High-frequency (140-GHz) time domain EPR and ENDOR spectroscopy: the tyrosyl radical-diiron cofactor in ribonucleotide reductase from yeast

High-frequency pulsed EPR and ENDOR have been employed to characterize the tyrosyl radical (Y*)-diiron cofactor in the Y2-containing R2 subunit of ribonucleotide reductase (RNR) from yeast. The present work represents the first use of 140-GHz time domain EPR and ENDOR to examine this system and demonstrates the capabilities of the method to elucidate the electronic structure and the chemical environment of protein radicals. Low-temperature spin-echo-detected EPR spectra of yeast Y* reveal an EPR line shape typical of a tyrosyl radical; however, when compared with the EPR spectra of Y* from E. coli RNR, a substantial upfield shift of the g(1)-value is observed. The origin of the shift in g(1) was investigated by 140-GHz (1)H and (2)H pulsed ENDOR experiments of the Y2-containing subunit in protonated and D(2)O-exchanged buffer. (2)H ENDOR spectra and simulations provide unambiguous evidence for one strongly coupled (2)H arising from a bond between the radical and an exchangeable proton of an adjacent residue or a water molecule. Orientation-selective 140-GHz ENDOR spectra indicate the direction of the hydrogen bond with respect to the molecular symmetry axes and the bond length (1.81 A). Finally, we have performed saturation recovery experiments and observed enhanced spin lattice relaxation rates of the Y* above 10 K. At temperatures higher than 20 K, the relaxation rates are isotropic across the EPR line, a phenomenon that we attribute to isotropic exchange interaction between Y* and the first excited paramagnetic state of the diiron cluster adjacent to it. From the activation energy of the rates, we determine the exchange interaction between the two irons of the cluster, J(exc) = -85 cm(-)(1). The relaxation mechanism and the presence of the hydrogen bond are discussed in terms of the differences in the structure of the Y*-diiron cofactor in yeast Y2 and other class I R2s.     

Metal-dependent ferro- versus antiferromagnetic interactions in molecular crystals of square Planar (M(II) imino-nitroxide radical) complexes (M = Pt,Pd)

The synthesis and structural, spectral, and magnetic characterizations of two new complexes of formula [Pt(IM(2)Py)Cl(2)] (A) and [Pd(IM(2)Py)Cl(2)] (B) are reported. IM(2)Py stands for the imino-nitroxide radical ligand 2-(ortho-pyridyl)-4,4,5,5-tetramethylimidazoline-1-oxyl. Their crystal structures were solved at room temperature and at 120 K revealing structural phase transitions from pseudo-orthorhombic to monoclinic systems for the two compounds which remain isostructural in the whole temperature range explored. Structural parameters for A: T = 293 K [120 K], monoclinic (P2(1)/n) [P2(1)/c], a = 7.906(2) [7.989(3)] A, b = 17.872(9) [10.168(4)] A, c = 10.357(3) [17.623(6)] A, beta = 90.732(13) degrees [95.940(2)] degrees, Z = 4 [4]. Structural parameters for B: T = 293 K [120 K], monoclinic (P2(1)/n) [P2(1)/c], a = 7.900(3) [7.9730(2)] A, b = 17.907(9) [10.1806(3)] A, c = 10.299(3) [17.7171(4)] A, beta = 90.524(14) degrees [95.747(2)] degrees, Z = 4 [4]. In both complexes, the metal coordination is essentially planar. The average Pt-N, Pt-Cl and Pd-N, Pd-Cl bond lengths are 1.996(6) [1.88], 2.295(2) [2.248(8)] A and 2.015(7) [2.029(8)], 2.287(3) [2.294(3)] A, respectively. The solid state structure is characterized by a pairlike molecular packing stacked in columns parallel to the a axis; this dimer character is reinforced at low temperature. Despite their structural similarity, the investigation of the magnetic properties revealed that dominant ferromagnetic interactions govern the behavior of the Pt derivative A, whereas antiferromagnetic interactions take place for the Pd compound B. A rationalization for this rather intriguing difference is proposed in light of the spin population deduced from density functional theory calculations. The electronic absorption spectra of A and B present structured absorption bands in the visible which are attributed to MLCT transitions. Both compounds are nonluminescent at room temperature. However, a weak emission is detected for A in butyronitrile glasses at 77 K, indicating that the MLCT excited state is strongly quenched at low temperature.     

Variable coordination geometries at the diiron(II) active site of ribonucleotide reductase R2

The R2 subunit of Escherichia coli ribonucleotide reductase contains a dinuclear iron center that generates a catalytically essential stable tyrosyl radical by one electron oxidation of a nearby tyrosine residue. After acquisition of Fe(II) ions by the apo protein, the resulting diiron(II) center reacts with O(2) to initiate formation of the radical. Knowledge of the structure of the reactant diiron(II) form of R2 is a prerequisite for a detailed understanding of the O(2) activation mechanism. Whereas kinetic and spectroscopic studies of the reaction have generally been conducted at pH 7.6 with reactant produced by the addition of Fe(II) ions to the apo protein, the available crystal structures of diferrous R2 have been obtained by chemical or photoreduction of the oxidized diiron(III) protein at pH 5-6. To address this discrepancy, we have generated the diiron(II) states of wildtype R2 (R2-wt), R2-D84E, and R2-D84E/W48F by infusion of Fe(II) ions into crystals of the apo proteins at neutral pH. The structures of diferrous R2-wt and R2-D48E determined from these crystals reveal diiron(II) centers with active site geometries that differ significantly from those observed in either chemically or photoreduced crystals. Structures of R2-wt and R2-D48E/W48F determined at both neutral and low pH are very similar, suggesting that the differences are not due solely to pH effects. The structures of these "ferrous soaked" forms are more consistent with circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopic data and provide alternate starting points for consideration of possible O(2) activation mechanisms.     

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.     

Isolation and identification of radical scavengers in olive tree (Olea europaea) wood

Several extracts of Olea europaea wood (Picual olive cultivar) were obtained with solvents of different polarity and their antioxidant activities determined. The active compounds were detected in fractions of an ethyl acetate extract using HPLC with on-line radical scavenging detection. After applying different separation techniques, hydroxytyrosol, tyrosol, cycloolivil, 7-deoxyloganic acid, oleuropein and ligustroside were isolated and characterized. Hydroxytyrosol showed a higher activity than the natural antioxidant rosmarinic acid in scavenging the DPPH model radical. Cycloolivil and oleuropein showed stronger activities than the synthetic antioxidant BHT against the same radical. Ligustroside, tyrosol and 7-deoxyloganic acid showed little activity. The latter compound has not been previously identified in the genus Olea.     

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.     

Understanding the interactions between artemisinin and cyclodextrins: spectroscopic studies and molecular modeling

Artemisinin extracted from Artemisia annua L. proved to be currently, with its derivatives, the most effective drugs against simple and severe malaria, and is also effective on the chloroquine-resistant forms. The advantageous effect of some cyclodextrins (CDs) on artemisinin solubilization was demonstrated by different authors. The present work aims to confirm the effect of several CDs on artemisinin solubilization and to analyse the complexes formed between these CDs and artemisinin in order to understand their solubilization capacities. In this context, solubility studies, liquid-state NMR spectroscopy (¹H NMR studies and ROESY experiments) as well as theoretical studies (molecular modeling) have been performed. Randomly methylated-βCD, Crysmeb^? and hydroxypropylated-γCD were also found to improve the aqueous solubilization of artemisinin as well as βCD, γCD and hydroxypropylated-βCD whose effects were already demonstrated. The best solubilization ability was found with Crysmeb^?. The spectroscopic studies showed a lot of interactions between artemisinin and all the CDs studied, but mainly outside the cavity. Molecular modeling confirmed that artemisinin and CDs formed non-inclusion complexes.     

Synthesis and molecular structures of mononitrosyl (N2S2)M(NO) complexes (M = Fe, Co)

A series of tetragonally distorted square pyramids of formula N2S2M(NO) (M = Fe, Co) is prepared and characterized by nu(NO) IR and EPR spectroscopies, magnetism and electrochemical properties, as well as solid-state crystal structure determinations. While the nu(NO) IR frequencies and the angleM-N-O angles indicate differences in the electronic environment of NO consistent with the Enemark-Feltham notation of [Fe(NO)]7 and [Co(NO)]8, the reduction potentials, assigned to [Fe(NO)]7 + e- <==> [Fe(NO)]8 and [Co(NO)]8 + e- <==> [Co(NO)]9 respectively, are very similar, and in cases identical, for most members of the series. Coupled with the potential for the M(NO) units to breathe out of and into the N2S2 core plane are unique S-M-N-O torsional arrangements and concomitant pi-bonding interactions which may account for the unusual coherence of reduction potentials within the series.     

The reaction of phenyl radical with molecular oxygen: a G2M study of the potential energy surface

Ab initio G2M calculations have been performed to investigate the potential energy surface for the reaction of C6H5 with O2. The reaction is shown to start with an exothermic barrierless addition of O2 to the radical site of C6H5 to produce phenylperoxy (1) and, possibly, 1,2-dioxaspiro[2.5]octadienyl (dioxiranyl, 8) radicals. Next, 1 loses the terminal oxygen atom to yield the phenoxy + O products (3) or rearranges to 8. The dioxiranyl can further isomerize to a seven-member ring 2-oxepinyloxy radical (10), which can give rise to various products including C5H5 + CO2, pyranyl + CO, o-benzoquinone + H, and 2-oxo-2,3-dihydrofuran-4-yl + C2H2. Once 10 is produced, it is unlikely to go back to 8 and 1, because the barriers separating 10 from the products are much lower than the reverse barrier from 10 to 8. Thus, the branching ratio of C6H5O + O against the other products is mostly controlled by the critical transition states between 1 and 3, 1 and 8, and 8 and 10. According to the calculated barriers, the most favorable product channel for the decomposition of 10 is C5H5 + CO2, followed by pyranyl + CO and o-benzoquinone + H. Since C6H5O + O and C5H5 + CO2 are expected to be the major primary products of the C6H5 + O2 reaction and thermal decomposition of C6H5O leads to C5H5 + CO, cyclopentadienyl radicals are likely to be the major product of phenyl radical oxidation, and so it results in degradation of the six-member aromatic ring to the five-member cyclopentadienyl ring. Future multichannel RRKM calculations of reaction rate constants are required to support these conclusions and to quantify the product branching ratios at various combustion conditions.     

Understanding the interactions between tris(pentafluoroethyl)-trifluorophosphate-based ionic liquid and small molecules from molecular dynamics simulation

Tris(pentafluoroethyl)trifluorophosphate ([FEP])-based ionic liquids have been widely applied in many fields. For better understanding the properties of [FEP]-based ionic liquids, the interactions between 1-hexyl-3-methylimidazolium ([hmim])[FEP] and small molecules were investigated by molecular dynamics simulations in this work. The small molecules are water, methanol and dimethyl ether. The united-atom (UA) force fields were proposed for methanol and dimethyl ether based on AMBER force field. The densities, enthalpies of vaporization, excess molar properties, and diffusion coefficients of the mixtures were calculated, as well as the microscopic structures characterized by radial distribution functions. Both of the results of the excess energies and microscopic properties show that the strongest interaction is between [hmim][FEP] and dimethyl ether, whereas the interaction between [hmim][FEP] and water is the weakest. Moreover, [hmim][FEP] is more hydrophobic than [hmim] hexafluorophosphate ([PF6]), and the three solutes are mainly distributed around [FEP] anion.     

A density functional evaluation of an Fe(III)-Fe(IV) model diiron cluster: comparisons with ribonucleotide reductase intermediate X

Using broken-symmetry density functional theory and spin-projection methods, we have examined the electronic structure and properties of a large mixed-valent Fe(III)-Fe(IV) diiron system that displays two bidentate carboxylates and a single mu-oxo moiety as bridging ligands. Two carboxylates and a single oxygen species have long been implicated as core elements of the elusive intermediate X in ribonucleotide reductase. Spectroscopic studies of X have also identified the presence of an additional terminal or bridging oxygen-based ligand. Introduction of a second oxygen and protonated variants thereof in the core of our structural model is favored as a bridging hydroxide based on the lowest energy structure. M?ssbauer measurements indicate clearly that the two iron sites of X are distinct and that there is significant electron delocalization onto the oxygen-based ligands. For several examined spin states of our model cluster, M?ssbauer parameters from density functional calculations are neither able to differentiate between the iron sites nor reproduce the strong spin delocalization onto the oxygen-based ligands observed experimentally. The combined comparison of the calculated geometries, spin states, spin densities, and M?ssbauer properties for our model clusters with available experimental data for X implies that intermediate X is significantly different from the diiron structural models examined herein.     

Preparation,characterization and reactivity of Cp2M(PMe3)2 complexes (M = Ti,Zr): the molecular structure of Cp2Zr(PMe3)2

The reduction of Cp₂MCl₂ (M = Ti, Zr) with magnesium in THF in the presence of PMe₃ affords the complexes Cp₂M(PMe₃)₂ in high yields. These compounds lose one or both PMe₃ ligands under very mild conditions. Cp₂Ti(PMe₃)₂ reacts readily with CH₃I, CH₃C(O)Cl, PhSSPh, Me₂PCH₂CH₂PMe₂, CO, RCN (R = Me, t-Bu) or (RN)₂S (R = t-Bu, Me₃Si) to give the corresponding titanocene products. The structure of Cp₂Zr(PMe₃)₂ has been determined by X-ray diffraction; the structural parameters are similar to those of the titanium analog Cp₂Ti(PMe₃)₂ except that the Zr-P and Zr-C distances are longer.     

Ferromagnetic interactions in Ru(III)-nitronyl nitroxide radical complex: a potential 2p4d building block for molecular magnets

The reaction between [Ru(salen)(PPh3)Cl] and the 4-pyridyl-substituted nitronyl nitroxide radical (NITpPy) leads to the [Ru(salen)(PPh3)(NITpPy)](ClO4)(H2O)2 complex while the reaction with the azido anion (N3-) leads to the [Ru(salen)(PPh3)(N3)] complex 2 (where salen2- = N,N'-ethan-1,2-diylbis(salicylidenamine) and PPh3 = triphenylphosphine). Both compounds have been characterized by single crystal X-ray diffraction. The two crystal structures are composed by a [Ru(III)(salen)(PPh3)]+ unit where the Ru(III) ion is coordinated to a salen2- ligand and one PPh3 ligand in axial position. In 1 the Ru(III) ion is coordinated to the 4-pyridyl-substituted nitronyl nitroxide radical whereas in 2 the second axial position is occupied by the azido ligand. In both complexes the Ru(III) ions are in the same environment RuO2N3P, in a tetragonally elongated octhaedral geometry. The crystal packing of 1 reveals pi-stacking in pairs. While antiferromagnetic intermolecular interaction (J2 = 5.0 cm(-1)) dominates at low temperatures, ferromagnetic intramolecular interaction (J1 = -9.0 cm(-1)) have been found between the Ru(III) ion and the coordinated NITpPy.     

DFT calculations for intermediate and active states of the diiron center with a tryptophan or tyrosine radical in Escherichia coli ribonucleotide reductase

Class Ia ribonucleotide reductase subunit R2 contains a diiron active site. In this paper, active-site models for the intermediate X-Trp48(?+) and X-Tyr122(?), the active Fe(III)Fe(III)-Tyr122(?), and the met Fe(III)Fe(III) states of Escherichia coli R2 are studied, using broken-symmetry density functional theory incorporated with the conductor-like screening solvation model. Different structural isomers and different protonation states have been explored. Calculated geometric, energetic, Mo?ssbauer, hyperfine, and redox properties are compared with available experimental data. Feasible detailed structures of these intermediate and active states are proposed. Asp84 and Trp48 are most likely the main contributing residues to the result that the transient Fe(IV)Fe(IV) state is not observed in wild-type class Ia E. coli R2. Asp84 is proposed to serve as a proton-transfer conduit between the diiron cluster and Tyr122 in both the tyrosine radical activation pathway and the first steps of the catalytic proton-coupled electron-transfer pathway. Proton-coupled and simple redox potential calculations show that the kinetic control of proton transfer to Tyr122(?) plays a critical role in preventing reduction from the active Fe(III)Fe(III)-Tyr122(?) state to the met state, which is potentially the reason why Tyr122(?) in the active state can be stable over a very long period.     

Density functional study of a micro-1,1-carboxylate bridged Fe(III)-O-Fe(IV) model complex. 2. Comparison with ribonucleotide reductase intermediate X

Using broken-symmetry density functional theory, we have studied an experimentally proposed model for ribonucleotide reductase (RNR) intermediate X, which contains a single oxo bridge, one terminal H(2)O or OH(-) ligand, a bidentate carboxylate from Glu115, and a mono-oxygen bridge provided by Glu238. For the models proposed here, the terminal H(2)O/OH(-) ligand binds to site Fe1 which is closer to Tyr122. The diiron centers are assigned as high-spin Fe(III)Fe(IV) and antiferromagnetically coupled to give the S(total) = (1)/(2) ground state. Calculations show that the model with a terminal hydroxide in the antiferromagnetic [S(Fe1) = 2, S(Fe2) = (5)/(2)] state (Fe1 = Fe(IV), Fe2 = Fe(III)) is the lowest energy state, and the calculated isomer shift and quadrupole splitting values for this cluster are also the best among the four clusters studied here when compared with the experimental values. However, the DFT-calculated (1)H proton and (17)O hyperfine tensors for this state do not show good agreement with the experiments. The calculated Fe1-Fe2 distances for this and the other three clusters at >2.9 A are much longer than the 2.5 A which was predicted by the EXAFS measurements. The mono-oxygen bridge provided by Glu238 tends to be closer to one of the Fe sites in all clusters studied here, and it does not function as a bridge in helping to produce a short Fe-Fe distance. Overall, the models tested here are not likely to represent the core structure of RNR intermediate X. The model with the terminal OH(-) binding to the Fe1(III) center shows the best calculated (1)H proton and (17)O hyperfine tensors compared with the experimental values. This supports the earlier proposal based on analysis of ENDOR spectra (Willems et al.(16)) that the terminal oxygen group binds to the Fe(III) site in RNR-X.     

Heteroatomic molecular clusters derived from Group 15 Zintl ion cages: synthesis and isolation of [M2(HP7)2](2-) (M=Ag, Au), two novel cluster anions exhibiting metallophilic interactions

Ethylenediamine (en) solutions of K(3)P(7) and 2,2,2-crypt (4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane) were reacted with the homoleptic group 11 complexes [M(nbe)(3)][SbF(6)] (M = Ag, Au; nbe = norbornene) yielding two novel cluster anions, [M(2)(HP(7))(2)](2-), both of which were isolated in low crystalline yields as [K(2,2,2-crypt)](2)[M(2)(HP(7))(2)] (M = Ag (1) and Au (2)). Optimization of the reaction conditions by incorporation of a proton source (ammonium tetraphenylborate) and the replacement of the light-sensitive nbe adducts of silver and gold with the chloride salts MCl (M = Ag, Au) was found to greatly increase the yield and purity in which 1 and 2 were isolated. Compounds 1 and 2 were characterized by single crystal X-ray diffraction, electrospray ionization mass-spectrometry (ESI- MS), elemental analysis, and (1)H and (31)P NMR spectroscopy. Density functional theory (DFT) calculations on the cluster anions were also conducted.     

Computational study of the reaction of chlorinated vinyl radical with molecular oxygen (C2Cl3 + O2)

Eight exothermic product channels of the reaction of chlorinated vinyl radical (C2Cl3) with molecular oxygen (O2) have been investigated using ab initio quantum chemistry methods. The energetics of the reaction pathways were calculated at the second-order Moller-Plesset Gaussian-3 level of theory (G3MP2) using the B3LYP/6-311G(d) optimized geometries. It has been shown that the C2Cl3 + O2 reaction takes place via a barrierless addition to form the chlorinated vinylperoxy radical complex, which can decompose or isomerize to various products via the complicated mechanisms. Two major reaction routes were revealed, i.e., the three-member-ring reaction mechanism leading to ClCO + CCl2O, CO + CCl3O, CO2 + CCl3, Cl + (ClCO)2, etc., and the OO bond cleavage mechanism leading to O(3P) + C2Cl3O. The other mechanisms are shown to be unimportant. The results are validated by the calculations using the restricted coupled cluster theory [RCCSD(T)] with the complete basis set extrapolation. Variational transition state theory was employed to calculate the individual and total rate coefficients as a function of temperature and pressure (helium). The theoretical rate coefficients are in good agreement with the available experimental data. It was found that the total rate coefficients show strong negative temperature dependence in the range 200-2000 K. At room temperature (297 K), the total rate coefficients are shown to be nearly pressure independent over a wide range of helium pressures (1-10(9) Torr). The deactivation of the initial adduct, C2Cl3O2, is only significant at pressures higher than 1000 Torr. The three-member-ring reaction mechanism is always predominant over the OO bond cleavage.     

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.     

Cation-π interactions of curved polycyclic systems: M (M=Li and Na) ion complexation with buckybowls

B3LYP/6-311+G** calculations on alkali metal ion (Li⁺ and Na⁺) complexation with corannulene and sumanene indicate stronger binding compared to [5]-radialene or benzene. The dependence of binding to the convex and concave site is marginal, albeit the preference was consistent for convex binding in the range of 1-4 kcal/mol. The bowl-to-bowl inversion barriers are only marginally affected, below 2 kcal/mol, by metal ion complexation.