EPR Studies on a Stable Sulfinyl Radical Observed in the Iron−Oxygen-Reconstituted Y177F/I263C Protein R2 Double Mutant of Ribonucleotide Reductase from Mouse (original) (raw)
Related papers
Journal of Biological Chemistry, 1999
Ribonucleotide reductase catalyzes all de novo synthesis of deoxyribonucleotides. The mammalian enzyme consists of two non-identical subunits, the R1 and R2 proteins, each inactive alone. The R1 subunit contains the active site, whereas the R2 protein harbors a binuclear iron center and a tyrosyl free radical essential for catalysis. It has been proposed that the radical properties of the R2 subunit are transferred ϳ35 Å to the active site of the R1 protein, through a coupled electron/proton transfer along a conserved hydrogen-bonded chain, i.e. a radical transfer pathway (RTP). To gain a better insight into the properties and requirements of the proposed RTP, we have used site-directed mutagenesis to replace the conserved tyrosine 370 in the mouse R2 protein with tryptophan or phenylalanine. This residue is located close to the flexible C terminus, known to be essential for binding to the R1 protein. Our results strongly indicate that Tyr 370 links the RTP between the R1 and R2 proteins. Interruption of the hydrogenbonded chain in Y370F inactivates the enzyme complex. Alteration of the same chain in Y370W slows down the RTP, resulting in a 58 times lower specific activity compared with the native R2 protein and a loss of the free radical during catalysis.
Inorganica Chimica Acta, 2002
The rates of reduction of the diferric/radical center in mouse ribonucleotide reductase protein R2 were studied by light absorption and EPR in the native protein and in three point mutants of conserved residues involved in the proposed radical transfer pathway (D266A, W103Y) or in the unstructured C terminal domain (Y370W). The pseudo-first order rate constants for chemical reduction of the tyrosyl radical and diferric center by hydroxyurea, sodium dithionite or the dihydro form of flavin adenine dinucleotide, were comparable with or higher (particularly D266A, by dithionite) than in native R2. Molecular modeling of the D266A mutant showed that the iron/radical site should be more accessible for external reductants in the mutant than in native R2. The results indicate that no specific pathway is required for the reduction. The dihydro form of flavin adenine dinucleotide was found to be a very efficient reductant in the studied proteins compared to dithionite alone. The EPR spectra of the mixed-valent Fe(II)Fe(III) sites formed by chemical reduction in the D266A and W103Y mutants were clearly different from the spectrum observed in the native protein, indicating that the structure of the diferric site was affected by the mutations, as also suggested by the modeling study. No difference was observed between the mixed-valent EPR spectra generated by chemical reduction in Y370W mutant and native mouse R2 protein.
Tyrosyl free radical formation in the small subunit of mouse ribonucleotide reductase
The Journal of biological chemistry, 1990
Each R2 subunit of mammalian ribonucleotide reductase contains a pair of high spin ferric ions and a tyrosyl free radical essential for activity. To study the mechanism of tyrosyl radical formation, substoichiometric amounts of Fe(II) were added to recombinant mouse R2 apoprotein under strictly anaerobic conditions and then the solution was exposed to air. Low temperature EPR spectroscopy showed that the signal from the generated tyrosyl free radical correlated well with the quantity of the Fe(II) added with a stoichiometry of 3 Fe(II) needed to produce 1 tyrosyl radical: 3 Fe(II) + P + O2 + Tyr-OH + H+----Fe(III)O2-Fe(III)-P + H2O. + Tyr-O. + Fe(III), where P is an iron-binding site of protein R2 and Tyr-OH is the active tyrosyl residue. The O-O bond of a postulated intermediate O2(2-)-Fe(III)2-P state is cleaved by the extra electron provided by Fe(II) leading to formation of OH., which in turn reacts with Tyr-OH to give Tyr-O.. In the presence of ascorbate, added to reduce the mo...
Biochimica Et Biophysica Acta-gene Structure and Expression, 1995
Mouse and Escherichia coli ribonucleotide reductases (RR) both belong to the same class of RR, where the enzyme consists of two non-identical subunits, proteins R1 and R2. A transient free radical was observed by EPR spectroscopy in the mouse RR reaction with the suicidal inhibitor 2'-azido-2'-deoxycytidine 5'-diphosphate. The detailed hyperfine structure of the EPR spectrum of the transient radical is somewhat different for the mouse and previously studied E. coli enzymes. When the positive allosteric effector ATP was replaced by the negative effector dATP, no transient radical was observed, showing that 'normal' binding of the inhibitor to the substrate binding site is required. Using the mouse protein R2 mutants W103Y and D266A, where the mutations have been shown to specifically block long range electron transfer between the active site of the R1 protein to the iron/radical site in protein R2, no evidence of transient radical was found. Taken together, the data suggest that the radical is located at the active site in protein R1, and is probably of the sulfenylimine type.
Biochemistry, 1998
We here report EPR studies that provide evidence for radical intermediates generated from the glycyl radical of activated pyruvate formate-lyase (PFL) during the process of oxygen-dependent enzyme inactivation, radical quenching, and protein fragmentation. Upon exposure of active PFL to air, a long-lived radical intermediate was generated, which exhibits an EPR spectrum assigned to a sulfinyl radical (RSO • ). The EPR spectrum of a sulfinyl radical was also generated from the activated C418A mutant of PFL, indicating that Cys 418 is not the site of sulfinyl radical formation. Exposure of the activated C419A mutant or C418AC419A double mutant to air on the other hand, resulted in a new EPR spectrum that we assign to the R-carbon peroxyl radical (ROO • ) of the active-site glycine, G734. These findings suggest that C419 is the site of sulfinyl radical formation and that replacement of this cysteine with alanine results in the accumulation of the carbon peroxyl radical. The results also support the proposal that the peroxyl radical and the sulfinyl radical are intermediates in the oxygen-dependent inactivation and cleavage of the protein. Moreover, these observations are consistent with the hypothesis that C419 and G734 are in close proximity in the activated enzyme and may participate in a glycyl/thiyl radical equilibrium. A mechanism that accounts for the formation of the radical intermediates is proposed. Abstract published in AdVance ACS Abstracts, December 15, 1997.
Journal of the American Chemical Society, 1995
Ribonucleotide reductase (RNR) from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two homodimeric subunits: R1 and R2. 2'-Azido-2'-deoxyuridine 5'-diphosphate (N3UDP) has previously been shown to be a stoichiometric mechanism based inhibitor of this enzyme. Inactivation of RNR is accompanied by loss of the tyrosyl radical on the R2 subunit concomitant with formation of a new nitrogen centered radical. The X-band EPR spectrum of this radical species exhibits a triplet hyperfine interaction of-25 G arising from one of the three nitrogens of the azide moiety of N3UDP and a doublet hyperfine interaction of 6.3 G which has been proposed to arise from a proton. High frequency (139.5 GHz) EPR spectroscopic studies of this nitrogen centered radical have resolved the peaks corresponding to all three principal g-values: gl1 = 2.01557, g22 = 2.00625, and g33 = 2.00209. In addition, the nitrogen hyperfine splitting along g33 is resolved (Atz/, = 31.0 G) and upper limits (-5 G) can be placed on both A; , and A& Comparison of these g-and A-values with those of model systems in the literature suggests a structure for the radical, XN'SCH2-, in which SCH2 is part of a cysteine residue of R1, and X is either a nonprotonated sulfur, oxygen, or carbon moiety. Use of an E. coli strain that is auxotrophic for cysteine and contains the nucleotide reductase gene allowed @2H]cysteine labeled RNR to be prepared. Incubation of this isotopically labeled protein with N3UDP produced the radical signal without the hyperfine splitting of 6.3 G, indicating that this interaction is associated with a proton from the-SCH2-component of the proposed structure. These results establish that the nitrogen centered radical is covalently attached to a cysteine, probably C225, of the R1 subunit of RNR. Site-directed mutagenesis studies with a variety of R1 mutants in which each cysteine (439,462, 754, and 759) was converted to a serine reveal that X cannot be a substituted sulfur. A structure for the nitrogen centered radical is proposed in which X is derived from 3'-keto-2'-deoxyuridine 5'-diphosphate, an intermediate in the inactivation of RNR by N3UDP. Specifically, X is proposed to be the 3'-hydroxyl oxygen of the deoxyribose moiety.
A Glycyl Radical Site in the Crystal Structure of a Class III Ribonucleotide Reductase
Science, 1999
Ribonucleotide reductases catalyze the reduction of ribonucleotides to deoxyribonucleotides. Three classes have been identified, all using free-radical chemistry but based on different cofactors. Classes I and II have been shown to be evolutionarily related, whereas the origin of anaerobic class III has remained elusive. The structure of a class III enzyme suggests a common origin for the three classes but shows differences in the active site that can be understood on the basis of the radical-initiation system and source of reductive electrons, as well as a unique protein glycyl radical site. A possible evolutionary relationship between early deoxyribonucleotide metabolism and primary anaerobic metabolism is suggested.
Journal of the American Chemical Society, 2014
The class III ribonucleotide reductases (RNRs) are glycyl radical (G•) enzymes that provide the balanced pool of deoxynucleotides required for DNA synthesis and repair in many facultative and obligate anaerobic bacteria and archaea. Unlike the class I and II RNRs, where reducing equivalents for the reaction are delivered by a redoxin (thioredoxin, glutaredoxin, or NrdH) via a pair of conserved active site cysteines, the class III RNRs examined to date use formate as the reductant. Here, we report that reaction of the Escherichia coli class III RNR with CTP (substrate) and ATP (allosteric effector) in the absence of formate leads to loss of the G• concomitant with stoichiometric formation of a new radical species and a "trapped" cytidine derivative that can break down to cytosine. Addition of formate to the new species results in recovery of 80% of the G• and reduction of the cytidine derivative, proposed to be 3′-keto-deoxycytidine, to dCTP and a small amount of cytosine. The structure of the new radical has been identified by 9.5 and 140 GHz EPR spectroscopy on isotopically labeled varieties of the protein to be a thiosulfuranyl radical [RSSR 2 ]•, composed of a cysteine thiyl radical stabilized by an interaction with a methionine residue. The presence of a stable radical species on the reaction pathway rationalizes the previously reported [ 3 H]-(k cat /K M) isotope effect of 2.3 with [ 3 H]-formate, requiring formate to exchange between the active site and solution during nucleotide reduction. Analogies with the disulfide anion radical proposed to provide the reducing equivalent to the 3′-keto-deoxycytidine intermediate by the class I and II RNRs provide further evidence for the involvement of thiyl radicals in the reductive half-reaction catalyzed by all RNRs.
J Am Chem Soc, 1994
The tyrosyl radicakliiron(II1) cofactor of E. coli ribonucleotide reductase assembles spontaneously in vitro when the iron-free (apo) form of the enzyme's R2 subunit is mixed with Fez+ and 02. In previous work (Bollinger, J. M., Jr. et al., Science, 1991, 253, 292-298), kinetic and spectroscopic evidence was presented that the cofactor assembly reaction partitions between two pathways and that the partition ratio depends on the availability of the "extra" reducing equivalent that is required to balance the four-electron reduction of 0 2. In this study, stopped-flow absorption, rapid freeze-quench electron paramagnetic resonance, and rapid freeze-quench Mijssbauer spectroscopies have been used to examine the kinetics of the reaction carried out with excess Fez+ (FeZ+/R2 1 5.0). The kinetic data are consistent with a mechanism involving two sequential first-order reactions, in which the diferric radical species, X(Ravi, N. et al. J. Am. Chem. SOC., previous paper in this issue), accumulates rapidly (kob = 5-10 s-l) and decays concomitantly with formation of 'Y122 and the oxo-bridged diferric cluster (kob = 0.7-1.0 s-1). The simplest interpretation of these data is that oxidation of Y 122 by X generates the prduct cofactor and therefore, that Y 122 oxidation is not carried out by a high valent iron species. The Mixssbauer kinetic data also suggest that a stable or slowly decaying Fe(II1)containing species, which is distinct from the diferric cluster, is produced concomitantly with X. It is proposed that this Fe(II1) species may represent the product of donation of the "extra" electron by Fez+.
Journal of the American Chemical Society, 1998
Ribonucleotide reductase (RDPR) from E. coli catalyzes the conversion of nucleotides to deoxynucleotides and contains an unusual tyrosyl radical-diferric cluster cofactor. (E)-2′-Fluoromethylene-2′-deoxycytidine 5′-diphosphate [(E)-1] obtained by phosphorylation of the clinically promising antitumor agent MDL 101,731, is a potent time-dependent inactivator of this protein. Electron paramagnetic resonance (EPR) spectroscopy reveals that inactivation is accompanied by loss of the essential tyrosyl radical cofactor and formation of a new radical species. The 9.4-GHz EPR spectrum of this new radical reveals two hyperfine splittings of approximately 1.5 mT producing a triplet-like signal. Incubation of the enzyme with [6′-2 H]-(E)-1 alters this EPR spectrum, providing the first evidence for a nucleotide-based radical species generated by RDPR. The observed spectrum is a 1:1 composite of a doublet and a triplet signal, the latter being identical to that obtained with unlabeled (E)-1. Studies with (E)-1 in 2 H 2 O also produce a 1:1 mixture of these two radical signals. The results of these isotope labeling experiments suggest wash-out or wash-in of ∼0.5 equiv of deuterium at the 6′ position, respectively. Incubation of the enzyme with [6′-2 H]-(E)-1 in 2 H 2 O produced only the doublet as would be expected on the basis of this hypothesis. EPR (139.5 GHz) spectra established the principal g values of the new radical species. Simulation of the 9.4-and 139.5-GHz EPR spectra yield a self-consistent set of principal hyperfine values. A structure is proposed for the radical intermediate that is consistent with the EPR data and kinetic data on release of the fluoride ion that accompanies inactivation. The proposed structure and a postulated mechanism for its formation provide further support for the hypothesis that catalysis is initiated by 3′-hydrogen atom abstraction from the nucleotide substrate.