Magnetic interaction between the tyrosyl free radical and the antiferromagnetically coupled iron center in ribonucleotide reductase

Sahlin, Margareta; Petersson, Leif; Graeslund, Astrid; Ehrenberg, Anders; Sjoeberg, Britt Marie; Thelander, Lars

doi:10.1021/bi00391a049

Cited by 131 publications

(104 citation statements)

References 24 publications

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“…Hirsh et al 53 have previously measured relaxation rates at 9-GHz for the stable tyrosyl radical of subunit β-2 in class I RNR from E. coli, which at low temperature has a relaxation rate near its intrinsic value (uncoupled). 54 To ensure similar conditions we repeated the measurement on a sample of stable tyrosyl radical of subunit β-2 in class I RNR from E. coli and compared it to that of the tyrosyl radical in PpoA (both samples including the literature data contained 20% glycerol). The data are displayed in Figure 6.…”

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Multifrequency Electron Paramagnetic Resonance Characterization of PpoA, a CYP450 Fusion Protein that Catalyzes Fatty Acid Dioxygenation

et al. 2011

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PpoA is a fungal dioxygenase that produces hydroxylated fatty acids involved in the regulation of the life cycle and secondary metabolism of Aspergillus nidulans. It was recently proposed that this novel enzyme employs two different heme domains to catalyze two separate reactions: within a heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid [(8R)-HPODE]; in the second reaction step (8R)-HPODE is isomerized within a P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. In the present study, pulsed EPR methods were applied to find spectroscopic evidence for the reaction mechanism, thought to involve paramagnetic intermediates. We observe EPR resonances of two distinct heme centers with g-values typical for Fe(III) S = 5/2 high-spin (HS) and Fe(III) S = 1/2 low-spin (LS) hemes. 14N ENDOR spectroscopy on the S = 5/2 signal reveals resonances consistent with an axial histidine ligation. Reaction of PpoA with the substrate leads to the formation of an amino acid radical on the early millisecond time scale concomitant to a substantial reduction of the S = 5/2 heme signal. High-frequency EPR (95- and 180-GHz) unambiguously identifies the new radical as a tyrosyl, based on g-values and hyperfine couplings from spectral simulations. The radical displays enhanced T 1-spin–lattice relaxation due to the proximity of the heme centers. Further, EPR distance measurements revealed that the radical is distributed among the monomeric subunits of the tetrameric enzyme at a distance of approximately 5 nm. The identification of three active paramagnetic centers involved in the reaction of PpoA supports the previously proposed reaction mechanism based on radical chemistry.

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Multifrequency Electron Paramagnetic Resonance Characterization of PpoA, a CYP450 Fusion Protein that Catalyzes Fatty Acid Dioxygenation

et al. 2011

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“…According to this model the C terminus of R2 binds into a cleft in R1, thus completing the specific electron transfer path between the two subunits required for radical shuttling. Unfortunately, no significant spectroscopic changes are observed upon complex formation between R1 and R2 (17,18). Moreover, in wild type RNR reactions the tyrosyl radical is always present, and no change in the radical spectroscopic properties is detected with naturally occurring substrates.…”

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Redox Studies of Subunit Interactivity in Aerobic Ribonucleotide Reductase from Escherichia coli

Zlateva

Quaroni²,

Que

et al. 2004

Journal of Biological Chemistry

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Ribonucleotide reductase is a heterodimeric (␣ 2 ␤ 2 ) allosteric enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides, an essential step in DNA biosynthesis and repair. In the enzymatically active form aerobic Escherichia coli ribonucleotide reductase is a complex of homodimeric R1 and R2 proteins. We use electrochemical studies of the dinuclear center to clarify the interplay of subunit interaction, the binding of allosteric effectors and substrate selectivity. Our studies show for the first time that electrochemical reduction of active R2 generates a distinct Met form of the diiron cluster, with a midpoint potential (؊163 ؎ 3 mV) different from that of R2 Met produced by hydroxyurea (؊115 ؎ 2 mV). The redox potentials of both Met forms experience negative shifts when measured in the presence of R1, becoming ؊223 ؎ 6 and ؊226 ؎ 3 mV, respectively, demonstrating that R1-triggered conformational changes favor one configuration of the diiron cluster. We show that the association of a substrate analog and specificity effector (dGDP/dTTP or GMP/dTTP) with R1 regulates the redox properties of the diiron centers in R2. Their midpoint potential in the complex shifts to ؊192 ؎ 2 mV for dGDP/dTTP and to ؊203 ؎ 3 mV for GMP/dTTP. In contrast, reduction potential measurements show that the diiron cluster is not affected by ATP (0.35-1.45 mM) and dATP (0.3-0.6 mM) binding to R1. Binding of these effectors to the R1-R2 complex does not perturb the normal docking modes between R1 and R2 as similar redox shifts are observed for ATP or dATP associated with the R1-R2 complex.Ribonucleotide reductases (RNRs) 1 catalyze the reduction of the four canonical ribonucleotides to the corresponding deoxyribonucleotides in all organisms, thus providing the precursors for DNA synthesis. Allosteric regulation ensures a balanced dNTP pool needed for DNA replication and repair. At present, three major classes of RNRs have been identified that differ in their protein structure and in the cofactors essential for catalysis (1). Despite these differences, all RNRs catalyze the reduction of ribonucleotides by similar radical-based mechanisms, including formation of a transient thiyl radical at the active site that in turn produces a transient 3Ј-substrate radical (2). In class I enzymes, it is proposed that the thiyl radical within the R1 subunit is formed during each catalytic cycle via long range electron/proton transfer from a stable tyrosyl radical located in the R2 subunit (1, 2).The enzymatic activity of aerobic Escherichia coli RNR, which belongs to the class I RNR like the mammalian enzyme, depends on the formation of the complex between the two different proteins, R1 (␣ 2 ) and R2 (␤ 2 ), and is regulated by the binding of dNTPs and ATP, which act as allosteric effectors. The R1 homodimer carries two types of regulatory sites in addition to the catalytic binding site. Overall activity is stimulated by ATP and inhibited by dATP through the binding of these effectors to the activity site (3). The regulation of su...

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“…This will give the investigator confidence that nonsingle-exponential saturation-recovery transients observed in the protein are the result of dipole-dipole interactions between the radical and the metal center. For a study of the enzyme RNR by the authors, the control sample consisted of a tyrosine radical generated by illuminating a frozen solution of tyrosine in borate buffer 4,42 . Figure 3 shows the saturation-recovery data confirming that both the UV-generated tyrosine radical and the tyrosyl radical of the B2 subunit of RNR are well fit by a single exponential at low temperatures.…”

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Measuring distances in proteins by saturation-recovery EPR

Hirsh

Brudvig

2007

Nat Protoc

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We describe a protocol for detecting electron spin-spin interactions between a radical and a metal ion in a protein or protein complex by saturation-recovery electron paramagnetic resonance (EPR). This protocol can be used with a protein containing an endogenous metal center and either an endogenous or synthetic radical species. We suggest a two-step approach whereby dipole-dipole or exchange interactions are first detected by continuous-wave EPR experiments and then quantified by saturation-recovery EPR. The latter measurements make it possible to measure long distances to within a few Å ngstroms. The protocol for making distance measurements by saturation-recovery EPR will take approximately 6 days to complete. INTRODUCTIONThe techniques that first come to mind when thinking about protein structure determination are usually solution-state NMR or X-ray crystallography. However, a complete protein structure is not always available or necessary to answer some of the most interesting biophysical questions. Sometimes, the measurement of one distance (or a few) is sufficient to establish the plausibility of a mechanism or corroborate a proposed structure. If a protein or protein complex contains a metal-binding site and a radical species, the magnetic interaction between the unpaired electrons at these two sites can be detected and quantified by saturation-recovery EPR and the resulting data used to measure distances of B10-40 Å (refs. 1,2). Enzymes with multiple redox-active cofactors such as photosystem II (PSII) and ribonucleotide reductase (RNR) are obvious targets for this methodology 3,4 . Indeed, in these systems, saturation-recovery EPR may also be used to measure exchange couplings between redox partners 4 . However, saturation-recovery EPR can also be used to measure distances between an endogenous metal-binding site and a strategically placed spin label to yield structural information 5 .Long distances in macromolecules have been successfully measured using fluorescence resonance energy transfer [6][7][8] and by pulsed EPR methods such as double quantum coherence, ''2 + 1,'' and DEER sequences that use a pair of spin labels (radicals) [9][10][11] . Distance measurement by saturation recovery has an advantage over these other methods when the protein contains an endogenous paramagnetic metal center in that the protein has a built-in ''label.'' The other label, that is, the radical, may be either endogenous or introduced through spin-labeling methods [12][13][14] . Additionally, in the case where the paramagnetic metal center is itself of interest, the radical can serve as a reporter of its magnetic properties. For example, in our experiments with the B2 subunit of RNR, the stable tyrosine radical was used to determine the exchange coupling within the dinuclear iron center 4 .As pulsed EPR instruments are not yet as common as pulsed (Fourier-transform) NMR instruments, we recommend that the investigator first attempts to detect the presence of electron spinspin interactions using the more common continuous...

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Magnetic interaction between the tyrosyl free radical and the antiferromagnetically coupled iron center in ribonucleotide reductase

Cited by 131 publications

References 24 publications

Multifrequency Electron Paramagnetic Resonance Characterization of PpoA, a CYP450 Fusion Protein that Catalyzes Fatty Acid Dioxygenation

Multifrequency Electron Paramagnetic Resonance Characterization of PpoA, a CYP450 Fusion Protein that Catalyzes Fatty Acid Dioxygenation

Redox Studies of Subunit Interactivity in Aerobic Ribonucleotide Reductase from Escherichia coli

Measuring distances in proteins by saturation-recovery EPR

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