(Received for publication, July 20, 1994; and in revised form, November 30, 1994)
From the
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.
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, ()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.
To investigate the overall effect on protein
stability by the mutations introduced, guanidine hydrochloride (GdnHCl) ()denaturation experiments were performed and compared with
those of active and apo-forms of the wild-type R2. Since R2 contains
more than 70%
-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.
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.
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.
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.
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 ()(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 -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 ()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
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
binding in this case. Thus, our protein engineering study cannot
distinguish between different O
binding possibilities but
can exclude the possibility that the presence of these hydrophobic
residues will have a major influence on the O
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), 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.