Circular dichroism and magnetic circular dichroism studies of the active site of p53R2 from human and mouse: iron binding and nature of the biferrous site relative to other ribonucleotide reductases

Ribonucleotide reductases (RNR) catalyze the rate-limiting step in the synthesis of deoxyribonucleotides from the corresponding ribonucleotides in the synthesis of DNA. Class I RNR has two subunits: R1 with the substrate binding and active site and R2 with a stable tyrosyl radical and diiron cluster. Biferrous R2 reacts with oxygen to form the tyrosyl radical needed for enzymatic activity. A novel R2 form, p53R2, is a 351-amino acid protein induced by the "tumor suppressor gene" p53. p53R2 has been studied using a combination of circular dichroism, magnetic circular dichroism, variable-temperature variable-field MCD, and EPR spectroscopies. The active site of biferrous p53R2 in both the human (hp53R2) and mouse (mp53R2) forms is found to have one five-coordinate and one four-coordinate iron, which are weakly antiferromagnetically coupled through mu-1,3-carboxylate bridges. These spectroscopic data are very similar to those of Escherichia coli R2, and mouse R2, with a stronger resemblance to data of the former. Titrations of apo-hp53R2 and apo-mp53R2 with Fe(II) were pursued for the purpose of comparing their metal binding affinities to those of other R2s. Both p53R2s were found to have a high affinity for Fe(II), which is different from that of mouse R2 and may reflect differences in the regulation of enzymatic activity, as p53R2 is mainly triggered during DNA repair. The difference in ferrous affinity between mammalian R2 and p53R2 suggests the possibility of specific inhibition of DNA precursor synthesis during cell division.     

Redox studies of subunit interactivity in aerobic ribonucleotide reductase from Escherichia coli

Ribonucleotide reductase is a heterodimeric (alpha(2)beta(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.     

Extended X-ray absorption fine structure studies on the iron-containing subunit of ribonucleotide reductase from Escherichia coli

Iron K-edge X-ray absorption spectra were obtained on the protein B2, the small subunit of ribonucleotide reductase from Escherichia coli. Protein B2 contains a binuclear iron center with many properties in common with the iron center of oxidized hemerythrins. The extended X-ray absorption fine structure (EXAFS) measurements on protein B2 were analyzed and compared with published data for oxyhemerythrin. In protein B2 there are, in the first coordination shell around each Fe atom, five or six oxygen or nitrogen atoms that are directly coordinated ligands. In oxyhemerythrin there are six ligands to each iron. As in oxyhemerythrin, one of the ligands in the first shell of protein B2 is at a short distance, about 1.78 A, confirming the existence of a mu-oxo bridge. The other atoms of the first shell are at an average distance of 2.04 A, which is about 0.1 A shorter than in oxyhemerythrin. In protein B2 the Fe-Fe distance is in the range 3.26-3.48 A, and the bridging angle falls between 130 and 150 degrees. On the basis of these data, there is no direct evidence for any histidine ligands in protein B2, but the noise level leaves way for the possibility of a maximum of about three histidines for each Fe pair. The X-ray absorption spectrum of a hydroxyurea-treated sample was not significantly different from that of the native protein B2, which implies that no significant alteration in the structure of the iron site occurs upon destruction of the tyrosine radical.     

Reduced forms of the iron-containing small subunit of ribonucleotide reductase from Escherichia coli

The B2 subunit of ribonucleotide reductase from Escherichia coli contains a stable tyrosyl free radical and an antiferromagnetically coupled dimeric iron center with high-spin ferric ions. The tyrosyl radical is an oxidized form of tyrosine-122. This study shows that the B2 protein has a fully reduced state, denoted reduced B2, characterized by a normal nonradical tyrosine-122 residue and a dimeric ferrous iron center. Reduced B2 can be formed either from active B2 by a three-electron reduction in the presence of suitable mediators or from apoB2 by addition of two equimolar amounts of ferrous ions in the absence of oxygen. The oxidized tyrosyl radical and the ferric iron center can be generated from reduced B2 by the admission of air. The tyrosyl radical can be selectively reduced by one-electron reduction in the presence of a suitable mediator, yielding metB2, a form that seems identical with the form resulting from treatment of active B2 with hydroxyurea. 1H NMR was used to characterize the paramagnetically shifted resonances associated with the reduced iron center. Prominent resonances were observed around 45 ppm (nonexchangeable with solvent) and 57 ppm (exchangeable with solvent) at 37 degrees C. From the temperature dependence of the chemical shifts of these resonances it was concluded that the ferrous ions in reduced B2 are only weakly, if at all, antiferromagnetically coupled. By comparison with data on the similar iron center of deoxyhemerythrin it is suggested that the 57 ppm resonance should be assigned to protons in histidine ligands of the iron center.     

Iron ligand mutants in protein R2 of Escherichia coli ribonucleotide reductase

 Ribonucleotide reductase protein R2 contains a diiron-oxo center with the ability to generate and stabilize a catalytically essential tyrosyl radical. The six protein-derived ligands (four carboxylates and two histidines) of the diiron site were, in separate experiments, mutated to alanines and in two cases also to histidines. We found that removal or exchange of an iron ligand did not in general abolish the formation of a diiron site in the mutant proteins, although all mutant proteins lost the bound metal ions with time upon storage. Iron bound to the mutant proteins was characterized by light absorption, EPR and resonance Raman spectroscopy. In addition, the ability of the mutant proteins to form a tyrosyl free radical and the catalytic competence of the latter were determined by EPR spectroscopy and activity measurements. The diiron sites of mutant proteins D84H and E238A were quite reminiscent of that in wild-type R2. Four of the other mutant proteins (H118A, E204A, E204H, H241A) could form the same number of metal sites as wild-type R2, but with different spectroscopic properties. The mutation E115A affecting the only μ-bridging ligand lowered the amount of bound iron to less than half. An important observation was that D84A, H118A and E204A formed transient tyrosyl radicals, but only the E204A mutant protein was enzymatically active. D84A and H118A affect iron ligands which have been suggested to participate in long-range electron transfer during catalysis. Our observation that these mutant proteins are catalytically inert, despite formation of a tyrosyl radical, underscores the necessity for an intact electron transfer pathway for catalytic activity in ribonucleotide reductase. Received: 31 August 1995 / Accepted: 14 February 1996     

Cloned mouse ribonucleotide reductase subunit M1 cDNA reveals amino acid sequence homology with Escherichia coli and herpesvirus ribonucleotide reductases

We have isolated and sequenced overlapping cDNA clones containing the entire coding region of mouse ribonucleotide reductase subunit M1. The coding region comprises 2.4 kilobases and predicts a polypeptide of 792 amino acids (Mr 90,234) which shows striking homology with ribonucleotide reductases from Escherichia coli and the herpesviruses, Epstein-Barr virus and herpes simplex virus. The homologies reveal three domains: an N-terminal domain common to the mammalian and bacterial enzymes, a C-terminal domain common to the mammalian and viral ribonucleotide reductases, and a central domain common to all three. We speculate on the functional basis of this conservation.     

Purification and characterization of recombinant mouse and herpes simplex virus ribonucleotide reductase R2 subunit.

Overexpression of recombinant mouse and herpes simplex virus ribonucleotide reductase small subunit (protein R2) has been obtained by using the T7 RNA polymerase expression system. Both proteins, which constitute about 30% of the soluble Escherichia coli proteins, have been purified to homogeneity by a rapid and simple procedure. At this stage, few of the molecules contain the iron-tyrosyl free-radical center necessary for activity; however, addition of ferrous iron and oxygen under controlled conditions resulted in a mouse R2 protein containing 0.8 radical and 2 irons per polypeptide chain. In this reaction, one oxygen molecule was needed to generate each tyrosyl radical. Both proteins had full enzymatic activity. EPR spectroscopy showed that iron-center/radical interactions are considerably stronger in both mouse and viral proteins than in E. coli protein R2. CD spectra showed that the bacterial protein contains 70% alpha-helical structure compared to only about 50% in the mouse and viral proteins. Light absorption spectra between 310 and 600 nm indicate close similarity of the mu-oxo-bridged binuclear iron centers in all three R2 proteins. Furthermore, the paramagnetically shifted iron ligand proton NMR resonances show that the antiferromagnetic coupling and ligand arrangement in the iron center are nearly identical in all three species.     

Kinetic studies on the reduction of the R2 subunit of mouse ribonucleotide reductase with hydroxyurea, hydrazine, phenylhydrazine and hydroxylamine

 Four reductions of the R2 subunit of mouse ribonucleotide reductase have been studied and found to exhibit different behaviour from that of Escherichia coli R2. An important difference is that there is no stable met-R2 (Fe₂ ^II I) form of mouse R2. With hydroxyurea, hydrazine and hydroxylamine uniphasic kinetics are observed for the combined reduction of radical Tyr ^˙ and Fe₂ ^II I components to tyrosine and Fe₂ ^II respectively. The rate constants, determined at 370 nm (emphasising Fe^III decay) and 417 nm (emphasising Tyr ^˙ decay), differ by factors of 2–3, allowing some mechanistic features to be defined. The studies with hydrazine are particularly important. In the case of E. coli R2, a first phase corresponding to two-equivalent reduction of the met-R2 component has been observed [18]. It is likely that the four times slower second phase reaction of active E. coli R2 also corresponds to the Fe₂ ^II I → Fe₂ ^II change and is followed by fast intramolecular Fe₂ ^II reduction of the higher potential Tyr ^˙. The latter changes are believed to hold also for (active) mouse R2. The Fe^IIFe^III semi forms have been detected at low levels by EPR for mouse R2 (9%) and E. coli (∼5%) in previous studies. Further substrate reduction of Fe^IIFe^III occurs at a comparable rate to account for the transient behaviour of Fe^IIFe^III. For mouse R2 the combined Fe^III decay processes (which we are unable to separate) give smaller uniphasic rate constants at 370 nm than at 417 nm. A fitted-base-line (FBL) treatment of absorbance changes at 417 nm targets more closely the Tyr ^˙ decay as a means of monitoring the rate-determining step. The FBL method gives rate constants k (M^–1 s^–1) at 25  °C and pH 7.5 for hydroxyurea (1.46), hydrazine (0.163) and hydroxylamine (4.4). Surprisingly, phenylhydrazine, with a less strong reduction potential (0.25 V), gives a substantially faster reduction of the Tyr ^˙ as the only redox step (rate constant 27 M^–1 s^–1). In this case a slower second phase at 370 nm is independent of reductant and corresponds to rate-controlling release of Fe^III. Overall the results indicate a more reactive redox centre for mouse R2 and help develop further an understanding of factors affecting the reactivity of R2. Received: 11 October 1996 / Accepted: 11 February 1997     

Carboxyl-terminal peptides as probes for Escherichia coli ribonucleotide reductase subunit interaction: kinetic analysis of inhibition studies

The active complex of Escherichia coli ribonucleotide reductase comprises two dissociable, nonidentical homodimeric proteins, B1 and B2. When B2 is the varied component, the reductase activity is competitively inhibited by synthetic peptides of varying lengths corresponding to the C-terminus of protein B2. This finding provides the first evidence that the C-terminal peptides and protein B2 share the same binding domain on protein B1. Our data also show that two molecules of peptide can bind to protein B1 with equal affinity. Similar inhibition constants (18 microM) were obtained for peptides containing the C-terminal 20, 30, and 37 residues. When the invariant residue Tyr 356 was omitted, a 2-fold decrease in peptide inhibitory ability was observed. A small peptide, lacking the last 11 residues, had virtually no inhibitory potency. These results, coupled with our previous observations that truncated protein B2, in which one or both polypeptide chains are missing approximately 24 C-terminal residues, had considerably lower or no affinity for B1, suggest that the C-terminal regions are the major determinants in the B1-B2 interaction. In the Appendix, two methods for treatment of kinetic situations pertinent to the ribonucleotide reductase system are presented. One method deals with the determination of kinetic parameters for two components present at comparable levels; the other is concerned with the differentiation of linear and nonlinear competitive inhibition involving the binding of two inhibitor molecules. Both methods should find application to other similar cases.     

Reduction of the small subunit of Escherichia coli ribonucleotide reductase by hydrazines and hydroxylamines.

Each polypeptide chain of protein R2, the small subunit of ribonucleotide reductase from Escherichia coli, contains a stable tyrosyl radical and an antiferromagnetically coupled diferric center. Recent crystallographic studies [Nordlund, P., Eklund, H., & Sj?berg, B.-M. (1990) Nature 345, 593-598] have shown that both the radical and the diiron site are deeply buried inside the protein and thus strongly support the hypothesis of long-range electron-transfer processes within protein R2. This study shows that monosubstituted hydrazines and hydroxylamines are able to reduce the tyrosyl radical and the ferric ions, under anaerobic conditions. It allows characterization of the site from which those compounds transfer their electrons to the iron/radical center. The efficiency of any given reducing agent is not solely governed by its redox potential but also by its size, its charge, and its hydrophobicity. We suggest, as a possible alternative to the long-range electron-transfer hypothesis, that conformational flexibility of the polypeptide chain might exist in solution and allow small molecules to penetrate the protein and react with the iron/radical center. This study also shows that two reduction mechanisms are possible, depending on which center, the radical or the metal, is reduced first. Full reduction of protein R2 yields reduced R2, characterized by a normal tyrosine residue and a diferrous center. Both the radical and the diferric center are regenerated from reduced R2 by reaction with oxygen, while only the diferric center is formed by reaction with hydrogen peroxide.     

Mutations in the R2 subunit of ribonucleotide reductase that confer resistance to hydroxyurea

Ribonucleotide reductase is an essential enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides for use in DNA synthesis. Ribonucleotide reductase from Escherichia coli consists of two subunits, R1 and R2. The R2 subunit contains an unusually stable radical at tyrosine 122 that participates in catalysis. Buried deep within a hydrophobic pocket, the radical is inaccessible to solvent although subject to inactivation by radical scavengers. One such scavenger, hydroxyurea, is a highly specific inhibitor of ribonucleotide reductase and therefore of DNA synthesis; thus it is an important anticancer and antiviral agent. The mechanism of radical access remains to be established; however, small molecules may be able to access Tyr-122 directly via channels from the surface of the protein. We used random oligonucleotide mutagenesis to create a library of 200,000 R2 mutants containing random substitutions at five contiguous residues (Ile-74, Ser-75, Asn-76, Leu-77, Lys-78) that partially comprise one side of a channel where Tyr-122 is visible from the protein surface. We subjected this library to increasing concentrations of hydroxyurea and identified mutants that enhance survival more than 1000-fold over wild-type R2 at high drug concentrations. Repetitive selections yielded S75T as the predominant R2 mutant in our library. Purified S75TR2 exhibits a radical half-life that is 50% greater than wild-type R2 in the presence of hydroxyurea. These data represent the first demonstration of R2 protein mutants in E. coli that are highly resistant to hydroxyurea; elucidation of their mechanism of resistance may provide valuable insight into the development of more effective inhibitors.     

A mixed valence form of the iron cluster in the B2 protein of ribonucleotide reductase from Escherichia coli.

A mixed valent form of the iron cluster (Fe(II)Fe(III) in the B2 protein of ribonucleotide reductase has been isolated and characterized. The irons in this state of the protein are ferromagnetically coupled as indicated by the observation of a novel S = 9/2 EPR spectrum. This is the first ferromagnetically coupled Fe(II)Fe(III) cluster reported for a protein and the first observation of the mixed valence form of ribonucleotide reductase.