order of magnitude higher P value.


Figure 4: EPR spectra at 9 K for protein R2 from wild-type (A), F208Y (B), F212Y (C), F212W (D), and I234N (E). The spectra were recorded under non-saturating conditions, and the number of accumulations (1-8 scans) were chosen as to give reasonable signal-to-noise ratio. Wild-type R2 and R2 I234N are as purified with 1.5 and 0.5 mM radical, respectively. Proteins R2 from F208Y, F212Y, and F212W were reactivated by addition of 4Fe per R2 to the apoprotein forms at room temperature and then frozen in liquid nitrogen (F208Y) or cold isopentane (170 K). The amount of radical was 5.5 µM per 0.1 mM F212Y after 11 s, 17 µM per 0.1 mM F212W after 7 s, and 36 µM per 0.2 mM F208Y after 85 s of incubation. mT, millitesla.



Stabilization of the Tyrosyl Radical

Reconstitutions of the tyrosyl radical in R2 F208Y, R2 F212W, and R2 F212Y resulted in transient radicals decaying 4-5 orders of magnitude faster than the wild-type radical. Kinetic measurements of the radical decay (Table 1) showed that the most short-lived species were found in the protein R2 F212Y with a half-life of 30 s (see Fig. 5) and that the most stable radical of these three mutants was that of R2 F208Y with a half-life of 10 min. The mutant R2 protein I234N also shows a perturbed ability to stabilize a tyrosyl radical. The protein was purified with less than normal amounts of radical, indicating a 65% loss during purification (approximately 4 days at 4 °C). A half-life of 6 h was obtained when radical decay was measured at 410 nm in a sample incubated for 24 h at 35 °C. This result, however, may be obtained by radical loss due to overall protein instability. On the other hand, in the presence of hydroxyurea, the radical half-life of the mutant protein was 6 min as compared with 9 min for the wild-type protein, which indicates that the effect in R2 I234N is on the radical stability per se. Thus all the mutant proteins showed to have a less stable tyrosyl radical than wild-type R2.


Figure 5: Light absorbance spectra of R2 F212Y after reactivation of iron center and tyrosyl radical by addition of ferrous iron. Protein concentration was 12.5 µM. A, apoprotein spectrum; B, spectra collected at 6, 8, 10, 12, 14, 18, 22, 30, 42, and 85 s after iron addition.



Measurements of R1 Interaction and Enzymatic Activity of R2 I234N

When a fixed concentration of R1 was assayed in the presence of increasing concentrations of R2 protein, maximal enzyme activities were obtained at the same R1:R2 stoichiometry for R2 I234N and R2 wild type. The reactivated R2 I234N has a maximal specific activity of 480 units/min/mg, which is only 20% of wild-type R2 activity. This implies that the R1 interaction constant for protein R2 I234N is similar to wild-type R2 protein, whereas the V(max) for ribonucleotide reduction has been changed. Model building suggests that an asparagine at position 234 could contact Tyr-122, the iron ligand His-118, or Trp-48. The two latter residues are postulated to take part in the electron transfer pathway between the active sites of R1 and R2 during catalysis (Nordlund et al., 1990; Sjöberg, 1994). Possible interactions with these residues could explain the lower k in R2 I234N, as it was earlier shown that mutational changes along the postulated electron pathway result in proteins with drastically impaired activity (Climent and Sjöberg, 1992).


DISCUSSION

Protein engineering experiments have the inherent shortcoming that observed changes in characteristics may be due to unexpected changes in overall structure, especially if substitutions affect the hydrophobic interior of a protein. The overall structure of the R2 protein, however, is surprisingly resistant to substitutions. In eight resolved three-dimensional structures of different mutant proteins and various metal-substituted forms of the wild-type protein, the root mean square values of the main chain atoms, compared to the wild-type structure, are less than 0.6 Å (^3)(Regnström et al., 1994; Atta et al., 1992; Åberg et al., 1993b, 1993c). Also, the R2 structure is not particularly dense in the vicinity of Tyr-122, and modeling experiments show that e.g. a tryptophan can readily be accommodated in the hydrophobic pocket surrounding Tyr-122.

To experimentally study the overall structural stability of the mutant R2 proteins, we compared their denaturation characteristics with those of wild-type R2. Denaturation of the wild-type protein was slow and biphasic. Such a behavior is generally connected with the unfolding of multidomain proteins like tryptophan synthase (Tweedy et al., 1990). However, under non-denaturing conditions, there is no indication of domain separations in the three-dimensional structure of R2 (Nordlund and Eklund, 1993). A more exaggerated biphasic form was observed with apoR2 during GdnHCl denaturation (Fig. 2B) and also during urea denaturation (Åberg et al., 1993c). A similar situation was reported for hemerythrin, where apohemerythrin has 31% less alpha-helical structure than the iron-containing form (Zhang and Kurtz, 1992). The apo-form of hemerythrin was therefore postulated to represent a molten globule structure. However, during non-denaturing conditions, the apo-form of R2 is identical in CD spectrum and overall three-dimensional structure to the iron-containing form (Åberg et al., 1993b, 1993c), suggesting that apoR2 does not represent a molten globule. Instead, the pronounced unfolding plateau at 2-3 M GdnHCl for both active R2 and apoR2 may reflect a common intermediate in the folding pathway, and since relatively mild denaturing conditions (1 M imidazole) are known to cause dissociation of R2 into monomers (Larsson et al., 1988), we suggest that the partially unfolded intermediate represents a monomeric molten globule of R2. The denaturation curves for the mutant proteins imply that F208Y and F212Y are as stable as wild-type R2, whereas the F212W and I234N substitutions have conferred subtle destabilization of their overall structures.

What is the function of the highly conserved hydrophobic pocket in R2? To answer this, one has to consider the different steps leading to the generation of the tyrosyl radical. Formation of an active R2 from apoR2, ferrous ion, and dioxygen is a fast process with rate constants in the order of a few per second at 5 °C (Bollinger et al., 1991). The main steps in the reaction involve diffusion and binding of ferrous iron to the apo-form, binding of dioxygen to the diferrous center, formation of a highly reactive intermediate, and generation of the tyrosyl radical and the diferric center (Sahlin et al., 1989; Bollinger et al., 1991; Ling et al., 1994). Since all four mutant proteins can bind comparable amounts of iron as the wild-type protein, there is no major effect on binding of ferrous ion. Also, the introduction of the bulky group in F212W did not change the overall rate (^4)of formation of the iron center, ruling out the possibility of a specific channel for iron at this site.

Oxidation of reduced R2 occurs via direct binding of dioxygen to the diferrous center (Ling et al., 1994). Two possible binding sites for dioxygen have been modeled into the three-dimensional structure of R2 (Nordlund et al., 1990; Nordlund and Eklund, 1993). Dioxygen bound at the water ligand position of Fe1 would be in close proximity to all three residues constituting the hydrophobic pocket, with the distal oxygen in van der Waals contact with the -oxygen of Tyr-122. Dioxygen bound at the water ligand position of Fe2 would still contact Phe-208, but not Tyr-122, Phe-212, or Ile-234. A third possibility is a bridging peroxide (Ling et al., 1994), in which case modeling suggests contacts with Glu-238. The substitutions introduced in this study would thus be expected to interfere with oxygen binding, were this the main function of the hydrophobic pocket. However, all mutant proteins readily formed an oxidized iron center, suggesting that they do not interfere with binding of dioxygen at Fe1. The competing dopa-generating reaction in F208Y would be in line with a perturbed O(2) binding at Fe2, but it was recently shown that the hydroxylating oxygen originates from solvent (Ling et al., 1994), suggesting disturbances in later steps than the initial O(2) binding in this case. Thus, our protein engineering study cannot distinguish between different O(2) binding possibilities but can exclude the possibility that the presence of these hydrophobic residues will have a major influence on the O(2) binding.

In wild-type R2, the highly reactive intermediate formed from ferrous ion and oxygen reacts exclusively with Tyr-122 but might in theory abstract an electron from any neighboring atom. Therefore, one obvious question concerns whether the radical of the mutant R2 proteins is localized to Tyr-122 or not. However, tyrosyl-specific radical characteristics (EPR signal and light absorbance) were observed in all four mutant R2 proteins. For R2 F208Y, a radical at Tyr-122 is corroborated by the EPR hyperfine pattern and the saturation data as well as the lack of the transient 410 nm absorbance in the double mutant R2 Y122F/F208Y (Åberg et al., 1993a). For the F212Y and F212W mutations, the hyperfine splitting of the EPR spectra, the rapid formation kinetics of the radicals (close to that of the wild-type protein),^4 and the essentially identical maximal yield of radical also indicate a radical at position 122. The saturation behavior of R2 F212W is similar to that of wild-type R2, whereas R2 F212Y is more similar to mammalian R2, in which the radical relaxes with a P of 2500 microwatts at 28 K (Sahlin et al., 1987). Model building of R2 F212Y shows that both Tyr-122 and Tyr-212 are at about 5 Å from the iron center and that Tyr-212 can adopt the same geometry as Tyr-122 without steric clashes. Thus, a radical localization at Tyr-212 in this mutant cannot be excluded on theoretical grounds. The I234N mutant protein was isolated with a relatively stable radical and substantial enzyme activity, making it unlikely that its tyrosyl radical would not be localized at Tyr-122. The hyperfine splitting of the EPR signal indicates an approximate 10° twist of the aromatic ring relative to the geometry of Tyr-122 in the wild-type protein, which may relate to the minor change in P in I234N. A similar geometry of the tyrosine radical has been observed in bacteriophage T4 R2 (Sahlin et al., 1982). In conclusion, direct analysis as well as several complementary pieces of evidence point to a tyrosyl radical at Tyr-122 in the three mutant R2 proteins, F208Y, F212W, and I234N, whereas the localization of the radical in F212Y can be either at the mutated residue or at Tyr-122.

Does the hydrophobic pocket direct the oxidative power of the reactive intermediate toward Tyr-122? In three of the four mutant proteins (F212Y, F212W, I234N) we observed formation of two-thirds to equal amounts of tyrosyl radical as in the wild-type protein, implying that the substituted residues do not play a role in directing the electron abstraction. In the mutant protein F208Y, the bulk of the reaction is directed toward Tyr-208. Concomitant formation of a transient tyrosyl radical (Åberg et al., 1993a) suggests that the F208Y protein may distribute between catechol formation at Tyr-208 and radical formation at Tyr-122.

What is the explanation for the remarkable stability of the R2 tyrosyl radical? One stabilizing factor may be the diferric center to which the radical is magnetically coupled (Sahlin et al., 1987). The EPR studies show that the microwave power saturation of the radical in F212Y differs from that of the wild-type radical and that the geometry of the tyrosyl radical in I234N is slightly perturbed. Another stabilizing factor is clearly the immediate surrounding of Tyr-122, since three of the four mutant proteins (F208Y, F212Y, F212W) had radical half-lives that are 4-5 orders of magnitude lower than that of the wild-type radical. A high degree of hydrophobic and aromatic residues in the vicinity of the radical-carrying residue seems to be a general motif for proteins harboring stable free radicals. In apogalactose oxidase, the radical positioned at tyrosine 272 and linked by a thioether bond to a cysteine residue is suggested to be stabilized by charge transfer interactions between Tyr-272 and nearby aromatic side chains, one of which is a stacking tryptophan (Ito et al., 1991). Another example is photosystem II, where the two radical-carrying residues denoted TyrZ and TyrD, located in proteins D1 and D2, respectively (Debus et al., 1988), show very different stability. The more stable TyrD is suggested by model building with the bacterial photocenter as a framework (Svensson et al., 1990) to have a more hydrophobic and aromatic environment than TyrZ. This difference between the two tyrosine residues is thought to be one of the reasons behind the large difference in stability between these radicals (Svensson et al., 1990, 1991).

The hydrophobic pocket in R2 was suggested to constitute a binding pocket for dioxygen and to direct the oxidation power of the ferryl intermediate toward Tyr-122 (Nordlund et al., 1990, 1993) or to channel dioxygen into the iron site (Fontecave et al., 1992). With the results presented here, the invariant residues Phe-208, Phe-212, and Ile-234 seem to have negligible effects on the binding of molecular oxygen. Instead, we would like to propose that the major function of these residues is to form an insulator together with Leu-77, Ile-125, and Ile-231 that shields the reduction-sensitive -oxygen of Tyr-122 from the solvent. There is a relatively high number of internal ordered water molecules found in the R2 structure, but only one is situated close to tyrosine 122 (Nordlund and Eklund, 1993). Because of the hydrophobic nature of the pocket, no water would be bound on this side of Tyr-122. The presence of a pocket would also prevent the tyrosyl radical from interacting with other side chains. Besides the above proposed function, Phe-208 may also play a role in directing the electron abstraction toward Tyr-122 during formation of an active R2 protein.


FOOTNOTES

*
This work was supported by grants 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 U. S. National Science Foundation Grant MCB-9405723 (to L. Q.). 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.

§
To whom correspondence should be addressed. Tel.: 46-8-164150 or 46-8-153628; Fax: 46-8-152350; bitte{at}molbio.su.se.

(^1)
B.-M. Sjöberg, unpublished observation.

(^2)
The abbreviation used is: GdnHCl, guanidine hydrochloride.

(^3)
A. Åberg, personal communication.

(^4)
M. Ormö, M. Sahlin, and G. Lassmann, unpublished observation.


ACKNOWLEDGEMENTS

We thank Kirsten Enander and Agneta Slaby-Ask for technical assistance and Björn Nilsson at Pharmacia Bioscience Center for generously making the CD spectrophotometer available.


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