PELDOR spectroscopy with DOPA-beta2 and NH2Y-alpha2s: distance measurements between residues involved in the radical propagation pathway of E. coli ribonucleotide reductase

Escherichia coli ribonucleotide reductase (RNR) catalyzes the reduction of nucleotides to 2'-deoxynucleotides. The active enzyme is a 1:1 complex of two homodimeric subunits, alpha2 and beta2. The alpha2 is the site of nucleotide reduction, and beta2 harbors a diferric tyrosyl radical (Y122*) cofactor. Turnover requires formation of a cysteinyl radical (C439*) in the active site of alpha2 at the expense of the Y122* in beta2. A docking model for the alpha2beta2 interaction and a pathway for radical transfer from beta2 to alpha2 have been proposed. This pathway contains three Ys: Y356 in beta2 and Y731/Y730 in alpha2. We have previously incorporated 3-hydroxytyrosine and 3-aminotyrosine into these residues and showed that they act as radical traps. In this study, we use these alpha2/beta2 variants and PELDOR spectroscopy to measure the distance between the Y122* in one alphabeta pair and the newly formed radical in the second alphabeta pair. The results yield distances that are similar to those predicted by the docking model for radical transfer. Further, they support a long-range radical initiation process for C439* generation and provide a structural constraint for residue Y356, which is thermally labile in all beta2 structures solved to date.     

pH Rate profiles of FnY356-R2s (n = 2, 3, 4) in Escherichia coli ribonucleotide reductase: evidence that Y356 is a redox-active amino acid along the radical propagation pathway

The Escherichia coli ribonucleotide reductase (RNR), composed of two subunits (R1 and R2), catalyzes the conversion of nucleotides to deoxynucleotides. Substrate reduction requires that a tyrosyl radical (Y(122)*) in R2 generate a transient cysteinyl radical (C(439)*) in R1 through a pathway thought to involve amino acid radical intermediates [Y(122)* --> W(48) --> Y(356) within R2 to Y(731) --> Y(730) --> C(439) within R1]. To study this radical propagation process, we have synthesized R2 semisynthetically using intein technology and replaced Y(356) with a variety of fluorinated tyrosine analogues (2,3-F(2)Y, 3,5-F(2)Y, 2,3,5-F(3)Y, 2,3,6-F(3)Y, and F(4)Y) that have been described and characterized in the accompanying paper. These fluorinated tyrosine derivatives have potentials that vary from -50 to +270 mV relative to tyrosine over the accessible pH range for RNR and pK(a)s that range from 5.6 to 7.8. The pH rate profiles of deoxynucleotide production by these F(n)()Y(356)-R2s are reported. The results suggest that the rate-determining step can be changed from a physical step to the radical propagation step by altering the reduction potential of Y(356)* using these analogues. As the difference in potential of the F(n)()Y* relative to Y* becomes >80 mV, the activity of RNR becomes inhibited, and by 200 mV, RNR activity is no longer detectable. These studies support the model that Y(356) is a redox-active amino acid on the radical-propagation pathway. On the basis of our previous studies with 3-NO(2)Y(356)-R2, we assume that 2,3,5-F(3)Y(356), 2,3,6-F(3)Y(356), and F(4)Y(356)-R2s are all deprotonated at pH > 7.5. We show that they all efficiently initiate nucleotide reduction. If this assumption is correct, then a hydrogen-bonding pathway between W(48) and Y(356) of R2 and Y(731) of R1 does not play a central role in triggering radical initiation nor is hydrogen-atom transfer between these residues obligatory for radical propagation.     

Site-specific replacement of Y356 with 3,4-dihydroxyphenylalanine in the beta2 subunit of E. coli ribonucleotide reductase

E. coli ribonucleotide reductase (RNR), composed of the homodimeric subunits alpha2 and beta2, catalyzes the conversion of nucleotides to deoxynucleotides via complex radical chemistry. The radical initiation process involves a putative proton-coupled electron transfer (PCET) pathway over 35 A between alpha2 and beta2. Y356 in beta2 has been proposed to lie on this pathway. To test this model, intein technology has been used to make beta2 semi-synthetically in which Y356 is replaced with a DOPA-amino acid. Analysis of this mutant with alpha2 and various combinations of substrate and effector by SF UV-vis spectroscopy and EPR methods demonstrates formation of a DOPA radical concomitant with disappearance of the tyrosyl radical, which initiates the reaction. The results reveal that Y356 lies on the PCET pathway and demonstrate the first kinetically competent conformational changes prior to ET. They further show that substrate binding brings about rapid conformational changes which place the complex into its active form(s) and suggest that the RNR complex is asymmetric.     

Equilibration of tyrosyl radicals (Y356•, Y731•, Y730•) in the radical propagation pathway of the Escherichia coli class Ia ribonucleotide reductase

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides using a diferric tyrosyl radical (Y(122)(?)) cofactor in β2 to initiate catalysis in α2. Each turnover requires reversible long-range proton-coupled electron transfer (PCET) over 35 ? between the two subunits by a specific pathway (Y(122)(?) ? [W(48)?] ? Y(356) within β to Y(731) ? Y(730) ? C(439) within α). Previously, we reported that a β2 mutant with 3-nitrotyrosyl radical (NO(2)Y(?); 1.2 radicals/β2) in place of Y(122)(?) in the presence of α2, CDP, and ATP catalyzes formation of 0.6 equiv of dCDP and accumulates 0.6 equiv of a new Y(?) proposed to be located on Y(356) in β2. We now report three independent methods that establish that Y(356) is the predominant location (85-90%) of the radical, with the remaining 10-15% delocalized onto Y(731) and Y(730) in α2. Pulsed electron-electron double-resonance spectroscopy on samples prepared by rapid freeze quench (RFQ) methods identified three distances: 30 ± 0.4 ? (88% ± 3%) and 33 ± 0.4 and 38 ± 0.5 ? (12% ± 3%) indicative of NO(2)Y(122)(?)-Y(356)(?), NO(2)Y(122)(?)-NO(2)Y(122)(?), and NO(2)Y(122)(?)-Y(731(730))(?), respectively. Radical distribution in α2 was supported by RFQ electron paramagnetic resonance (EPR) studies using Y(731)(3,5-F(2)Y) or Y(730)(3,5-F(2)Y)-α2, which revealed F(2)Y(?), studies using globally incorporated [β-(2)H(2)]Y-α2, and analysis using parameters obtained from 140 GHz EPR spectroscopy. The amount of Y(?) delocalized in α2 from these two studies varied from 6% to 15%. The studies together give the first insight into the relative redox potentials of the three transient Y(?) radicals in the PCET pathway and their conformations.     

Photoactive peptides for light-initiated tyrosyl radical generation and transport into ribonucleotide reductase

The mechanism of radical transport in the alpha2 (R1) subunit of class I E. coli ribonucleotide reductase (RNR) has been investigated by the phototriggered generation of a tyrosyl radical, *Y356, on a 20-mer peptide bound to alpha2. This peptide, Y-R2C19, is identical to the C-terminal peptide tail of the beta2 (R2) subunit and is a known competitive inhibitor of binding of the native beta2 protein to alpha2. *Y356 radical initiation is prompted by excitation (lambda >or= 300 nm) of a proximal anthraquinone, Anq, or benzophenone, BPA, chromophore on the peptide. Transient absorption spectroscopy has been employed to kinetically characterize the radical-producing step by time resolving the semiquinone anion (Anq*-), ketyl radical (*-BPA), and Y* photoproducts on (i) BPA-Y and Anq-Y dipeptides and (ii) BPA/Anq-Y-R2C19 peptides. Light-initiated, single-turnover assays have been carried out with the peptide/alpha2 complex in the presence of [14C]-labeled cytidine 5'-diphosphate substrate and ATP allosteric effector. We show that both the Anq- and BPA-containing peptides are competent in deoxycytidine diphosphate formation and turnover occurs via Y731 to Y730 to C439 pathway-dependent radical transport in alpha2. Experiments with the Y730F mutant exclude a direct superexchange mechanism between C439 and Y731 and are consistent with a PCET model for radical transport in which there is a unidirectional transport of the electron and proton transport among residues of alpha2.     

Kinetics of radical intermediate formation and deoxynucleotide production in 3-aminotyrosine-substituted Escherichia coli ribonucleotide reductases

Escherichia coli ribonucleotide reductase is an α2β2 complex and catalyzes the conversion of nucleoside 5'-diphosphates (NDPs) to 2'-deoxynucleotides (dNDPs). The reaction is initiated by the transient oxidation of an active-site cysteine (C(439)) in α2 by a stable diferric tyrosyl radical (Y(122)?) cofactor in β2. This oxidation occurs by a mechanism of long-range proton-coupled electron transfer (PCET) over 35 ? through a specific pathway of residues: Y(122)?→ W(48)→ Y(356) in β2 to Y(731)→ Y(730)→ C(439) in α2. To study the details of this process, 3-aminotyrosine (NH(2)Y) has been site-specifically incorporated in place of Y(356) of β. The resulting protein, Y(356)NH(2)Y-β2, and the previously generated proteins Y(731)NH(2)Y-α2 and Y(730)NH(2)Y-α2 (NH(2)Y-RNRs) are shown to catalyze dNDP production in the presence of the second subunit, substrate (S), and allosteric effector (E) with turnover numbers of 0.2-0.7 s(-1). Evidence acquired by three different methods indicates that the catalytic activity is inherent to NH(2)Y-RNRs and not the result of copurifying wt enzyme. The kinetics of formation of 3-aminotyrosyl radical (NH(2)Y?) at position 356, 731, and 730 have been measured with all S/E pairs. In all cases, NH(2)Y? formation is biphasic (k(fast) of 9-46 s(-1) and k(slow) of 1.5-5.0 s(-1)) and kinetically competent to be an intermediate in nucleotide reduction. The slow phase is proposed to report on the conformational gating of NH(2)Y? formation, while the k(cat) of ~0.5 s(-1) is proposed to be associated with rate-limiting oxidation by NH(2)Y? of the subsequent amino acid on the pathway during forward PCET. The X-ray crystal structures of Y(730)NH(2)Y-α2 and Y(731)NH(2)Y-α2 have been solved and indicate minimal structural changes relative to wt-α2. From the data, a kinetic model for PCET along the radical propagation pathway is proposed.     

A hot oxidant, 3-NO2Y122 radical, unmasks conformational gating in ribonucleotide reductase

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides and requires a diferric-tyrosyl radical (Y(?)) cofactor to initiate catalysis. The initiation process requires long-range proton-coupled electron transfer (PCET) over 35 ? between the two subunits by a specific pathway (Y(122)(?)→W(48)→Y(356) within β to Y(731)→Y(730)→C(439) within α). The rate-limiting step in nucleotide reduction is the conformational gating of the PCET process, which masks the chemistry of radical propagation. 3-Nitrotyrosine (NO(2)Y) has recently been incorporated site-specifically in place of Y(122) in β2. The protein as isolated contained a diferric cluster but no nitrotyrosyl radical (NO(2)Y(?)) and was inactive. In the present paper we show that incubation of apo-Y(122)NO(2)Y-β2 with Fe(2+) and O(2) generates a diferric-NO(2)Y(?) that has a half-life of 40 s at 25 °C. Sequential mixing experiments, in which the cofactor is assembled to 1.2 NO(2)Y(?)/β2 and then mixed with α2, CDP, and ATP, have been analyzed by stopped-flow absorption spectroscopy, rapid freeze quench EPR spectroscopy, and rapid chemical quench methods. These studies have, for the first time, unmasked the conformational gating. They reveal that the NO(2)Y(?) is reduced to the nitrotyrosinate with biphasic kinetics (283 and 67 s(-1)), that dCDP is produced at 107 s(-1), and that a new Y(?) is produced at 97 s(-1). Studies with pathway mutants suggest that the new Y(?) is predominantly located at 356 in β2. In consideration of these data and the crystal structure of Y(122)NO(2)Y-β2, a mechanism for PCET uncoupling in NO(2)Y(?)-RNR is proposed.     

Deciphering radical transport in the large subunit of class I ribonucleotide reductase

Incorporation of 2,3,6-trifluorotyrosine (F(3)Y) and a rhenium bipyridine ([Re]) photooxidant into a peptide corresponding to the C-terminus of the β protein (βC19) of Escherichia coli ribonucleotide reductase (RNR) allows for the temporal monitoring of radical transport into the α2 subunit of RNR. Injection of the photogenerated F(3)Y radical from the [Re]-F(3)Y-βC19 peptide into the surface accessible Y731 of the α2 subunit is only possible when the second Y730 is present. With the Y-Y established, radical transport occurs with a rate constant of 3 × 10(5) s(-1). Point mutations that disrupt the Y-Y dyad shut down radical transport. The ability to obviate radical transport by disrupting the hydrogen bonding network of the amino acids composing the colinear proton-coupled electron transfer pathway in α2 suggests a finely tuned evolutionary adaptation of RNR to control the transport of radicals in this enzyme.     

Biosynthetic approach to modeling and understanding metalloproteins using unnatural amino acids

Metalloproteins have inspired chemists for many years to synthesize artificial catalysts that mimic native enzymes.As a complementary approach to studying native enzymes or making synthetic models,biosynthetic approach using small and stable proteins to model native enzymes has offered advantages of incorporating non-covalent secondary sphere interactions under physiological conditions.However,most biosynthetic models are restricted to natural amino acids.To overcome this limitation,incorporating unnatural amino acids into the biosynthetic models has shown promises.In this review,we summarize first synthetic,semisynthetic and biological methods of incorporates unnatural amino acids(UAAs)into proteins,followed by progress made in incorporating UAAs into both native metalloproteins and their biosynthetic models to fine-tune functional properties beyond native enzymes or their variants containing natural amino acids,such as reduction potentials of azurin,O_2 reduction rates and percentages of product formation of HCO models in Mb,the rate of radical transport in ribonucleotide reductase(RNR)and the proton and electron transfer pathways in photosystemⅡ(PSⅡ).We also discuss how this endeavour has allowed systematic investigations of precise roles of conserved residues in metalloproteins,such as Metl21 in azurin,Tyr244 that is cross-linked to one of the three His ligands to CuB in HCO,Tyr122,356,730 and 731 in RNR and TyrZ in PSⅡ.These examples have demonstrated that incorporating UAAs has provided a new dimension in our efforts to mimic native enzymes and in providing deeper insights into structural features responsible high enzymatic activity and reaction mechanisms,making it possible to design highly efficient artificial catalysts with similar or even higher activity than native enzymes.     

Direct interfacial Y731 oxidation in α2 by a photoβ2 subunit of E. coli class Ia ribonucleotide reductase

Proton-coupled electron transfer (PCET) is a fundamental mechanism important in a wide range of biological processes including the universal reaction catalysed by ribonucleotide reductases (RNRs) in making de novo, the building blocks required for DNA replication and repair. These enzymes catalyse the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs). In the class Ia RNRs, NDP reduction involves a tyrosyl radical mediated oxidation occurring over 35 Å across the interface of the two required subunits (β₂ and α₂) involving multiple PCET steps and the conserved tyrosine triad [Y₃₅₆(β₂)–Y₇₃₁(α₂)–Y₇₃₀(α₂)]. We report the synthesis of an active photochemical RNR (photoRNR) complex in which a Re(i)-tricarbonyl phenanthroline ([Re]) photooxidant is attached site-specifically to the Cys in the Y₃₅₆C-(β₂) subunit and an ionizable, 2,3,5-trifluorotyrosine (2,3,5-F₃Y) is incorporated in place of Y₇₃₁ in α₂. This intersubunit PCET pathway is investigated by ns laser spectroscopy on [Re₃₅₆]-β₂:2,3,5-F₃Y₇₃₁-α₂ in the presence of substrate, CDP, and effector, ATP. This experiment has allowed analysis of the photoinjection of a radical into α₂ from β₂ in the absence of the interfacial Y₃₅₆ residue. The system is competent for light-dependent substrate turnover. Time-resolved emission experiments reveal an intimate dependence of the rate of radical injection on the protonation state at position Y₇₃₁(α₂), which in turn highlights the importance of a well-coordinated proton exit channel involving the key residues, Y₃₅₆ and Y₇₃₁, at the subunit interface.     

Perturbations of aromatic amino acids are associated with iron cluster assembly in ribonucleotide reductase

The β2 subunit of class Ia ribonucleotide reductases (RNR) contains an antiferromagnetically coupled μ-oxo bridged diiron cluster and a tyrosyl radical (Y122?). In this study, an ultraviolet resonance Raman (UVRR) difference technique describes the structural changes induced by the assembly of the iron cluster and by the reduction of the tyrosyl radical. Spectral contributions from aromatic amino acids are observed through UV resonance enhancement at 229 nm. Vibrational bands are assigned by comparison to histidine, phenylalanine, tyrosine, tryptophan, and 3-methylindole model compound data and by isotopic labeling of histidine in the β2 subunit. Reduction of the tyrosyl radical reveals Y122? Raman bands at 1499 and 1556 cm(-1) and Y122 Raman bands at 1170, 1199, and 1608 cm(-1). There is little perturbation of other aromatic amino acids when Y122? is reduced. Assembly of the iron cluster is shown to be accompanied by deprotonation of histidine. A p(2)H titration study supports the assignment of an elevated pK for the histidine. In addition, structural perturbations of tyrosine and tryptophan are detected. For tryptophan, comparison to model compound data suggests an increase in hydrogen bonding and a change in conformation when the iron cluster is removed. pH and (2)H(2)O studies imply that the perturbed tryptophan is in a low dielectric environment that is close to the metal center and protected from solvent exchange. Tyrosine contributions are attributed to a conformational or hydrogen-bonding change. In summary, our work shows that electrostatic and conformational perturbations of aromatic amino acids are associated with metal cluster assembly in RNR. These conformational changes may contribute to the allosteric effects, which regulate metal binding.     

Proton Coupled Electron Transfer and Redox Active Tyrosines: Structure and Function of the Tyrosyl Radicals in Ribonucleotide Reductase and Photosystem II

Proton coupled electron transfer (PCET) reactions are important in many biological processes. Tyrosine oxidation/reduction can play a critical role in facilitating these reactions. Two examples are photosystem II (PSII) and ribonucleotide reductase (RNR). RNR is essential in DNA synthesis in all organisms. In E. coli RNR, a tyrosyl radical, Y122(?), is required as a radical initiator. Photosystem II (PSII) generates molecular oxygen from water. In PSII, an essential tyrosyl radical, YZ(?), oxidizes the oxygen evolving center. However, the mechanisms, by which the extraordinary oxidizing power of the tyrosyl radical is controlled, are not well understood. This is due to the difficulty in acquiring high-resolution structural information about the radical state. Spectroscopic approaches, such as EPR and UV resonance Raman (UVRR), can give new information. Here, we discuss EPR studies of PCET and the PSII YZ radical. We also present UVRR results, which support the conclusion that Y122 undergoes an alteration in ring and backbone dihedral angle when it is oxidized. This conformational change results in a loss of hydrogen bonding to the phenolic oxygen. Our analysis suggests that access of water is an important factor in determining tyrosyl radical lifetime and function. TOC graphic.     

2,3-difluorotyrosine at position 356 of ribonucleotide reductase R2: a probe of long-range proton-coupled electron transfer

Escherichia coli class I ribonucleotide reductase catalyzes the conversion of ribonucleotides to deoxyribonucleotides and consists of two subunits: R1 and R2. R1 possesses the active site, while R2 harbors the essential diferric-tyrosyl radical (Y*) cofactor. The Y* on R2 is proposed to generate a transient thiyl radical on R1, 35 A distant, through amino acid radical intermediates. To study the putative long-range proton-coupled electron transfer (PCET), R2 (375 residues) was prepared semisynthetically using intein technology. Y356, a putative intermediate in the pathway, was replaced with 2,3-difluorotyrosine (F2Y, pKa = 7.8). pH rate profiles (pH 6.5-9.0) of wild-type and F2Y-R2 were very similar. Thus, a proton can be lost from the putative PCET pathway without affecting nucleotide reduction. The current model involving H* transfer is thus unlikely.     

Electrophoretic purification of the alpha and beta subunits of phosphorylase kinase and evidence in support of the deduced amino acid sequences

A simple, rapid sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method is presented for isolating the alpha, alpha' and beta subunits of rabbit muscle phosphorylase kinase. The SDS-PAGE procedure can yield milligram amounts of alpha and beta from a single preparative gel and also allows isolation of the alpha' isozyme free of alpha. Notably the method provides the purified subunits in a form amenable to structural analysis. Edman degradation of alpha and alpha' reveal identical NH2-terminal structures. Amino acid analysis of the electrophoretically purified alpha and beta subunits are in good agreement with their deduced primary structures. The amino acid sequence of 488 residues in alpha and 713 residues in beta were determined by gas phase Edman degradation. The data support the recently deduced primary structures of alpha (Zander et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 9381-9385).     

Spectroscopic and electronic structure studies of intermediate X in ribonucleotide reductase R2 and two variants: a description of the FeIV-oxo bond in the FeIII-O-FeIV dimer

Spectroscopic and electronic structure studies of the class I Escherichia coli ribonucleotide reductase (RNR) intermediate X and three computationally derived model complexes are presented, compared, and evaluated to determine the electronic and geometric structure of the FeIII-FeIV active site of intermediate X. Rapid freeze-quench (RFQ) EPR, absorption, and MCD were used to trap intermediate X in R2 wild-type (WT) and two variants, W48A and Y122F/Y356F. RFQ-EPR spin quantitation was used to determine the relative contributions of intermediate X and radicals present, while RFQ-MCD was used to specifically probe the FeIII/FeIV active site, which displayed three FeIV d-d transitions between 16,700 and 22,600 cm(-1), two FeIV d-d spin-flip transitions between 23,500 and 24,300 cm(-1), and five oxo to FeIV and FeIII charge transfer (CT) transitions between 25,000 and 32,000 cm(-1). The FeIV d-d transitions were perturbed in the two variants, confirming that all three d-d transitions derive from the d-pi manifold. Furthermore, the FeIV d-pi splittings in the WT are too large to correlate with a bis-mu-oxo structure. The assignment of the FeIV d-d transitions in WT intermediate X best correlates with a bridged mu-oxo/mu-hydroxo [FeIII(mu-O)(mu-OH)FeIV] structure. The mu-oxo/mu-hydroxo core structure provides an important sigma/pi superexchange pathway, which is not present in the bis-mu-oxo structure, to promote facile electron transfer from Y122 to the remote FeIV through the bent oxo bridge, thereby generating the tyrosyl radical for catalysis.     

Structure of the nitrogen-centered radical formed during inactivation of E. coli ribonucleotide reductase by 2'-azido-2'-deoxyuridine-5'-diphosphate: trapping of the 3'-ketonucleotide

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides providing the monomeric precursors required for DNA replication and repair. The class I RNRs are composed of two homodimeric subunits: R1 and R2. R1 has the active site where nucleotide reduction occurs, and R2 contains the diiron tyrosyl radical (Y*) cofactor essential for radical initiation on R1. Mechanism-based inhibitors, such as 2'-azido-2'-deoxyuridine-5'-diphosphate (N(3)UDP), have provided much insight into the reduction mechanism. N(3)UDP is a stoichiometric inactivator that, upon interaction with RNR, results in loss of the Y* in R2 and formation of a nitrogen-centered radical (N*) covalently attached to C225 (R-S-N*-X) in the active site of R1. N(2) is lost prior to N* formation, and after its formation, stoichiometric amounts of 2-methylene-3-furanone, pyrophosphate, and uracil are also generated. On the basis of the hyperfine interactions associated with N*, it was proposed that N* is also covalently attached to the nucleotide through either the oxygen of the 3'-OH (R-S-N*-O-R') or the 3'-C (R-S-N*-C-OH). To distinguish between the proposed structures, the inactivation was carried out with 3'-[(17)O]-N(3)UDP and N* was examined by 9 and 140 GHz EPR spectroscopy. Broadening of the N* signal was detected and the spectrum simulated to obtain the [(17)O] hyperfine tensor. DFT calculations were employed to determine which structures are in best agreement with the simulated hyperfine tensor and our previous ESEEM data. The results are most consistent with the R-S-N*-C-OH structure and provide evidence for the trapping of a 3'-ketonucleotide in the reduction process.     

Pulsed ELDOR spectroscopy measures the distance between the two tyrosyl dadicals in the R2 subunit of the E. coli ribonucleotide reductase

Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs). This RNR is composed of two homodimeric subunits: R1 and R2. R1 binds the NDPs in the active site, and R2 harbors the essential di-iron tyrosyl radical (Y*) cofactor. In this paper, we used PELDOR, a method that detects weak electron-electron dipolar coupling, to make the first direct measurement of the distance between the two Y*'s on each monomer of R2. In the crystal structure of R2, the Y*'s are reduced to tyrosines, and consequently R2 is inactive. In R2, where the Y*'s assume a well-defined geometry with respect to the protein backbone, the PELDOR method allows measurement of a distance of 33.1 +/- 0.2 A that compares favorably to the distance (32.4 A) between the center of mass of the spin density distribution of each Y* on each R2 monomer from the structure. The experiments provide the first direct experimental evidence for two Y*'s in a single R2 in solution.     

Mono-, di-, tri-, and tetra-substituted fluorotyrosines: new probes for enzymes that use tyrosyl radicals in catalysis

A set of N-acylated, carboxyamide fluorotyrosine (F(n)()Y) analogues [Ac-3-FY-NH(2), Ac-3,5-F(2)Y-NH(2), Ac-2,3-F(2)Y-NH(2), Ac-2,3,5-F(3)Y-NH(2), Ac-2,3,6-F(3)Y-NH(2) and Ac-2,3,5,6-F(4)Y-NH(2)] have been synthesized from their corresponding amino acids to interrogate the detailed reaction mechanism(s) accessible to F(n)()Y*s in small molecules and in proteins. These Ac-F(n)()Y-NH(2) derivatives span a pK(a) range from 5.6 to 8.4 and a reduction potential range of 320 mV in the pH region accessible to most proteins (6-9). DFT electronic-structure calculations capture the observed trends for both the reduction potentials and pK(a)s. Dipeptides of the methyl ester of 4-benzoyl-l-phenylalanyl-F(n)()Ys at pH 4 were examined with a nanosecond laser pulse and transient absorption spectroscopy to provide absorption spectra of F(n)()Y*s. The EPR spectrum of each F(n)()Y* has also been determined by UV photolysis of solutions at pH 11 and 77 K. The ability to vary systematically both pK(a) and radical reduction potential, together with the facility to monitor radical formation with distinct absorption and EPR features, establishes that F(n)()Ys will be useful in the study of biological charge-transport mechanisms involving tyrosine. To demonstrate the efficacy of the fluorotyrosine method in unraveling charge transport in complex biological systems, we report the global substitution of tyrosine by 3-fluorotyrosine (3-FY) in the R2 subunit of ribonucleotide reductase (RNR) and present the EPR spectrum along with its simulation of 3-FY122*. In the companion paper, we demonstrate the utility of F(n)()Ys in providing insight into the mechanism of tyrosine oxidation in biological systems by incorporating them site-specifically at position 356 in the R2 subunit of Escherichia coli RNR.     

Ketene-forming eliminations from aryl phenylacetates promoted by R2NH/R2NH2+ in aqueous MeCN. Mechanistic borderline between E2 and E1cb

Elimination reactions of 2-X-4-NO2C6H3CH2C(O)OC6H3-2-Y-4-NO2 [X = H (1), NO2 (2)] promoted by R2NH/R2NH2+ in 70 mol % MeCN(aq) have been studied kinetically. The base-promoted eliminations from 1 proceeded by the E2 mechanism when Y = Cl, CF3, and NO2. The mechanism changed to the competing E2 and E1cb mechanisms by the poorer leaving groups (Y = H, OMe) and to the E1cb extreme by the strongly electron-withdrawing beta-aryl group (2, X = NO2). The values of beta = 0.14 and beta(lg) = 0.10-0.21 calculated for elimination from 1 (Y = NO2) indicate a reactant-like transition state with small extents of proton transfer and C(alpha)-OAr bond cleavage. The extent of proton transfer increased with a poorer leaving group, and the degree of leaving group bond cleavage increased with a weaker base. Also, the changes in the k(1) and k(-1)/k(2) values with the reactant structure variation are consistent with the E1cb mechanism. From these results, a plausible pathway of the change of the mechanism from E2 to the E1cb extreme is proposed.