The Iron-Oxygen Reconstitution Reaction in Protein R2-Tyr-177 Mutants of Mouse Ribonucleotide Reductase
EPR AND ELECTRON NUCLEAR DOUBLE RESONANCE STUDIES ON A NEW TRANSIENT TRYPTOPHAN RADICAL*

Stephan PötschDagger §, Friedhelm Lendzian, Rolf Ingemarsonparallel , Andreas Hörnbergparallel , Lars Thelanderparallel , Wolfgang Lubitz, Günter Lassmann, and Astrid GräslundDagger **

From the Dagger  Department of Biophysics, Stockholm University, Arrhenius Laboratories, S-106 91 Stockholm, Sweden, the  Max-Volmer-Institut für Biophysikalische Chemie und Biochemie, Technische Universität Berlin, D-10623 Berlin, Germany, and the parallel  Department of Medical Biochemistry and Biophysics, Umeå University, S-901 87 Umeå, Sweden

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The ferrous iron/oxygen reconstitution reaction in protein R2 of mouse and Escherichia coli ribonucleotide reductase (RNR) leads to the formation of a stable protein-linked tyrosyl radical and a µ-oxo-bridged diferric iron center, both necessary for enzyme activity. We have studied the reconstitution reaction in three protein R2 mutants Y177W, Y177F, and Y177C of mouse RNR to investigate if other residues at the site of the radical forming Tyr-177 can harbor free radicals. In Y177W we observed for the first time the formation of a tryptophan radical in protein R2 of mouse RNR with a lifetime of several minutes at room temperature. We assign it to an oxidized neutral tryptophan radical on Trp-177, based on selective deuteration and EPR and electron nuclear double resonance spectroscopy in H2O and D2O solution. The reconstitution reaction at 22 °C in both Y177F and Y177C leads to the formation of a so-called intermediate X which has previously been assigned to an oxo (hydroxo)-bridged Fe(III)/Fe(IV) cluster. Surprisingly, in both mutants that do not have successor radicals as Trp· in Y177W, this cluster exists on a much longer time scale (several seconds) at room temperature than has been reported for X in E. coli Y122F or native mouse protein R2. All three mouse R2 mutants were enzymatically inactive, indicating that only a tyrosyl radical at position 177 has the capability to take part in the reduction of substrates.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The reduction of ribonucleotides to their corresponding deoxyribonucleotides is an essential reaction for all living cells and provides precursors for DNA synthesis. This reaction is catalyzed by the enzyme ribonucleotide reductase (RNR).1 A number of RNRs of different species have been discovered and classified into three major classes (1). A common feature of all RNRs within these three classes is that they use radical mechanisms to reduce ribonucleotides. RNR of class Ia, found in mammals, plants, DNA viruses, and some procaryotes like Escherichia coli, consists of two components, proteins R1 and R2 (2, 3).

The three-dimensional structures of proteins R2 of E. coli (4) and mouse (5) and of E. coli protein R1 (6) provide a basis for understanding the enzymatic reaction. A main step in ribonucleotide reduction, which takes place in the large subunit, protein R1, is the abstraction of the 3'-hydrogen from the ribose moiety of the substrate by a protein-linked radical (probably on Cys-439) (7, 8). Since Tyr-122 in the small subunit, protein R2 of E. coli (Tyr-177 in protein R2 of mouse RNR), is deeply buried inside the protein structure, it cannot take part directly in the substrate reduction in protein R1. A structure model of the enzyme (based on the known R1 and R2 structures) shows that Tyr-122 is connected to the substrate-binding site in protein R1 over a total length of about 35 Å via a network of conserved hydrogen-bonded amino acids, the so-called radical transfer pathway (2). It is believed that coupled electron/proton (H· radical) transfer takes place via this pathway during the enzymatic reaction (9). The enzyme loses activity when this network is disturbed, e.g. by mutations (10, 11).

The tyrosyl radical and its adjacent µ-oxo-bridged diferric iron center is formed in vitro in the so-called reconstitution reaction, when ferrous iron reacts with apoprotein R2 and molecular oxygen. This reaction, which results in the reduction of molecular oxygen to water, requires four reducing equivalents. Three are provided by the oxidation of the tyrosyl residue and the two ferrous irons. The fourth may come from an external ferrous iron ion via the radical transfer pathway. Schmidt et al. (12) showed recently that the radical transfer pathway in mouse protein R2 is involved in the generation of the tyrosyl radical/diferric iron site. In the E. coli protein R2 mutant Y122F, where the tyrosyl radical cannot be formed, other aromatic amino acids in the vicinity of the diferric iron center become oxidized, like tryptophans (13, 14) or a tyrosine (15). These radicals did not yield enzyme activity but show the existence of side pathways for the radical transfer in protein R2 when Tyr-122 is not available.

The unique high stability of the protein-linked tyrosyl free radical on Tyr-122 in protein R2 of E. coli RNR has been discussed since its discovery and spectroscopic assignment in 1972 (2, 16). As shown by site-directed mutagenesis, the tyrosyl radical might be protected by a cluster of hydrophobic amino acids, like in E. coli protein R2; changing only one hydrophobic amino acid for a hydrophilic one leads to a drop in the lifetime of the radical by several orders of magnitude (17).

In the present study we addressed the question whether another redox-active amino acid (for example tryptophan) may take over the role and enzymatic function of a tyrosine. In solution both tyrosine and tryptophan have similar redox potentials, 0.94 and 1.05 V, respectively (18). We have constructed protein R2 mutants from mouse and E. coli RNR,2 where the radical carrying Tyr-177 (122 in E. coli) is replaced by other oxidizable amino acids with side chains of different sizes, like tryptophan, cysteine, and histidine or a non-oxidizable amino acid like phenylalanine. The latter was constructed to look for side pathways or suppressed radicals as previously observed in E. coli protein R2 Y122F (13, 14). We have used EPR and ENDOR to study the paramagnetic species formed in the Fe2+/oxygen reconstitution reaction of the mouse protein R2 mutants Y177W, Y177C, and Y177F. The results underline the uniqueness of the tyrosyl radical in the catalytic reaction of the native enzyme.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- L-Tryptophan (indole-d5, 98%) and 57Fe (95.2%) were purchased from Larodan Fine Chemicals AB, Sweden; D2O, 99.97%, was obtained from Studsvik Energiteknik AB, Sweden. Tris buffer in D2O was prepared by freeze-drying a solution of 50 mM Tris-HCl, 0.1 M KCl, pH 7.5, and dissolving it in D2O. This procedure was repeated twice.

Plasmids-- The mouse R2 protein is produced in E. coli by expression from its cDNA using the plasmid pETM2 (19, 48). A new modified plasmid was constructed expressing the mouse R2 mutants Y177W, Y177F, and Y177C. By using the R2-expressing pETM2 plasmid as a template, a 246-base pair DNA fragment was amplified by the polymerase chain reaction with the mutated primer. The following upstream primers were used: for Y177W, 5'-CAA ATT GCC ATG GAA AAC ATA CAC TCT GAA ATG TGG AGT CTC CTTA-3'; for Y177F, 5'-CAA ATT GCC ATG GAA AAC ATA CAC TCT GAA ATG TTC AGT CTC CTTA-3'; and for Y177C, 5'-CAA ATT GCC ATG GAA AAC ATA CAC TCT GAA ATG TGC AGT CTC CTTA-3'. The downstream primer in each reaction was 5'-GAG CCA GAA TAT CGA TGC AA-3'. The changing amino acid codon 177 is underlined. The second downstream primer contained the restriction cleavage site of MluI. The resulting PCR fragment was cleaved with the restriction enzymes NcoI and MluI and introduced into the plasmid pETM2 cleaved in the same two unique restriction sites. The new constructions pETM2-Y177W, pETM2-Y177F, and pETM2-Y177C were verified with dideoxyribonucleotide sequencing.

Expression and Purification of the Mutant R2 Proteins-- The plasmid pETM2-Y177W (pETM2-Y177F or pETM2-Y177C) was transfected into E. coli strain BL21(DE3)pLysS that contains an IPTG-inducible chromosomal copy of the T7 RNA polymerase gene and a plasmid with the T7 lysozyme gene (20). LB medium (5 liters) containing carbenicillin 50 mg/ml and chloramphenicol 25 mg/ml was inoculated with 10 ml of overnight cultures, shaken (275 rpm) at 37 °C, and supplemented with IPTG (0.5 mM) at A590 = 0.7. After a further 4 h at 37 °C, the cultures were chilled, centrifuged at 2,500 × g for 20 min at 2 °C, and gently resuspended in 100 ml of 50 mM Tris-HCl, pH 7.6. The suspensions were frozen in liquid nitrogen and stored at -70 °C.

For preparation of protein R2 Y177W with indole-d5-Trp, the bacteria were grown in a minimal medium (21) including the antibiotics described above. At the time for induction with IPTG the medium was supplemented with a solution of indole-d5-Trp to a final concentration of 0.003% (w/v). The time between induction with IPTG and harvest was prolonged to 6 h.

Frozen bacteria were gently thawed at 25 °C in a water bath, and the viscous solution was cleared by centrifugation at 45,000 rpm for 1 h at 2 °C (Beckman L-90 ultracentrifuge, Ti70 rotor). The purification was performed as described earlier for the mouse R2 recombinant protein (19, 48). The R2 proteins were in the apo form (metal-free) after purification. Protein purity was analyzed by SDS-polyacrylamide gel electrophoresis. The protein R2 concentrations were measured by light absorbance and calculated using the molar extinction coefficient E280-310 = 124,000 M-1 cm-1 (19, 48). The proteins were kept and investigated in 50 mM Tris-HCl, 0.1 M KCl, pH 7.5. The enzymatic activity of the native and mutated R2 proteins was measured in the presence of pure recombinant mouse protein R1 using the [3H]CDP reduction as described earlier (22).

In order to exchange the H2O to D2O buffer 200 µl of apoprotein was kept in 2 ml of 50 mM Tris-HCl, 0.1 M KCl in D2O, pH 7.5, at 5 °C. After 12 h incubation the diluted apoprotein was concentrated with a Centricon 10 (Amicon) (10-kDa exclusion size). When the volume was approximately 200 µl, an additional 2 ml of 50 mM Tris-HCl, 0.1 M KCl in D2O, pH 7.5, were added to the apoprotein, and the solution was again concentrated to approximately 200 µl. The concentrating procedure was repeated twice.

Reconstitution Reaction of Apoprotein R2 with Fe2+ and Oxygen-- The reconstitution of the iron site in the mutant proteins R2 in H2O or D2O buffer at room temperature (22 °C) was carried out as follows: apoprotein R2 in oxygen-saturated 50 mM Tris-HCl, 0.1 M KCl, pH 7.5, was mixed with an equal volume of an anaerobic (NH4)2Fe(SO4)24H2O solution (six times molar excess) dissolved in 50 mM Tris-HCl, 0.1 M KCl, pH 7.5. A rapid freeze quench technique (System 1000 from Update Instruments) was used to obtain time points from 8 ms to 2 s. Both the aerobic apoprotein R2 and anaerobic Fe2+ solutions were rapidly mixed and sprayed into a cold isopentane bath (-120 °C) to quench the reaction. The frozen spray was then tightly packed into an EPR tube (12). For reaction times >2 s, the anaerobic ferrous iron solution was mixed with the apoprotein R2 in an EPR tube using a gas-tight Hamilton syringe. The reaction was stopped by immersing the EPR tube into cold n-pentane (-120 °C). Radical decay was monitored by measuring the amount of free radical versus the reaction time of the sample warmed up to room temperature and refrozen again.

The amount of bound iron in protein R2 was colorimetrically determined after removing unspecifically bound iron by running the protein R2 through a Sephadex G-25 column (23, 24). The apoproteins contained approximately 0.2 iron ions/protein R2 after purification. An acidic 57Fe stock solution was prepared as described in Sturgeon et al. (25). The reconstitution of the protein with 57Fe was done as described above except that the protein was dissolved in 200 mM HEPES, pH 7.5.

EPR and ENDOR Instrumentation-- X band EPR spectra were recorded on a Bruker ESP 300E spectrometer using a standard rectangular Bruker EPR cavity (ER4102T) equipped with an Oxford helium flow cryostat or on a Bruker ESP 380E spectrometer using a Bruker dielectric ring flex line resonator (ER 4118-X-MD-5W2) equipped with an Oxford helium bath cryostat. Continuous wave (cw) ENDOR spectra were measured on a Bruker ESP 300E spectrometer using a self-built ENDOR accessory, which consists of a Rhode and Schwarz RF synthesizer (SMT02), an ENI A200L solid state RF amplifier, and a self-built high-Q TM110 ENDOR cavity (26). The cavity was adapted to an Oxford helium flow cryostat (ESR 910). The measured g values were calibrated using the known g value standard Li/LiF, with g = 2.002293 ± 0.000002 (27). Spin concentrations were determined by comparison of double integrals of EPR spectra with that of the tyrosyl radical in the mouse R2 wild type Tyr-177 which had been previously calibrated with a 1 mM Cu2+/EDTA standard. Evaluation of the microwave power saturation behavior of the radical was done by using the plot described in Ref. 28. The microwave saturation and the kinetic curves were fitted using the program Grafit 3.01.

Mass Spectrometry-- The correct incorporation of isotope-labeled tryptophan into protein R2 was proved by electrospray ionization or matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Approximately 3 nmol of apoprotein R2 was dissolved in 1 ml of 1% acetic acid and injected into an liquid chromatography/mass spectrometry-coupled electrospray single quadruple mass spectrometer (Applied Biosystems, Sciex, Perkin-Elmer Corp., Canada). For determining the mass by matrix-assisted laser desorption ionization-time-of-flight (Voyager; Perspective Biosystems Inc.), approximately 1 pmol of apoprotein R2 was crystallized in an excess of sinapinic acid as matrix. The following molecular masses of mouse R2 proteins in Da were determined (calculated polypeptide molecular mass in parentheses): native R2, 45,101.5 (45,099); Y177W R2 indole-h5-Trp, 45,129 (45,122.6); Y177W R2 indole-d5-Trp, 45155 (45,157.6).

Simulation of EPR Powder Spectra-- The EPR powder spectra have been analyzed using a program for simulation and fitting of EPR spectra with anisotropic g and hyperfine tensors based on the work of Rieger (29). Resonant field positions were calculated to second order for arbitrary orientations of the g and hyperfine tensor principal axes (14).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mouse Protein R2 Mutants-- All three mutated R2 proteins, Y177W, Y177C, and Y177F, behaved like the native protein R2 during the growth and purification procedure; the final yield was approximately 4-6 mg of pure protein R2/liter of bacterial culture. The light absorption spectra of all three mutants after 6Fe2+/R2 reconstitution are shown in Fig. 1. The absorbance of the mutants is significantly increased in the 300-400-nm region after the reconstitution reaction. However, the two bands at 320 and 370 nm, which are characteristic for a µ-oxo-bridged diferric iron center like in the active form of the native mouse protein R2 dimer (Fig. 1e), are not resolved in the mutant proteins. The Met form of native mouse protein R2 (19, 48) and other mouse R2 mutants (10) shows similar badly resolved light absorption spectra in the iron absorption region as seen for the mutants in Fig. 1, b-d. Upon reconstitution of Y177W, a broad transient light absorption band in the range 500-600 nm with a maximum at 550 nm and a half-width about 60 nm appeared initially (Fig. 1, inset). This band is completely gone after 2 min reaction time, which corresponds approximately with the decay of the transient EPR doublet signal in Y177W R2 (Fig. 3, discussed below). In an earlier work (13) the light absorption spectrum of a neutral tryptophan radical in E. coli Y122F R2 was described as a broad band centered at 545 nm. From the similar characteristics of the transient absorption bands centered at 550 nm in mouse Y177W R2 and E. coli R2 Y122F (13), we assigned the observed transient here to the tryptophan radical.


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Fig. 1.   Light absorption spectra of non-reconstituted apo form (a) and reconstituted mouse R2 proteins Y177F (b), Y177C (c), Y177W (d), and native (e) at 10 °C (22 µM protein R2). Inset, transient light absorption band upon reconstitution of Y177W measured after 15 s (a), 2 min (b), and 5 min (c) reaction time; spectrum recording time, 15 s.

Further characterization of the amount of bound iron in the protein R2 mutants by spectrophotometric analysis showed that all three mutants were able to bind approximately 3.5-4 iron ions/protein R2. In holoenzyme studies the interaction between native protein R1 and mutant protein R2 was analyzed by BIAcore studies as described earlier (30). The binding efficiency of Y177W, Y177C, and Y177F to protein R1 was about the same as found for native protein R2 (data not shown).

The enzyme activity of the mutated R2 proteins was measured by the reduction of [3H]CDP in the presence of an excess of pure recombinant mouse R1 protein and ATP as a positive effector (22). Neither of the mutated proteins showed any significant activity compared with native R2 protein (Table I). As demonstrated earlier and again clearly seen here for the native R2 protein, reactivation of apoR2 protein occurs continuously and with high efficiency during the assay in the presence of iron-dithiothreitol (31). Therefore, we should have been able to detect activity even from an R2 protein with a radical less stable than the normal tyrosyl radical.

                              
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Table I
Ribonucleotide reductase activity of native and mutated protein R2
Enzyme activity was measured in the presence of 14 µg of R1 protein (native and Y177W) or 10 µg of R1 protein (Y177C and Y177F). For the native and Y177W R2 proteins, no reactivation of the apoprotein was made prior to the assay, whereas for the Y177C and the Y177F R2 proteins, reactivation was performed before the assay as described (19).

Reconstitution of Protein R2 Y177W with 6Fe2+ and Oxygen

EPR Spectroscopy-- After addition of ferrous iron to aerobic apoY177W and a reaction time of 3 s at room temperature, an EPR spectrum was observed at 40 K with giso = 2.0029 (see Table II) and a dominating doublet hyperfine structure (hfs) (Fig. 2, top). The radical decays with a half-life time of 49 s at 22 °C (Fig. 3). Quantitation of this radical showed a maximum value of 0.34 unpaired spins/protein R2 dimer. The measured microwave power at half-saturation of the new radical at 30 K was at least 1 order of magnitude lower (P50 = 0.2 mW) than that of the tyrosyl radical at Tyr-177 in native protein R2 (P50 = 2-3 mW) at the same temperature, indicating a weaker magnetic interaction of the new radical with the diferric site in R2 Y177W.

                              
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Table II
Experimental electronic g tensor (gi) and hyperfine tensor (Ai (mT)) principal components of the tryptophan radical Trp-177 in mouse R2 Y177W and Trp-111 in E. coli R2 Y122F
The tryptophan radical was obtained after a reaction time of 3 s. The reconstitution reaction was performed with 6 Fe2+/protein R2 at 22 °C. g and hyperfine tensor principal components were obtained from fits of the EPR powder spectra (Fig. 2) and from ENDOR spectra (Fig. 4) of the tryptophan radical in protein R2 Y177W indole-h5-Trp and Y177W indole-d5-Trp. Axes correspond for 14N, H(5), and H(7) to the molecular axes system (Fig. 5). Note that 14N and g tensors have axial symmetry in the given error margins.


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Fig. 2.   X band EPR spectra at T = 40 K of the tryptophan radical in mutant Y177W. Top, in protein R2 with indole-h5-Trp (0.15 mM) in H2O buffer; bottom, in protein R2 with indole-d5-Trp (0.17 mM) in H2O buffer, both frozen after 3 s reaction time with Fe2+ and O2 at 22 °C. Solid line, experiment; dotted line, best simulation (fit). The obtained g and hyperfine parameters are given in Table II. The parameters for g, 14N, and the large beta -proton coupling were obtained from R2 with indole-d5-Trp. For the simulation of Trp-indole-h5 these parameters were kept constant, and in addition the parameters for H-5 and H-7 from proton- and deuterium-ENDOR were used (see text). A single component Gaussian line width of 0.19 and 0.23 mT was used for indole-h5-Trp and indole-d5-Trp, respectively. Experimental conditions are as follows: microwave power, 10 µW; modulation frequency, 12.5 kHz; modulation amplitude, 0.15 mT.


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Fig. 3.   Decay of the tryptophan radical in protein R2 Y177W at 22 °C after 6Fe2+/R2 reconstitution. The data points were fitted with a single exponential decay equation (kdecay = 0.015 s-1).

To identify the origin of the new radical in protein R2 Y177W we exchanged all tryptophans in the protein with selectively deuterated indole-d5-Trp. The incorporation of indole-d5-Trp into protein R2 Y177W was checked by mass spectroscopy (see "Experimental Procedures"). A comparison with the calculated molecular mass of Y177W R2 indole-d5-Trp shows that the isotopically labeled amino acid has been incorporated properly into the protein. A significant change of the hfs of the EPR spectrum was observed with the mutant R2 Y177W containing ring-deuterated tryptophans, indole-d5-Trp (Fig. 2, bottom). The small sub-splittings of each doublet component (Fig. 2, top) into three lines disappeared in the indole-d5-Trp labeled sample, proving that these lines resulted from a hyperfine interaction with the ring alpha -protons of Trp. Furthermore, well resolved lines became visible at the outer wings of the spectrum (Fig. 2, bottom). These lines are assigned to the Az component of an anisotropic hyperfine powder pattern from a 14N coupling. A similar line shape was observed also for tryptophan radical Trp-111· in mutant Y122F of E. coli R2 (14).

The dominating doublet splitting of 2.25 mT (Fig. 2, top), which is not affected by indole-d5-Trp labeling is assigned to a large isotropic hfc of a beta -proton. In the spectra simulations, the best fit of the spectrum in Fig. 2, bottom, was obtained by using a large isotropic proton hfc (Aiso = 2.25 mT) and an axially symmetric hyperfine tensor of 14N (Ax = 0.07 mT, Ay = 0.07 mT, and Az = 0.94 mT, see Table II). Due to the large Az tensor component of the 14N hyperfine tensor, we expect a significant spin density on the nitrogen of about 20%. An N-H proton coupled to the indole nitrogen, as would be expected for a tryptophan cation radical, would exhibit a hyperfine tensor component of about 0.5 mT (32). Such a large coupling is, however, not observed in the EPR spectrum of Y177W indole-d5-Trp in H2O buffer (Fig. 2, bottom). Therefore, we conclude that there is no NH proton involved in the hfs. The observed tryptophan radical must therefore be neutral with the nitrogen atom deprotonated.

ENDOR Spectroscopy-- cw-ENDOR experiments were performed at X band on Y177W with indole-h5-Trp or Y177W indole-d5-Trp in H2O or in D2O buffer. The ENDOR resonance condition is given to first order by Equation 1.
&ngr;<SUB><UP>±</UP></SUB>(<UP>ENDOR</UP>)=‖&ngr;<SUB>n</SUB>±A/2‖, (Eq. 1)
where nu ± are the high and low frequency ENDOR transitions; nu n is the respective nuclear Larmor frequency (13.9 MHz for protons, 2.1 MHz for deuterons, and 1.0 MHz for 14N at a magnetic field of 326 mT); and A is the hfc constant of the respective nucleus. For each hfc, A, two ENDOR lines are expected that are spaced symmetrically around nu n and separated by A in case of |A/2| < nu n (Equation 1). In frozen disordered solution, ENDOR resonances are expected over the full range of anisotropic hyperfine values. Pronounced features are often observed in the first derivative cw-ENDOR spectra from which the principal values Ai (i = x, y, z) of the hyperfine tensor of each nucleus can be obtained (33).

The large hfcs of the beta -proton and the Az component of the nitrogen were already determined by EPR. We attempted to observe ENDOR lines corresponding to the large beta -proton coupling, which are expected at 17.6 and 45.4 MHz, but with no success. For such large beta -proton couplings large ENDOR line widths are expected, in particular for methylene protons for which a variation of the side chain geometry leads to a distribution of the observed hyperfine couplings (see Equation 2). This effect and the low radical yield of 0.34 in Y177W reduce signal amplitudes for these couplings beyond detection.

The smaller hfcs, which are not resolved in EPR, were obtained from the ENDOR spectra. Fig. 4A shows the X band cw-ENDOR spectrum of Y177W with indole-h5-Trp in H2O buffer measured at 8 K. The spectrum is characterized by a strong matrix signal at the proton Larmor frequency nu H (13.9 MHz) and several pairs of lines that are symmetrically placed around 13.9 MHz and discussed below. Another pair of rather broad lines, at 4.8 MHz and at 23 MHz, yielding a proton hfc corresponding to 0.65 mT, is absent in the ENDOR spectrum of Y177W with indole-d5-Trp in H2O buffer (Fig. 4B). We conclude that this coupling represents one hyperfine tensor principal value of one (or two) indole ring alpha -protons. Proton ENDOR signals corresponding to the other hyperfine principal values were not observed. This is due to the expected large hyperfine anisotropy of alpha -protons, which broadens the ENDOR signals in frozen solution beyond detection.


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Fig. 4.   cw-ENDOR spectra (X band) at T = 8 K of the tryptophan radical after 3 s reaction time at 22 °C. Spectra were recorded with a field setting at the maximum of the EPR low field absorption line, corresponding to 344.85 mT in Fig. 2. Since the ENDOR cavity exhibits a different resonance frequency than the EPR cavity used for recording the spectra in Fig. 2, the actual ENDOR spectra were recorded at 325.4 mT. The ENDOR splittings and the nuclear Zeeman frequencies for protons and deuterons (vH and vD) are indicated by dashed lines. A, protein R2 Y177W indole-h5-Trp in H2O buffer (0.15 mM). The dotted trace 15-31 MHz shows a proton ENDOR simulation using solely the tensor principal values of two alpha -protons given in Table II and assuming an inequivalence of the two smaller tensor values for both protons within the given error margins (±0.06 and ±0.04 mT). The simulation indicates that the most intense peak results from the largest tensor component which is assumed to be equivalent for both protons. The same behavior is observed in the deuteron ENDOR spectrum, trace C. The ENDOR simulation program is described in Ref. 47. B, protein R2 Y177W indole-d5-Trp in H2O buffer (0.17 mM) (left, deuterium-ENDOR with expanded frequency axis); C, protein R2 Y177W indole-d5-Trp in D2O buffer (0.2 mM). Dotted trace, 2.1-5 MHz, expanded scale, is an ENDOR simulation with two alpha -deuterons using the same values as for protons in trace A except for a scaling factor 1/6.5 accounting for the smaller magnetic moment of the deuteron. D, difference spectrum: indole-d5-Trp in H2O buffer minus indole-d5-Trp in D2O buffer. Splittings of a proton hydrogen bonded to N-1 are indicated (see text). Experimental conditions are as follows: microwave power, 5 mW; radiofrequency power, 100-150 W; modulation frequency 12.5 kHz; modulation amplitude, ±140 kHz. Total accumulation time 8-10 h each trace.

Corresponding hyperfine components are, however, observed for deuterons at the ring alpha -positions in Y177W with indole-d5-Trp. Fig. 4B shows additional lines between 2 and 4 MHz. These signals belong to alpha -deuterons of the indole ring of ring-deuterated tryptophan. Their positions are shifted by a factor of 6.5 to lower frequencies, and their ENDOR spectrum is compressed by the same factor in comparison with the alpha -protons in normal tryptophan due to the smaller magnetic moment of D as compared with H. The amplitudes of the ENDOR signals are correspondingly increased. The lines are symmetrically spaced around the deuterium Zeeman frequency, nu D = 2.1 MHz. Only the high frequency part of the deuterium-ENDOR spectrum appears at useful ENDOR frequencies (>1.5 MHz, Fig. 4B, see inset 2.1-5 MHz with expanded frequency scale). Three couplings (A1 = 0.6 MHz, A2 = 2.1 MHz, and A3 = 2.8 MHz) have been obtained. The second component is seen mostly as an inflection on the third component (Fig. 4, B and C). The couplings were assigned to the three principal values of one hyperfine tensor, since for alpha -deuterons, as for alpha -protons, a rhombic hyperfine tensor is expected. The values for the corresponding alpha -proton are 3.9, 13.7, and 18.2 MHz or 0.14, 0.49, and 0.65 mT. The largest value agrees well with that obtained from the proton-ENDOR spectrum of the protein with indole-h5-Trp (see above, and Fig. 4A). This interpretation is supported by ENDOR simulations (dotted traces in Fig. 4A, 15-31 MHz, and Fig. 4C, 2.1-5 MHz) using solely the tensor principal values of the two alpha -protons and assuming an inequivalence for the two smaller components of both tensors within the error margins given in Table II. The simulations show that for this case the largest component gives rise to the most intense peak as is observed in the proton- and deuterium-ENDOR spectra.

The three alpha -deuteron and derived proton hyperfine tensor principal values agree remarkably well with those obtained earlier for the tryptophan neutral radical Trp-111· in E. coli protein R2 Y122F (14), see Table II. In this earlier work the tensor principal values were corroborated by EPR simulations and by McConnel-Strathdee based dipolar tensor calculations accounting for all carbon and nitrogen spin densities in the radical (14). The EPR simulations performed for Trp-177· (Fig. 2, top) showed, as in the earlier work (14), that two alpha -protons have to be assigned to the observed three hyperfine tensor values and that their hyperfine tensor axes of the largest value (0.65 mT) are rotated by 90° relative to each other in order to obtain the observed pattern in the experimental spectrum (see Table II). Based on earlier density functional calculations, these two proton hyperfine tensors were assigned to ring protons H-5 and H-7 (14) (see Fig. 5 for numbering of tryptophan ring). Since the largest hyperfine tensor component of an alpha -proton is expected for the magnetic field oriented perpendicular to the C-H bond and in the tryptophan ring plane, the largest component (0.65 mT) is assigned to Ax (H-5) and Ay (H-7) and the smallest component (0.14 mT) to Ay (H-5) and Ax (H-7), whereas Az is assumed to be similar (0.49(6) mT) for both alpha -protons (compare Fig. 4 and Table II).


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Fig. 5.   Molecular structure, molecular axis system, numbering scheme, and spin densities derived from the hyperfine coupling constants (see text) for the tryptophan radical in mouse R2 mutant Y177W.

The ENDOR spectra of Fig. 4, B and C, show two lines at 11.8 and 16.0 MHz corresponding to a 4.2-MHz coupling which is not affected by indole-D5-Trp nor by D2O buffer. Since a proton weakly coupled to the nitrogen would exchange in D2O buffer, we assign this small coupling of 0.15 mT to the second methylene beta -proton. This coupling is not resolved in the EPR spectrum in Fig. 2, bottom.

Exchangeable proton hfcs were not observed in the EPR spectra. However, from the ENDOR difference spectrum Y177W indole-d5-Trp in H2O minus Y177W indole-d5-Trp in D2O buffer (Fig. 4D), three small hyperfine tensor values of -0.12, +0.18, and -0.06 mT were obtained and assigned to one exchangeable proton. Previous ENDOR experiments on the neutral tryptophan radical Trp-111· in E. coli Y122F yielded very similar tensor components of an exchangeable proton of -0.12, +0.19, and -0.08 mT. Based on spin density and dipolar hyperfine tensor calculations, they were assigned to a hydrogen-bonded proton at the indole nitrogen (14). Since the tryptophan radical in Y177W exhibits a very similar 14N hyperfine tensor as Trp-111· in Y122F, we likewise assign the three values -0.12, +0.18, and -0.06 mT to the three hyperfine tensor principal values of a proton hydrogen-bonded to the nitrogen in the tryptophan indole ring (for signs, see Table II, legend and footnotes).

Reconstitution of Y177F and Y177C R2 with 6Fe2+ and Oxygen

EPR Spectroscopy-- When aerobic apoprotein R2 Y177F was mixed with 6Fe2+ at 22 °C, an EPR singlet signal at g = 2.00 with a peak-to-peak line width of 1.8 mT was observed at 20 K. Fig. 6A shows this signal when the reaction was quenched after 3 s. A very similar EPR singlet signal, with regard to g value, line width, and microwave saturation behavior, appeared also after reconstitution reaction in protein R2 Y177C under the same conditions (Table III). The EPR singlet in Y177F appeared with a rate constant of about 0.5 s-1 and decays with a rate of 0.25 s-1. The EPR singlet in Y177C had similar kinetics and appeared with a rate of 0.48 s-1 and decayed with a rate of 0.23 s-1. Both EPR singlets had completely disappeared after 20 s reaction time (Fig. 7). Upon reconstitution with apoprotein Y177F and 57Fe, we observed a doublet splitting (A = 2.6 mT) of the EPR signal due to the nuclear spin of 1/2 for 57Fe (Fig. 6C). A similar transient EPR signal has also been observed in the reconstitution of wild-type and Y122F protein R2 of E. coli (34-36) and native mouse R2 (12) and has been assigned to an oxo (hydroxo)-bridged Fe(III)/Fe(IV) cluster, intermediate X (25). For comparison, Fig. 6, B and D, shows the corresponding spectra of E. coli R2 Y122F after a freeze-quenched reaction with 56Fe and 57Fe, respectively. The dotted trace is a simulation. Microwave saturation studies of the singlet EPR spectrum in mouse Y177F R2 and of intermediate X in E. coli Y122F R2 gave coinciding values of P50, 0.02 mW at 5 K and 0.2 mW at 20 K (data not shown). From the almost identical g values, line shapes, microwave saturation behavior, and 57Fe hfcs, we assign also the transient EPR signals in Y177F and Y177C to such a bridged Fe(III)/Fe(IV) cluster, similar to intermediate X observed earlier in E. coli protein R2.


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Fig. 6.   X band EPR spectra at T = 20 K of intermediate X formed in the reconstitution reaction of mouse protein R2 Y177F (0.1 mM) (A and C) and E. coli protein R2 Y122F (1 mM) (B and D). Apoprotein R2 in 200 mM HEPES buffer, pH 7.5, was 1:1 mixed with 0.6 mM 56Fe (A), 4 mM 56Fe (B), and 0.6 mM 57Fe in 2.5 mM H2SO4 (C), and 4 mM 57Fe in 2.5 mM H2SO4 (D, dotted trace simulation, see text). The reaction was stopped after 3 s at 22 °C (A), 300 ms at 22 °C (B), and 2 s at 5 °C (C and D), see text. Experimental conditions are as follows: microwave power, 0.03 mW; modulation frequency, 100 kHz; modulation amplitude, 0.2 mT.

                              
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Table III
EPR and kinetic data of intermediate X in protein R2 mutants Y177C and Y177F of mouse and Y122F of E. coli RNR


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Fig. 7.   Time course of formation and decay of intermediate X in mouse protein R2 Y177C and Y177F upon addition of 6 Fe2+/R2 at 22 °C as measured by EPR. The data points were fitted with a double exponential equation assuming a consecutive reaction. Rates for formation and decay of X are presented in Table III.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Among free radical enzymes (3), RNR from E. coli was the first enzyme in which a protein-linked tyrosyl free radical was identified. In a kinetic study of the formation of the tyrosyl radical, Bollinger et al. (36) have shown that an intermediate X, an Fe(III)/Fe(IV) cluster with spin delocalization, serves as precursor to the tyrosyl radical in the E. coli protein R2. When the tyrosyl radical cannot be formed due to a mutation like in E. coli protein R2 Y122F, intermediate X becomes longer lived and can oxidize other neighboring aromatic residues like tryptophans (13, 37). Two tryptophan radicals (located at Trp-111 and Trp-107) have previously been studied in detail and assigned to oxidized neutral radicals (13, 14).

An Oxidized Neutral Tryptophan Radical on Trp-177-- We have observed a transient tryptophan radical in mouse protein R2 upon the reconstitution of Y177W with ferrous iron and oxygen. Its light absorption centered around 550 nm agrees with reported properties of photochemically produced tryptophan radicals (38, 49). Its dominant EPR doublet signal arises from the hyperfine splitting of one beta -methylene proton, whereas two alpha -protons at the indole ring cause the small hfs as seen in Fig. 2, top. In addition to the proton hfcs, the full 14N hyperfine tensor was obtained with the help of EPR simulations (see Fig. 2 and Table II). Finally, ENDOR difference spectra of Y177W with indole-d5-Trp in H2O and D2O revealed the hyperfine tensor of an exchangeable proton assigned to a hydrogen-bonded proton at the indole-nitrogen.

The tryptophan radical in Y177W can now be compared with the previously reported oxidized neutral tryptophan radical at Trp-111 in E. coli R2 Y122F (13, 14). The tryptophan radical in Y177W exhibits only one large beta -proton hfc. The hfc of the second beta -proton is very small and only resolved in the ENDOR spectrum. In the tryptophan radical Trp-111· of Y122F, there are two large beta -proton hfs. This indicates a difference in the dihedral angles of the beta -methylene protons for the two tryptophan residues, whereas the spin densities in the indole rings are very similar.

The spin density on C-3 in the tryptophan radical in Y177W can be estimated from the observed hfcs of the beta -protons using the empirical relationship (Equation 2)
A<SUB>&bgr;H</SUB>=&rgr;<SUP>&pgr;</SUP><SUB>c</SUB>(B′+B″<UP>cos</UP><SUP>2</SUP>&thgr;) (Eq. 2)
where rho pi c is the pi  spin density of the neighboring carbon, B' and B" are empirical constants, and theta  is the dihedral angle between the axis of the pz orbital and the projected Cbeta Hbeta bond (39). Since the hfc of one beta -proton in Y177W is close to 0, its dihedral angle theta  must be close to 90°. For a tetragonal geometry this implies theta  = -30° for the second beta -proton (theta 1 - theta 2 = 120°). Using Abeta H = 2.25 mT, B' = 0, B" = 5.8 mT (14), and theta  = -30°, a spin density rho pi c of 0.52 is obtained for C-3 (see Fig. 5). The same spin density was obtained for C-3 in Trp-111· of Y122F from E. coli (14). Based on empirical constants between hyperfine tensor values and spin densities and assignments from density functional calculations, for positions N-1, C-5, and C-7 (Fig. 5) in Trp-111· of E. coli R2 Y122F spin densities of 0.20, 0.17, and 0.15 were determined, respectively (14). By using the same assignment for the two ring protons (H-5 and H-7) as in Ref. 14 and a Q factor of -2.48 mT for alpha -protons (40), a spin density of 0.17 is obtained for each C-5 and C-7 in Y177W based on the hyperfine tensor values given in Table II. For N-1 a spin density of 0.18 is obtained for the tryptophan radical in Y177W using the hyperfine tensor values of Table II and B = 1.706 mT (41) for 14N (rho pi (N) = 1; B = (Az - Aiso)/2). This shows that the spin densities in the indole rings of these two tryptophan radicals are indeed very similar. Furthermore, in both radicals the expected large coupling from an N---H proton is missing, and the observed hyperfine tensors from H-bonded exchangeable protons clearly indicate that the radicals are in their neutral state, deprotonated but H-bonded at N-1.

Another common feature of both tryptophan radicals in mouse R2 Y177W and E. coli R2 Y122F is that they exhibit a rather small g anisotropy (gx - gz congruent  8 × 10-4, see Table II). This is typical for pi  radicals with high spin densities only on carbon and nitrogen atoms, like tryptophan. For comparison, tyrosyl radicals with a high spin density on an oxygen atom (larger spin-orbit interaction than nitrogen or carbon) exhibit a larger g anisotropy (for native mouse R2 gx - gz = 54 × 10-4 (42)). It should also be pointed out that in these two mutant R2 proteins we have established that protein-linked neutral tryptophan radicals exhibit a light absorption band centered around 550 nm (Fig. 1, inset, and see Ref. 13).

Which is the site of the observed oxidized neutral tryptophan radical in protein R2 Y177W? Including Trp-177, there are 7 tryptophans per polypeptide chain in mouse R2 Y177W. We expect the transient radical to be located relatively close to the iron site (cf. Ref. 13), and we expect the geometry of the beta -protons with respect to the indole plane (dihedral angles theta ) as derived from the crystal structure (5) to agree with the experimental observations. Tryptophans 103, 214, 210, 92, 246, and 117 have distances to their indole-nitrogen from Fe2 (the iron ion most distant from Tyr-177) of 11, 15, 17.4, 21, 24.7, and 25.5 Å, respectively. Only tryptophans 92 and 246 have dihedral angles of the beta -methylene protons theta 1 = -27°, theta 2 = 93°, and theta 1 = -37°, theta 2 = 83° (5), which agree within ±10° with those obtained for the tryptophan radical in Y177W (-30 and 90°). Trp-92 and Trp-246 are, however, 21 and 24.7 Å from the Fe2 atom, far away from the iron site. Tryptophans 111 and 107 in E. coli, which carry radicals in the mutant Y122F (13, 14), are much closer to the iron site (5 and 7 Å). The closest tryptophan to the iron site in native mouse R2 (Trp-103) is 11 Å from Fe2, but its dihedral angles (theta 1 = -43° and theta 2 = 77°) do not agree well with the experimental data. Therefore, we assign the observed neutral tryptophan radical to residue Trp-177, which should be the tryptophan closest to the iron site. Reconstitution with 57Fe did not change the EPR spectrum of the radical (data not shown), indicating only weak interaction with the iron site like in native enzymes with tyrosyl radicals (43) or the Trp-111· radical in E. coli Y122F (13). The Trp-177 radical is not enzymatically active (Table I).

The Long-lived Intermediate X in Protein R2 Mutants Y177F and Y177C-- The reconstitution of apoY177F and Y177C R2 with an excess of Fe2+ showed the formation of an S = 1/2 paramagnetic species. The X band EPR spectrum of 57Fe-labeled R2-Y177F protein could be successfully simulated using the g tensor and the two hyperfine tensors of 57Fe from the Q band studies of intermediate X (25) (Fig. 6D). We therefore assigned the intermediates in mouse R2 Y177F and Y177C to Fe(III)/Fe(IV) clusters, like intermediate X in E. coli R2, with similar structures as reported for intermediate X in E. coli R2 Y122F (25). However, the intermediates X in the two mouse mutants differ kinetically from that in E. coli Y122F in the rates of formation and decay, i.e. in their time window of appearance. Intermediate X in E. coli R2 Y122F became reduced after 2 s at 25 °C, whereas in the two mouse mutants intermediates X are still detectable after 10 s reaction time at 22 °C (Fig. 7).

In E. coli R2 Y122F intermediate X becomes reduced by the oxidation of the non-conserved tryptophans 111 and 107 (13). E. coli Trp-111 and Trp-107 correspond to Gln-167 and Phe-163, respectively, in mouse protein R2, which cannot be easily oxidized and may explain the missing successor radicals in Y177F. Besides Tyr-177 in native mouse protein R2, there are no other aromatic amino acids in the close vicinity of the iron site, which can reduce intermediate X. This may be the reason for its long lifetime.

Whereas phenylalanine in Y177F is not expected to be oxidized, cysteine in Y177C was expected to form a cysteine radical upon reconstitution. The crystal structure of the analogous mutant E. coli R2 Y122C shows a much larger distance of the -SH group to Fe1, the iron ion closest to Cys-122, (8.9 Å) than the OH group of tyrosine 122 in E. coli wild type R2 (5.3 Å) and a hole at the site of the missing phenyl ring.3 In neither of the R2 mutants E. coli Y122C and mouse Y177C did we detect any EPR signal at 77 K in the time window of 8 ms to 20 s (concomitant with the decay of X) up to 20 min reaction time at room temperature after the reconstitution. We have to conclude that either due to the larger distance between Cys-177 and Fe1 no cysteine radical has been formed or, if it was formed, it is short lived, or it has a low signal intensity caused by its large g anisotropy (44). Functionally related cysteine-based radicals have been detected in a class II RNR with strong magnetic interaction with a cobalt complex (45) and very recently in an E. coli protein R1 mutant with a lifetime of several seconds (46).

Concluding Remarks-- The present study shows that the iron/oxygen reconstitution reaction in protein R2 is able to create protein-linked free radicals in the close vicinity of the diferric iron center in mouse protein R2. When the tyrosine 177 is mutated for another redox-active amino acid with a suitable side chain and an appropriate redox potential, like tryptophan, this residue can be oxidized. Despite the formation of a transient tryptophan radical, the loss of the enzymatic activity in all three mutants, Y177W, Y177F, and Y177C, suggests strongly that the tyrosyl radical 177 cannot be replaced by other amino acids. The long lifetime and high relative yield of intermediate X in mouse Y177F and Y177C mutants with no observable successor radicals may facilitate further studies of the properties of this important intermediate in iron-oxygen chemistry of diiron-oxygen proteins.

    ACKNOWLEDGEMENT

We thank Per Ingvar Olsson of the Department of Medical Chemistry and Biophysics, Umeå University, for providing the results of the mass spectrometric analysis.

    FOOTNOTES

* This work was supported by grants from the Deutscher Akademischer Austauschdienst (to S. P.), the Swedish Natural Science Research Council (to A. G.), the C. Trygger Foundation (to A. G.), Deutsche Forschungsgemeinschaft DFG:La 751-2/1 (to F. L. and G. L.), and Fonds der Chemischen Industrie (to G. L. and W. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Institute for Physiological Chemistry and Biochemistry, University of Mainz, D-55099 Mainz, Germany.

** To whom correspondence should be addressed. Tel.: 46-8-162450; Fax: 46-8-155597; E-mail: astrid{at}biophys.su.se.

2 S. Pötsch, F. Lendzian, R. Ingemarson, A. Hörnberg, L. Thelander, W. Lubitz, G. Lassmann, and A. Gräslund, manuscript in preparation.

3 S. Pötsch and P. Nordlund, unpublished data.

    ABBREVIATIONS

The abbreviations used are: RNR, ribonucleotide reductase; cw, continuous wave; EPR, electron paramagnetic resonance; ENDOR, electron nuclear double resonance; hfc, hyperfine coupling; hfs, hyperfine splitting; IPTG, isopropyl-1-thio-beta -D-galactopyranoside; mW, milliwatt; mT, millitesla.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Reichard, P. (1993) Science 260, 1773-1777[Medline] [Order article via Infotrieve]
  2. Sjöberg, B.-M. (1997) Struct. Bonding 88, 139-173
  3. Stubbe, J., and van der Donk, W. A. (1998) Chem. Rev. 98, 705-762[CrossRef][Medline] [Order article via Infotrieve]
  4. Nordlund, P., Sjöberg, B.-M., and Eklund, H. (1990) Nature 345, 593-598[CrossRef][Medline] [Order article via Infotrieve]
  5. Kauppi, B., Nielsen, B. B., Ramaswamy, S., Kjøller-Larsen, I., Thelander, M., Thelander, L., and Eklund, H. (1996) J. Mol. Biol. 262, 706-720[CrossRef][Medline] [Order article via Infotrieve]
  6. Uhlin, U., and Eklund, H. (1994) Nature 370, 533-539[CrossRef][Medline] [Order article via Infotrieve]
  7. Mao, S. S., Holler, T. P., Yu, G. X., Bollinger, J. M., Booker, S., Johnston, M. I., and Stubbe, J. (1992) Biochemistry 31, 9733-9743[Medline] [Order article via Infotrieve]
  8. Siegbahn, P. E. M. (1998) J. Am. Chem. Soc. 120, 8417-8429[CrossRef]
  9. Siegbahn, P. E. M., Blomberg, M. R. A., and Crabtree, R. H. (1997) Theor. Chem. Acc. 97, 289-300[CrossRef]
  10. Rova, U., Goodtzova, K., Ingemarson, R., Behravan, G., Gräslund, A., and Thelander, L. (1995) Biochemistry 34, 4267-4275[Medline] [Order article via Infotrieve]
  11. Ekberg, M., Sahlin, M., Eriksson, M., and Sjöberg, B.-M. (1996) J. Biol. Chem. 271, 20655-20659[Abstract/Free Full Text]
  12. Schmidt, P. P., Rova, U., Katterle, B., Thelander, L., and Gräslund, A. (1998) J. Biol. Chem. 273, 21463-21472[Abstract/Free Full Text]
  13. Sahlin, M., Lassmann, G., Pötsch, S., Sjöberg, B.-M., and Gräslund, A. (1995) J. Biol. Chem. 270, 12361-12372[Abstract/Free Full Text]
  14. Lendzian, F., Sahlin, M., MacMillan, F., Bittl, R., Fiege, R., Pötsch, S., Sjöberg, B.-M., Gräslund, A., Lubitz, W., and Lassmann, G. (1996) J. Am. Chem. Soc. 118, 8111-8120[CrossRef]
  15. Katterle, B., Sahlin, M., Schmidt, P. P., Pötsch, S., Logan, D., Gräslund, A., and Sjöberg, B.-M. (1997) J. Biol. Chem. 272, 10414-10421[Abstract/Free Full Text]
  16. Ehrenberg, A., and Reichard, P. (1972) J. Biol. Chem. 247, 3485-3488[Abstract/Free Full Text]
  17. Ormö, M., Regnström, K., Wang, Z. G., Que, L., Jr., Sahlin, M., and Sjöberg, B.-M. (1995) J. Biol. Chem. 270, 6570-6576[Abstract/Free Full Text]
  18. DeFelippis, M. R., Murthy, C. P., Faraggi, M., and Klapper, M. H. (1989) Biochemistry 28, 4847-4853[Medline] [Order article via Infotrieve]
  19. Mann, G. J., Gräslund, A., Ochiai, E.-I., Ingemarson, R., and Thelander, L. (1991) Biochemistry 30, 1939-1947[Medline] [Order article via Infotrieve]
  20. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorf, J. W. (1990) Methods Enzymol. 185, 60-89[Medline] [Order article via Infotrieve]
  21. Åberg, A., Ormö, M., Nordlund, P., and Sjöberg, B.-M. (1993) Biochemistry 26, 5541-5548
  22. Engström, Y., Eriksson, S., Thelander, L., and Åkerman, M. (1979) Biochemistry 18, 2941-2948[Medline] [Order article via Infotrieve]
  23. Atkin, C. L., Thelander, L., Reichard, P., and Lang, G. (1973) J. Biol. Chem. 248, 7464-7472[Abstract/Free Full Text]
  24. Sahlin, M., Sjöberg, B.-M., Backes, G., Loehr, T. M., and Sanders-Loehr, J. (1990) Biophys. Biochem. Res. Commun. 167, 813-818
  25. Sturgeon, B. E., Burdi, D., Chen, S., Huynh, B.-H., Edmondson, D.-E., Stubbe, J., and Hoffman, B. M. (1996) J. Am. Chem. Soc. 118, 7551-7557[CrossRef]
  26. Zweygart, W., Thanner, R., and Lubitz, W. (1994) J. Magn. Reson. 109, 172-177[CrossRef]
  27. Stesmans, A., and Van Gorp, G. (1989) Rev. Sci. Instrum. 60, 2949-2952[CrossRef]
  28. Brudvig, G. W. (1995) Methods Enzymol. 246, 536-554[Medline] [Order article via Infotrieve]
  29. Rieger, P. H. (1982) J. Magn. Reson. 50, 485-489
  30. Ingemarson, R., and Thelander, L. (1996) Biochemistry 35, 8603-8609[CrossRef][Medline] [Order article via Infotrieve]
  31. Nyholm, S., Mann, G. J., Johansson, A. G., Bergeron, R. J., Gräslund, A., and Thelander, L. (1993) J. Biol. Chem. 268, 26200-26205[Abstract/Free Full Text]
  32. Huyett, J. E., Doan, P. E., Gurbiel, R., Houseman, A. L. P., Sivaraja, M., Goodin, D. B., and Hoffman, B. M. (1995) J. Am. Chem. Soc. 117, 9033-9041
  33. Lubitz, W., and Lendzian, F. (1995) in Advances in Photosynthesis (Ames, J., and Hoff, A. J., eds), Vol. 3, pp. 255-275, Kluwer Academic Publishers, Norwell, MA
  34. Bollinger, J. M., Edmondson, D. E., Huynh, B. H., Filley, J., Norton, J. R., and Stubbe, J. (1991) Science 253, 292-298[Medline] [Order article via Infotrieve]
  35. Bollinger, J. M., and Stubbe, J. (1991) J. Am. Chem. Soc. 113, 6289-6291
  36. Bollinger, J. M ., Tong, W. H., Ravi, N., Huynh, B. H., Edmondson, D. E., and Stubbe, J. (1994) J. Am. Chem. Soc. 116, 8015-8023
  37. Tong, W., Burdi, D., Riggs-Galasco, P., Chen, S., Edmondson, D., Huynh, B. H., Stubbe, J., Han, S., Arvai, A., and Trainer, J. (1998) Biochemistry 37, 5840-5848[CrossRef][Medline] [Order article via Infotrieve]
  38. Baugher, J. F., and Grossweiner, L. I. (1977) J. Phys. Chem. 81, 1349-1354
  39. Stone, E. W., and Maki, A. H. (1962) J. Chem. Phys. 37, 1326-1333
  40. Bender, C. J., Sahlin, M., Babcock, G, T., Barry, B. A., Chandrashekar, T. K., Salowe, S. P., Stubbe, J., Lindström, B., Peterson, L., Ehrenberg, A., and Sjöberg, B.-M. (1989) J. Am. Chem. Soc. 111, 8076-8093
  41. Wertz, J. E., and Bolton, J. R. (1986) Electron Spin Resonance, Elementary Theory and Practical Application, Chapman & Hall, New York
  42. Schmidt, P. P., Andersson, K. K., Barra, A.-L., Thelander, L., and Gräslund, A. (1996) J. Biol. Chem. 271, 23615-23618[Abstract/Free Full Text]
  43. Sahlin, M., Petersson, L., Gräslund, A., Ehrenberg, A., Sjöberg, B. M., and Thelander, L. (1987) Biochemistry 26, 5541-5548[Medline] [Order article via Infotrieve]
  44. Nelson, D. J., Petersen, R. L., and Symons, M. C. R. (1977) J. Chem. Soc. Perkin II, 2005-2015
  45. Gerfen, G. J., Licht, S., Willems, J. P., Hoffman, B. M., and Stubbe, J. (1996) J. Am. Chem. Soc. 118, 8192-8197[CrossRef]
  46. Persson, A. L., Sahlin, M., and Sjöberg, B. M. (1998) J. Biol. Chem. 273, 31016-31020[Abstract/Free Full Text]
  47. Gessner, C. (1997) NiFe Hydrogenases: Contributions by EPR Spectroscopy to the Structural Elucidation of the Active Center.Ph.D. thesis, Technical University, Berlin
  48. Davydov, A., Schmidt, P. P., and Gräslund, A. (1996) Biophys. Biochem. Res. Commun. 219, 213-218[CrossRef]
  49. Land, E. J., and Prütz, W. A. (1979) Int. J. Radiat. Biol. 36, 513-520


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