INTRODUCTION

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The three established classes of ribonucleotide reductases catalyze the de novo synthesis of all four deoxyribonucleotides, the building blocks of DNA (1). The iron-containing class I is the one best characterized (2-4) and has been found e.g. in mammalian cells, some prokaryotes such as Escherichia coli and Salmonella typhimurium, and is coded for by DNA viruses of the herpes group. The common protein structure of class I ribonucleotide reductase is ₂₂, i.e. it is composed of two homodimeric proteins, R1 and R2. Protein R2 contains a diferric iron center and a stable free radical on a tyrosine residue necessary for enzymatic activity, whereas protein R1 contains the substrate binding site. The sequence homology in both proteins is >90% between human, mouse, and hamster ribonucleotide reductases, but only about 25% between mouse and E. coli (3). Nevertheless, the general enzymatic properties are almost the same.

The three-dimensional structures of R1 and R2 from E. coli have been determined, and the holoenzyme has been model-built (5-7). In the model, the radical-forming residue Tyr-122^· is situated in R2 in a hydrophobic pocket close to the µ-oxo-bridged diiron center, about 35 Å distant from the active site in R1. A long range electron transfer pathway consisting of conserved amino acids has been proposed to be essential for enzymatic activity of class I ribonucleotide reductases (2, 5-8). Recently, mouse R2 protein was crystallized, and its three-dimensional structure was determined (9). Despite the low sequence homology between mouse and E. coli R2 (3), the overall structure and the enzymatically important features are very similar (9). The ligands of the iron center as well as the R2 part of the proposed electron transfer pathway can be superimposed. Whereas the hydrogen bonds of the Fe1 ligand His¹⁷³ involved in the proposed electron transfer pathway are the same as in E. coli R2, His²⁷⁰, a ligand of Fe2, has fewer hydrogen bonds and is, therefore, less constrained in mouse R2.

The electron transfer pathway was suggested to consist of the conserved amino acids (Fe1) His¹⁷³-Asp²⁶⁶-Trp¹⁰³-Tyr³⁷⁰ in R2 and Tyr⁷³⁸-Tyr⁷³⁷-Cys⁴²⁹ in R1 (mouse numbering) forming a hydrogen-bonded chain between the iron center in R2 and the active site in R1. Mutation of D266A and W103(Y/F) in mouse R2 leads to a complete loss of enzymatic activity (10), as is the case for the corresponding mutations in E. coli R2.¹ The mutant mouse R2 proteins have been well characterized (10) and are identical to wild type R2 regarding secondary structure probed by CD spectroscopy (W103F) and the association/dissociation kinetics and binding constant for R1. Additionally, the unchanged Mn²⁺ binding constants, as a probe for Fe²⁺ binding, and the UV-visible spectra strongly indicate a stiochiometric iron binding in all mutant R2 proteins, including W103F (10).

Reconstitution of active R2 can easily be achieved by mixing apoR2 with ferrous iron and oxygen (11-14). Four electrons reduce the molecular oxygen: one is delivered from the tyrosine residue, and two are supplied from the ferrous iron forming the diferric center. An external electron has to come from a third iron under our conditions. In wild type mouse and E. coli R2 (11-15) as well as in the mouse mutants D266A and W103Y (10), similar yields of about 1 tyrosyl free radical (Tyr^·)²/R2 (±0.5) were achieved by various similar reconstitution procedures. However, the low amount of about 0.01 Tyr^·/R2 in apoW103F could not be increased by reconstitution with Fe²⁺ (10).

The kinetics of the reconstitution reaction of E. coli R2 with  Fe(II)/R2 has been studied intensively (13-15). Rapid freeze-quench (RFQ)-EPR and -Mössbauer studies at 5 °C yielded an EPR- and Mössbauer-visible transient, denoted species X, that was kinetically competent to be a direct precursor of Tyr^· (14, 15). Starting from aerobic apoR2, the formation rate of species X was 8 s¹ (14), whereas preloading of the anaerobic R2 with ferrous iron yielded about 60 s¹ (15), suggesting that iron binding to the aerobic R2 is the second slowest step in R2 reconstitution (Scheme 1). The slowest step in both procedures was found to be the formation of the stable Tyr^·, with an unchanged rate of 1 s¹. Mössbauer studies revealed no other iron center intermediates beside species X starting from apoR2 (14), whereas in the studies with preloaded R2, 10-20% of the iron showed unusual isomer shifts (15), possibly reflecting small amounts of accumulated iron center intermediates appearing before species X (e.g. peroxo or diferryl as proposed in Scheme 1). Our results on the reconstitution kinetics of mouse R2 proteins as well as the earlier findings in E. coli wild type R2 can be summarized in the reaction pathway sketched in Scheme 1.

View larger version (12K): [in this window] [in a new window]   Scheme 1.   Proposed reaction mechanism for the reconstitution reaction of ribonucleotide reductases. For certain steps, rate constants are known (Refs. 14 and 15 and this work). The binding of the two substrates (Fe2+ and O2) in ribonucleotide reductases is assumed to be random (k1,1' and k2,2'). The starting point for the redox reactions is assumed as an O2-R2-[Fe2]4+ intermediate, corresponding to intermediate O in methane monooxygenase (26). This will be converted to a peroxo species (k3), which in turn forms a diferryl compound R2-[Fe2O2]4+ (k4, compound Q in methane monooxygenase) reactive enough to perform the proposed H· transfer along the hydrogen-bonded chain (k5, Fig. 7). The source of the external reducing equivalent (a H· in our model) in step k5 is proposed to be a (hydrated) Fe2+ (11, 13), which is likely to be bound to Trp103/Tyr370 (38). The resulting Fe3+-Fe4+ intermediate R2-[Fe2O2H]4+, species X, eventually abstracts the H· from the tyrosine (k6). Note that not all ligands to the iron centers are shown in the structures.

Here, we have investigated the kinetics of the reconstitution reaction in wild type mouse R2 protein and in three mutants (D266A, W103Y, and W103F) on the R2 part of the proposed electron transfer pathway, thus probing its role in the reconstitution reaction. Our kinetic results show that the external electron is delivered via the proposed electron transfer pathway and give strong evidence for the participation of an intact hydrogen-bonded chain in the reconstitution reaction. To explain our findings, we propose a novel reaction scheme consisting of the delivery of electron/proton pairs, i.e. hydrogen radicals, to the iron site. Theoretical calculations (16) have recently shown that movement of an uncharged H^· in a hydrogen-bonded system is energetically favorable compared with electron transfer, which involves charge separation. Thus we will refer to the electron transfer pathway as radical transfer pathway (RTP).

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

All experiments were done in 50 mM Tris/HCl buffer, pH 7.5, 100 mM KCl with a ratio of ferrous iron to protein R2 dimer of 4 or 6.

Protein Purification-- The mouse proteins were overexpressed in logarithmically growing BL21(DE3) pLysS bacteria. They contained pETR2 plasmids encoding wild type mouse R2 protein (11) or the mutant mouse R2 proteins D266A, W103Y and W103F (10). The mutant proteins were purified according to Rova et al. (10). Wild type protein was purified as in Mann et al. (11) with slight modifications. Grinding with aluminum oxide could be omitted due to the pLysS strain. Mutant and wild type mouse R2 were mainly obtained as apo protein. If necessary Tyr^· and the iron center in wild type mouse R2 were removed by hydroxyurea/EDTA treatment and subsequent passage through a G-25 column. Fractionation yielded a protein pool with less than 10% N-terminal degraded protein monitored by SDS-polyacrylamide gel electrophoresis, whereas up to 50% N-terminal degraded protein were detected in a second protein pool. Reconstitution kinetics from both protein pools under identical conditions exhibited no significant difference (data not shown). E. coli R2 proteins were prepared as described for wild type in (17). E. coli apoproteins were produced by growing cells in iron-depleted medium (18).

Preparation of the Anaerobic Fe²⁺ Solution-- About 150 ml of buffer was thoroughly degassed in a septum-sealed 250-ml bulb for several h. A second bulb containing 5 to 15 mg of Mohr's salt crystals (NH₄)₂Fe(SO₄)₂ was sealed with a septum and evacuated. The bulbs were repeatedly flushed with oxygen-free argon (L'air liquide, Alphagaz, Sorbal 500) and evacuated. The bulbs were left under a slight excess pressure of argon. A 60-ml plastic syringe was made anaerobic by washing twice with argon gas and twice partly filled with degassed buffer and argon gas. Then the appropriate volume of anaerobic buffer to prepare the desired iron concentration was transferred into the bulb containing Mohr's salt.

RFQ-- Rapid freeze-quench (19) was performed with a System 1000 apparatus from Update Instruments by mixing 1:1 aerobic protein solution (usually 100 µM) and anaerobic Fe²⁺ solution (400-600 µM). An EPR tube connected with a funnel fixed in a home-built holder was immersed completely into an isopentane bath. The stirred isopentane in the bath was cooled by a steady flow of gaseous N₂, which in turn was cooled by liquid N₂. For sample preparation stirring was switched off to avoid movement of the funnel and to allow its proper placement beneath the nozzle. Funnels were made from tips for 5-ml Gilson pipetman by cutting off the small end and widening it.

Slow Freeze Quench (SFQ)-- Slow freeze-quench samples were obtained by hand-mixing. EPR tubes filled with 80 µl of apoprotein and the septum-sealed bulb with the anaerobic Fe²⁺ solution were thermostated at the reaction temperature. A gas-tight Hamilton syringe with a long needle was washed several times with the anaerobic Fe²⁺ solution to remove oxygen. Then, 80 µl of Fe²⁺ solution was thermostated inside the syringe at the reaction temperature by immersing the syringe but not the end of the needle into the water bath. After temperature equilibration, the iron solution was swiftly injected into the apoprotein solution, and the reaction was stopped by immersing the EPR tube in cold n-pentane (120 °C). For very short time points like 4 s, the mixing was done quickly above the n-pentane bath. The results confirmed that this was done rapidly enough to avoid changes in temperature.

Reconstitution of Active R2 from Fe(II)-preloaded R2 Protein-- To investigate if iron binding is the rate-limiting step in the reconstitution reaction or if it is affected by the mutation, both wild type and W103Y R2 proteins were preincubated with ferrous iron before oxygen addition. An EPR tube fitted with a stopcock was filled with 200 µl of protein solution (approximately 23-30 µM). Anaerobic protein solution was obtained by equilibration of the EPR tube with argon for 30 min on ice. Then a 3-4-fold molar excess of ferrous iron (a freshly prepared, argon-purged solution of Mohr's salt) was added anaerobically to the protein. The mixture was then preincubated at 4 °C (wild type R2) or 16 °C (W103Y R2) for 5 min. Then a 3-fold molar excess of oxygen was added in form of 50 µl of oxygen-saturated water equilibrated at the reaction temperature. The reaction was stopped as described above (SFQ).

EPR Measurements-- The EPR measurements were made at 9 GHz (X-band) on a Bruker ESP 300 spectrometer equipped with an Oxford Instruments continuous flow cryostat for temperatures below 77 K or a cold finger Dewar for 77 K. Signals were measured at three different microwave powers to ensure that they were not saturated with microwave power. An average spin concentration was calculated by comparison with a 1 mM copper standard (12 mM H₃ClO₄, pH 1.8) or a mouse R2 standard with a well known radical content calibrated earlier against the copper standard. Composite spectra containing the signal of the stable tyrosyl radical (Tyr^·) and the new EPR singlet (mouse X) were evaluated by subtracting fractions of the Tyr^· spectrum of the corresponding 15-min sample using the ESP 300 software. The concentration of mouse X given by the resulting spectra was determined by comparison with the copper standard. The concentration of Tyr^· in these composite spectra was calculated in two ways, from the multiplication factor used and the Tyr^· content of the 15-min samples and by the difference between total radical and mouse X concentration.

Evaluation of Kinetics-- The Tyr^· concentrations measured were corrected for the Tyr^· content of the blank. The packing factor was estimated by comparing the Tyr^· concentration in samples frozen directly and those that were produced by RFQ, both aged for long reaction times. The Tyr^· concentrations were divided by the protein concentration corrected for the packing factor. Tyr^·/R2 versus time plots were first evaluated with a first order rate equation including a variable for the dead time, which varied between 13 and 30 ms. For presentation in the figures, the dead time was added to the reaction times calculated from the length of the reaction tube.

	RESULTS

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Formation of the Stable Tyr^· in Wild Type Mouse R2-- The reconstitution reaction of wild type mouse R2 with 4 or 6 Fe(II)/R2 was investigated at several temperatures between 5 and 32 °C by RFQ and SFQ methods. At 5 and 10 °C reaction temperatures none of the EPR spectra recorded between 4 and 77 K from RFQ or SFQ samples quenched from 8 ms reaction times up did reveal any EPR visible species beside Tyr^·. As shown in the inset of Fig. 1, all spectra recorded have about the same shape as the final spectrum after 15 min, showing only the stable Tyr^·.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Time course of the reconstitution reaction of wild type mouse R2 at 5 °C with 4 Fe(II)/R2. Only formation of Tyr· (Y·) is detectable. Rapid freeze quench () and slow freeze quench () time points show the same first order rate kinetics kform = 0.14 s1 (Equation 1; solid line), indicating that both methods work equally well. Reconstitution of Fe(II)-preloaded mouse wild type R2 protein showed a very similar time course (, dashed line), indicating that in mouse R2 the uptake of Fe(II) is not the limiting step for the formation of intermediates as it is in E. coli R2 (15). The SFQ data ( and ) are average values from at least three experiments; their variance is indicated by the error bars. The inset shows selected EPR spectra at different time points of the kinetics: apoprotein, 313 ms, 4 s (SFQ), 5 s (RFQ), 10 s, 30 s, and 15 min (RFQ and SFQ), in order of increasing amplitude. Recording conditions: frequency 9.43 GHz, temperature 20 K, microwave power 10 µW, modulation frequency 100 kHz, modulation amplitude 3 G, time constant 43 ms, scan time 84 s, and receiver gain 3.2 × 104.

1

(Eq. 1)

t,

View this table: [in this window] [in a new window]   Table I Kinetic constants (rate kform and final yield (Tyr·/R2)) for the reconstitution reaction of wild type, D266A, and W103Y mouse R2 protein at various temperatures and iron/R2 ratios Values and errors were achieved by fits with GraFit®.

(Eq. 2)

k_bt

k_at

k

Suppression of the Reconstitution Reaction upon Breakage of the Hydrogen-bonded Chain-- The mutation W103F interrupts the hydrogen-bonded chain formed by conserved residues from the surface of the R2 protein to the iron site. Any change in the reconstitution reaction of such a mutant protein may be regarded as a hint that this particular hydrogen bond is involved in the delivery of the external electron and may elucidate some aspects of the reaction mechanism. Up to now it was not clear whether W103F forms an unstable Tyr^· or does not at all form Tyr^· upon reconstitution, whereas its light absorption spectra indicates formation of a normal iron center (10). Therefore, we scanned the reconstitution kinetics of W103F completely down to the limit of time resolution by reaction times equally spaced on a logarithmic scale (inset of Fig. 2). Combined RFQ and SFQ experiments between 20 ms and 18 min at 20 °C 6Fe(II)/R2 show that W103F does not form any transient Tyr^· under our conditions. An unchanged background amount of about 0.01 Tyr^·/R2 was measured in all samples (10).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Reconstitution kinetics of D266A mutant mouse R2 protein with 6 Fe(II)/R2 at 25 °C. Formation of Tyr· (Y·) is also pseudo first order (Equation 1, kform = 0.29 s1) but 24 times slower (Table I) than in mouse wild type R2 under the same conditions (Fig. 6). The inset shows that in W103F no tyrosyl radical above the background is formed under the same conditions as above. A constant contamination of 0.01 Tyr·/R2 was detected in all samples including the apoprotein.

Slowing Down of the Reconstitution Reaction upon Alteration of the Hydrogen-bonded Chain-- The mutant mouse R2 proteins D266A and W103Y have been shown to develop a stable Tyr^· up to a yield of 1 Tyr^·/R2 with an anaerobic reconstitution procedure (10). We measured the kinetics of the reconstitution reaction of these mutant proteins with 6 Fe(II)/R2 at various temperatures. The W103Y and D266A R2 proteins developed the stable tyrosyl radical only to 0.3-0.5 Tyr^·/R2 under our conditions. The time course of the generated stable Tyr^· is shown in Fig. 2 for D266A at 25 °C. All time courses for D266A and W103Y at different reaction temperatures exhibit the same kinetic pattern (data not shown) with different formation rates k_form (Table I). No EPR visible intermediates could be detected at any of the reaction temperatures investigated, neither in D266A nor in W103Y. The formation kinetics of the stable Tyr^· in these two mutant proteins are still first order without a detectable lag period but on average a factor of 20 slower than in wild type mouse R2 (Table I).

Reconstitution Kinetics of R2 Proteins Preloaded with Ferrous Iron-- To elucidate if iron binding is a rate-limiting step in the reconstitution or if the mutations affect the kinetics of iron binding, we performed reconstitution experiments with Fe(II)-preloaded R2 proteins. SFQ measurements with wild type R2 at 4 °C yielded k_form of 0.10 s¹ (Fig. 1, open squares), which is not significantly different from k_form = 0.14 s¹ obtained without preloading of iron at 5 °C (Fig. 1, closed symbols). As in the nonpreloading measurements for wild type R2, no other EPR visible intermediate could be detected. Taken together these findings indicate that Fe(II) binding is not a limiting step in the reconstitution reaction of mouse wild type R2.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Kinetics of Fe(II)-preloaded W103Y mouse R2 at 16 °C () compared with apo-W103Y mouse R2 at 15 °C (). For the experiments with Fe(II)-preloaded W103Y mouse R2, error bars were included to visualize the variance in the measurements. Points are averages of at least two experiments beside 12 s (). The time courses are identical in the error range of the measurements, indicating that the 20 times slower formation of Tyr· (Y·) in the mutants is not due to a different iron uptake kinetics. The Tyr· formation in the wild type R2 at 18 °C is included for comparison (). Kinetic data ([Tyr·]/[R2])t were normalized to the corresponding final yield ([Tyr·]/[R2]) for a better comparison of the curves.

Temperature Dependence of the Reconstitution Reactions-- The temperature dependence of the Tyr^· formation rates k_form of wild type and mutant mouse R2 proteins is shown in Fig. 4. At 5 °C the Tyr^· yield in the mutants D266A and W103Y was very low, and therefore the kinetics was not measurable. Above 30 °C the mutant apoproteins started to precipitate. However, between 10 and 25 °C the reconstitution kinetics of the mutant proteins gave reproducible results and could be evaluated according to the Arrhenius law (Equation 3).

(Eq. 3)

The 1/T dependence of ln(k_form) of wild type mouse R2 is linear between 5 and 32 °C and gave similar results for 4 () or 6 () Fe(II)/R2. The activation enthalpy H_a of about 140 ± 20 kJ/mol for wild type mouse R2 is only slightly affected by the mutations, leading to 110 ± 20 kJ/mol and 90 ± 20 kJ/mol for D266A and W103Y, respectively.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Arrhenius plots of kform from wild type (4 () and 6 () Fe(II)/R2), D266A (), and W103Y () mouse R2 proteins. The reconstitution reaction of all three mouse proteins is strongly dependent on temperature and yields activation enthalpies of about 140, 110, and 90 kJ/mol, respectively (Equation 3).

Appearance of a New EPR Singlet Species, Mouse X-- Reconstitution of apo wild type mouse R2 with Fe²⁺ resulted in the appearance of small amounts of a new EPR active intermediate at reaction temperatures between 18 and 32 °C (Fig. 5). Composite EPR spectra were obtained from RFQ samples up to 200 ms of reaction time, e.g. after 61 ms reaction time at 25 °C, the second EPR component is clearly detectable (Fig. 5A) compared with the final EPR signal of Tyr^· after 15 min of reaction time (Fig. 5B). After subtraction of a fraction of a pure Tyr^· spectrum, an EPR singlet-like spectrum denoted as mouse X became visible (Fig. 5C). The peak-to-trough line width of mouse X was determined to 18 ± 2 G and was found to be invariant between 4 and 77 K. The line width is apparently identical to the 18 ± 2 G of species X from E. coli R2 Y122F (Fig. 5D) at 8 K (13). Upon reconstitution with ⁵⁷Fe and subtraction as above, broadening and hyperfine coupling to the nuclear spin of ⁵⁷Fe is observed in both mouse X and species X of E. coli R2 Y122F (13 and data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Comparison of EPR spectra from mouse X and E. coli X. The reconstitution of mouse R2 and E. coli R2 was performed at 25 °C with 6 Fe(II)/R2 and 4 Fe(II)/R2, respectively. The EPR spectra of mouse X were obtained after subtraction of appropriate amounts of the stable tyrosyl radical spectrum (see text). A, 61 ms, mouse R2, 22 µM total radical; B, 15 min, mouse R2, 60 µM Tyr·; C, 61 ms, mouse X obtained by subtraction of a fraction of B from A, 4.2 µM; D, 200 ms, E. coli R2 Y122F species X, 12 µM. The line shape and line width (18 ± 2 G) of E. coli R2 Y122F species X and mouse X are identical. Recording conditions: frequency 9.43 GHz, temperature 8 K, microwave power 10 µW, modulation frequency 100 kHz, modulation amplitude 3 G, time constant 43 ms, scan time 84 s, and receiver gain 3.2 × 104.

Kinetics of Mouse X-- The concentration of mouse X after different reaction times was determined after spectra subtraction as described above. In Fig. 6 the time course for mouse X and the stable Tyr^· in the reconstitution reaction of wild type mouse R2 with 6 Fe(II)/R2 at 25 °C is shown. The formation of Tyr^· could still be described by the first order rate Equation 1 yielding k_form = 7.1 s¹. However, subtracting simulated curves of Equations 1 and 2 from each other using the values A = 1, k₅ = 7 s¹ (= k_form with respect to k_a), and e.g. k₆ = 10 · k₅ = 70 s¹ (= k_b in Equation 2) yielded a maximal difference of 0.08 at 35 ms. Such a small difference is not detectable with a point to point kinetic method like RFQ, and 8% is less than the usual error for spin concentration determination in EPR. Thus, a k₆ only 10 times faster than k₅ is already sufficient to not lead to any detectable lag period in the time course of Tyr^· formation. Inserting the parameters above in the time dependence of the intermediate B of a consecutive reaction as shown in Equation 4 allowed an accumulation of B to about 0.08. This is comparable with the amount of mouse X found in the reconstitution of wild type mouse R2 with 6 Fe(II)/R2 at 25 °C shown in Fig. 6.

(Eq. 4)

where ([X]/[R2])_t, are the concentration ratios at time t and at long times if no decay took place, respectively, and k_5,6 are rates from Scheme 1.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Reconstitution kinetics of mouse R2 wild type with 6 Fe(II)/R2 at 25 °C. The formation of Tyr· is consistent with a first order rate kinetics, kform = 7.1 s1. The inset shows a blow-up of the data obtained for mouse X. Here, the error bars show the variations that result from judging the amount of Tyr· spectra that was subtracted from the composite spectra. The curves represent mathematical fits assuming different models (see "Results, Kinetics of Mouse X") except the dashed and dotted line, which is fitted by eye, changing the values for the parameters of Equation 5 manually. The much better description of the data by Equation 5 with ([X]/[R2]) = 0.22, k5 = 54 s1, k6 = 45 s1, and t = 35 ms compared with the other models indicates that mouse X is an intermediate of a side reaction as proposed in Scheme 2.

Is Mouse X a Kinetically Competent Precursor of Tyr^·?-- Assuming k₆  k₅ and that mouse X is a precursor of Tyr^·, then ([X]/[R2]) and k₅ in Eq. 4 have to be the same as A₀ and k_a in Equation 2. As we know ([Tyr^·]/[R2]) = 1.1 and k_form = 7 s¹ from fitting ([Tyr^·]/[R2])_t with Equation 1, which is a very good approximation for Equation 2, we can use these values as constant parameters in Equation 4. Determination of the variable k₆ by fitting Equation 4 to the mouse X data in Fig. 6 yielded k₆ = 112 ± 38 s¹, which is much bigger than k₅, as expected, but the shape of the curve (solid line in the inset of Fig. 6) fits badly to the experimental points.

1

1

1

1

(Eq. 5)

1

1

1

View larger version (12K): [in this window] [in a new window]   Scheme 2.   Proposed reaction mechanism for the formation of the diferric site without formation of Tyr·, a side reaction of the reconstitution in vitro. For this reaction, evidenced by the finding of more diferric sites than Tyr· radicals (14, 28), we likely detected mouse X as an intermediate. The difference from Scheme 1 is the source of the second H· (k6), which here might come from a second external [Fe(H2O)6]2+ instead of the tyrosine.

	DISCUSSION

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In respiratory and photosynthetic processes, unidirectional electron transport occurs between redox centers of different redox potentials (22-24). Two models for electron transport, one proposing a uniform traveling barrier for electrons unaffected by protein structure (23), the other including guidance of an electron through covalent and hydrogen bonds (24), are commonly discussed for distances up to 20 Å. For the enzymatic reaction in class I ribonucleotide reductases an electron has to be transferred over 35 Å, since it is believed that Tyr^· in R2 abstracts an electron from a cysteine at the active site of R1, and the radical transfer must be reversible (2, 7). These features are clearly different from those usually found in other redox proteins and raise the question what kind of transfer actually takes place during catalysis in ribonucleotide reductases. The presence of a conserved hydrogen-bonded chain between the two sites, Tyr^177· in mouse R2 and Cys-429 in mouse R1, the fact that Tyr^· is a neutral radical lacking a H^·, and that the first step of the enzymatic reaction is proposed to be H^· abstraction from the substrate suggests a transport of a proton concomitant with the transfer of an electron, i.e. H^· transfer. Therefore, the hydrogen-bonded chain might serve as a RTP. This is in good agreement with recent theoretical calculations leading to high activation energies for a pure electron transfer from one uncharged amino acid (R1-Cys⁴²⁹) to another (R2-Tyr^177·) due to the charge separation necessary (16).

Reactions at the Iron Center-- For the following discussion we assume the reaction pathway for the reconstitution as outlined in Scheme 1 (13-15, 25). Binding of the two substrates Fe²⁺ (k₁ and k_1') and O₂ (k₂ and k_2') is random, since both aerobic and anaerobic reconstitution procedures are possible. A probably very short lived, O₂-R2-[Fe₂]⁴⁺ intermediate is proposed in analogy with the early O₂-[Fe₂]⁴⁺ complex in the enzymatic cycle of soluble methane monooxygenase (26). The irons are oxidized via a peroxo intermediate (k₃) to a diferryl compound (k₄, Q in methane monooxygenase), which is converted to species X by one external reducing equivalent (k₅). The source of the external reducing equivalent was shown to be either Fe²⁺, as under our conditions, or other reductants like ascorbic acid (12-14). The last step is the formation of Tyr^· by abstraction of an H^· (k₆) from the tyrosine residue adjacent to the iron center.

1

1

1

Nature of the External Reduction Equivalent-- In contrast to W103Y, the mutant W103F does not even form a transient Tyr^· during reconstitution, although a diferric iron center seems to be formed (10). The only molecular difference between W103Y and W103F is the phenyl hydroxyl group, which is apparently crucial for the reconstitution. This observation cannot be explained by either of the two currently most discussed electron transport models (22-24).

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Proposed model for the last steps (k5 and k6) of the reconstitution of mouse R2 proteins under nonlimiting iron conditions. The arrows indicate the shift of a hydrogen radical along the RTP (k5). The highly reactive [Fe2O2]4+ species might abstract a H· from the N of His173, which in turn takes a H· from Asp266, which takes a H· from Trp103. Eventually the Trp103· is quenched by the simultaneous oxidation/deprotonation of hydrated Fe2+ bound to the aromatic system of Trp103 by cation- interaction (38). This consecutive series of H· abstractions is supposed to be very fast if not simultaneous in the sense that the abstraction of the initially covalently bound hydrogen is supported by the delivery of the initially hydrogen-bonded hydrogen. Finally, mouse X is thought to abstract a second H· from Tyr177, forming the stable Tyr· (k6). The latter process is proposed to be faster than the delivery of the external H· (k6  k5) so that the paramagnetic iron intermediate mouse X cannot accumulate.

Effect of Alteration of the Hydrogen-bonded Chain-- Trp¹⁰³ and Asp²⁶⁶ have been shown to participate in radical transfer during catalysis (10). Our present result shows that they are also involved in the reconstitution reaction, since the kinetics are on average slowed down by a factor of 20 compared with wild type mouse R2 (Table I). Their activation enthalpies are very similar to that of wild type mouse R2 (Fig. 5). This suggests that the overall reaction mechanism is only little affected by these mutations.

From What Species Is the External Reducing Equivalent Delivered?-- It is unlikely that a hydrogen radical is delivered from the (reduced) iron site of another R2 subunit without creation of an EPR visible signal, e.g. the mixed valent state (33, 34). It is more likely that some external iron binds to Trp¹⁰³ (Fig. 7) or maybe to Tyr³⁷⁰ and delivers the electron/proton pair. An almost concomitant hydrogen transfer from a water ligand of an Fe²⁺ bound to the surface of R2 as well as from the tyrosine to the proposed [Fe₂O₂]⁴⁺ intermediate of the iron center is consistent with all findings in the mouse R2 reconstitution reaction so far (Fig. 7).

What Regulates the Functional State of the RTP?-- The entire RTP seems to depend on intact hydrogen bonds, since it can be knocked out by mutations similar to W103F on residues in the E. coli R1 protein corresponding to mouse R1 residues Y737F and Y738F (36).