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Reduction of Escherichia coli ribonucleotide reductase subunit R2 with eight water-soluble ferrocene derivatives
Abstract
Water soluble ferrocenes [Fe(Cp)(CpL)], where Cp− is the η5-cyclopentadienide ligand and the side chain L is (a) the carboxylic acid group –(CH2)xCO2H with x=0–4 (I–V); (b) the complex x=2 with the β-methylene mono-methyl substituted (VI); (c) the amine hydrochloride derivative with L=CH(Me) NH3+ (VII); and (d) the complex with two Cp rings bridged by the amine hydrochloride –CH(NH3+)CH2CH2− (VIII); have been prepared, and are used as one-equivalent reductants for the active-R2 subunit of Escherichia coli ribonucleotide reductase. Formal reduction potentials E1°′ (25°C) of the carboxylates of acids I–VI in 20 mM NaOH, and of the amine hydrochlorides VII and VIII in water were determined by cyclic voltammetry, and are in the range 0.308–0.550 V versus nhe, I=0.100 M (NaCl). Second-order rate constants k12 (25°C) for the reduction of active-R2 were determined by UV–Vis spectrophotometry, and are in the range 0.15–0.50 M−1 s−1 at I=0.100 M. A free-energy plot of logk12 versus E°′ values gives no clearcut unidirectional trend. Since from present information the electron self-exchange rate constant for the [Fe(Cp)2]+/[Fe(Cp)2] couple is favourable (>7×106 M−1 s−1 in methanol at 25°C), it would appear that electron-transfer from the ferrocenes via Trp-48, Asp-237, His-118 to the FeIII2 site on R2 is much slower than expected, and smaller than with the organic radical reductants previously studied. Electron-transfer from some other position on the protein surface to the Tyr is considered as an alternative.
... Water-soluble ferrocene derivatives have become a research focus as cathodic active materials in AORFBs, with their water solubility and redox potential being the main targets of research. The introduction of an electron-withdrawing group, such as trimethylammonium [11,27] and sulfonate [20,[28][29][30], may effectively improve the cathodic potential, and hydrophilic groups, such as hydroxyl [12,13], carboxy [31] and phosphate [32] may help to increase the solubility. Furthermore, the existence of certain substituents may increase steric effects and improve the structural stability of the redox-active ferrocene [27]. ...
Ferrocene derivatives are amongst the most promising electroactive organic electrolytes. The bottleneck problems of their application in aqueous redox flow batteries are their poor solubility and lower potential as well as the complexity of the modification methods to solve these problems. In this study, a benzenesulfonic acid group is easily introduced into the ferrocene structure by a mature diazotization reaction, and the synthesized sodium m-phenylferrocene sulfonate BASFc is used as the novel cathodic electroactive electrolyte for AORFB. The hydrophilicity and the electron-absorbing effect of the introduced benzenesulfonic group can effectively improve the water solubility and redox potential of ferrocene. Moreover, the introduction of phenyl extends the conjugated structure of ferrocene and increases its structural dimension, which may be conducive to reducing its membrane permeability and improving the structural stability to some extent. The physical structure and the electrochemical properties of BASFc are studied systematically; the feasibility of its application as a cathodic electrolyte in AORFBs is verified by assembling the half-cell and full-cell. The results verify the good electrochemical reaction kinetics of BASFc in acid electrolyte and the corresponding AORFB shows satisfactory efficiency and stability.
... Gratifyingly, the mixture of [Cu(L-H)Cl 2 ] and Fc in the presence of SO 2 showed the appearance of a new band at 625 nm that is characteristic of the Fe 3+ ion of ferrocenium species with the concurrent formation of sulfate anions (Fig. 8). 57,58 Hence, undoubtedly, the electron transfer from ferrocene was triggered by the binding of SO 2 to the back-bone of the Cu-koneramine complex (Scheme 3). The SO 2 -binding to the Cu(II) ion can be ruled out as it can only occur when the metal ion is electron rich such as in Vaska Table S5; CCDC 1895748 ‡). ...
Reported is an unprecedented, simple and sustainable strategy to catalytically convert SO2 gas into sulfate dianions at ambient conditions utilizing ferrocene (SO2 + 2Fc + aerial O2 Fc2SO4) and aerial oxygen whereby ferrocenium sulfate was formed without any unwanted byproducts. Frequent urban haze and increase in respiratory health issues connected to atmospheric SO2 concentration attracted interest in the sequestration and valorization of SO2. Even though a number of methods have been reported in the past decade for the stoichiometric sequesteration and activation of SO2, effective catalytic activation of SO2 has not been realized before. The copper(II) koneramine complex [Cu(L-H)Cl2] designed to have exclusive basic sites for SO2 binding, which complemented efficient electron transfer from ferrocene to SO2 that underwent S-oxygenation to yield sulfate dianions. In this proof of concept study, SO2 binding coupled electron transfer (SOCET) from ferrocene was established with the assistance of control experiments, electronic absorption spectroscopy, cyclic voltammetry and electrospray ionization mass spectrometry.
... At 0.12 V, there is 99% probability that the molecule is in the oxidized state and we attribute 0.13 G 0 as the average conductance of a 3C-Fc molecule in this state. The increase in the single-molecule conductance associated with oxidation accords with previous reports (16,24) and is supported by UV-Vis spectroscopy, which reveals an increased absorption wavelength (smaller energy gap between the highest occupied molecular orbital and lowest excited state) (29) for the oxidized form than for the reduced form. To study the electron transfer reaction of the molecule further, we varied the potential systematically from low to high values and plotted the average conductance against potential, which reveals a sigmoidal dependence of the conductance on the electrochemical potential (Fig. 2C). ...
Significance
Electron transfer (ET) is an elementary step in many chemical reactions and biological processes. Measuring ET at the single-molecule level opens new prospects for studying individual reaction steps and heterogeneity in ET. Despite recent advances, the connection between the single-molecule behavior and the ensemble averages remains unclear. By performing direct electrical measurements on ferrocene with variable lengths of molecular linkers, we found that the ET process is stochastic and follows first-order kinetics at the single-molecule level, but ensemble averaging of the single-molecule measurements recovers the thermodynamic behavior. This study provides a framework to connect stochastic single-molecule behavior and deterministic ensemble thermodynamics.
... At 0.12 V, there is 99% probability that the molecule is in the oxidized state and we attribute 0.13 G 0 as the average conductance of a 3C-Fc molecule in this state. The increase in the single-molecule conductance associated with oxidation accords with previous reports (16,24) and is supported by UV-Vis spectroscopy, which reveals an increased absorption wavelength (smaller energy gap between the highest occupied molecular orbital and lowest excited state) (29) for the oxidized form than for the reduced form. To study the electron transfer reaction of the molecule further, we varied the potential systematically from low to high values and plotted the average conductance against potential, which reveals a sigmoidal dependence of the conductance on the electrochemical potential (Fig. 2C). ...
Providing a mechanically stable and electronically efficient coupling between a molecule and an electrode is critical to the study of charge transfer and conductance of the molecule. A common method is to link the molecule to Au electrodes via a linker (e.g., thiol terminal of the molecule). Here we study the mechanical stability and electronic coupling of S-Au bond in single molecule junctions over a broad range of electrode potential. Our results show that the mechanical and electromechanical properties of molecule-electrode contact undergo a systematic change with the potential involving Au oxidation at positive potentials and S protonation at negative potentials. The study establishes the potential range for a stable S-Au bond and determines the potential dependence of the mechanical and electromechanical properties of the molecule-electrode contact, which is crucial to the interpretation of potential dependent charge transfer in electrochemistry and electrochemical gating of charge transport in molecular electronics.
... In the second mechanism, the ferrocenyl group itself acts as a reducing agent when it reduces the tyrosyl radical of the R2 subunit of the enzyme ribonucleotide reductase. [30] The active site of dimeric R2 consists of a tyrosil radical and two Fe III centers which are μ-oxo bridged. This enzyme catalyses the reduction of ribonucleotides to deoxyribonucleotides, a key step in DNA syntheses, and it's inactivation is therefore a goal in chemotherapy. ...
Ferrocene-containing rhodium(I) complexes of the type [Rh(FcCOCHCOR)(CO)(PPh3)] with R = Fc (ferrocenyl), 1, Ph (phenyl), 2, CH3, 3, and CF3, 4, have been studied by cyclic voltammetry (CV) and bulk electrolysis techniques in CH3CN. Two isomers for complexes 2–4 were detected. Results are consistent with RhI being oxidised first in an electrochemically irreversible two-electron transfer process, followed by the electrochemical reversible oxidation of each ferrocenyl group in a one-electron transfer process at slightly larger potentials. Only the Epa(Rh) CV peaks resolved into two separate waves, each one being associated with an isomer. Slow isomerisation kinetics allowed the detection of two isomers of 2–4. Epa(Rh) and other compound physical parameters relates to the second-order rate constant for the oxidative addition of CH3I to complexes 1–4 in acetone. Rhodium(III) reduction of oxidised 1, 2, 3 and 4 was observed at large negative potentials, while for [Rh(FcCOCHCOCF3)(CH3)(I)(CO)(PPh3)] (9) it was observed at –1.492 V vs. Fc/Fc+. Complex 2 was slightly more cytotoxic than the free FcCOCH2COPh ligand but less cytotoxic than cisplatin.
Ferrocenylbutoxy-bearing dodecylated phthalocyanines, MPc(C12H25)x(OC4H8Fc)y with M = 2H (compound series 6 and 8) or Zn (compound series 5, 7 and 9), x ≤ 8 and y ≤ 4, were synthesized through either metal-free statistical condensation between 3,6-bis(dodecyl)phthalonitrile, 2, and 4- (1), or 3-(4'-ferrocenylbutoxy)phthalonitrile, 4, or a zinc template statistical condensation between 4,5-bis(dodecyl)phthalonitrile, 3, and 1 in the presence of anhydrous zinc acetate, or by zinc insertion into metal-free phthalocyanines. Compounds were designed to have eight nonperipheral dodecyl substituents, six nonperipheral dodecyl, either one peripheral or one nonperipheral 4'-ferrocenylbutoxy substituent, four nonperipheral dodecyl and two peripheral 4'-ferrocenylbutoxy substituents, or four peripheral 4'-ferrocenylbutoxy substituents. The compound having six peripheral dodecyl and one peripheral 4'-ferrocenylbutoxy substituents was also synthesized. Metal-free and zinc complex Q-band maximum absorption wavelengths increased nonlinearly from 704 to 725 nm for the Qy-band of metal-free compounds, or from 676 to 699 nm for the Q-band of zinc complexes in moving from all peripheral-substituted to all non-peripheral-substituted complexes. A rare case of accidental Q-band degeneracy where only one electronic Q-band is observed for asymmetrical zinc complexes NOT having D4h symmetry, compounds 5, 7b-e, and 9b, is also described. X-ray photoelectron spectroscopy (XPS) differentiated between four types of phthalocyanine nitrogen atoms; binding energies were ca. 399.8 (N-H), 398.1 (Nmeso), 397.8 (Ncore), and 398.7 eV (N-Zn), respectively. An electrochemical study of these compounds revealed up to five different redox processes in dichloromethane but only three in tetrahydrofuran (THF). The first ring-based oxidation of both metal-free compounds 6a-e and zinc phthalocyanines 7a-e exhibited a near-linear increase in peak anodic potentials, Epa, with the systematic replacement of two nonperipheral dodecyl substituents with one peripheral 4'-ferrocenylbutoxy group. When four 4'-ferrocenylbutoxy groups were substituted on the phthalocyanine macrocycle, aggregation of the first oxidized species was observed. Zinc insertion into metal-free phthalocyanines lowered formal redox potentials. An electrochemical scheme consistent with electrochemical results is provided.
Ferrocenylcarboxylic acid Fe, Co, and Ni complexes were synthesized as model complexes to investigate how many ferrocene units of multi-ferrocenyl system can react with the electrode. The molecular structures of model complexes were characterized by X-ray single-crystal diffraction, the diffusion coefficient of the complexes and ferrocene was determined by the diffusion-ordered spectroscopy and Einstein–Stokes equation, the electrode reaction numbers of ferrocene units were determined by cyclic voltammetry and the Randles–Sevcik equation. A model of electrode reaction of multi-ferrocenyl system was proposed in this paper, which was used to explore how many ferrocene units of multi-ferrocenyl complexes can react with the electrode.
There are many different perspectives about the sustainable agriculture, which had been proposed since the last three decades in the world. While China's ecologists and agronomists proposed a similar concept named 'ecological agriculture'. Although ecological agriculture in China has achieved substantial progress, including theory, models and supporting technologies nearly several decades of practice and development, its application guidance still is not yet clear. The organic agriculture model proposed by European Union is popular, but it is limited in the beneficiary groups and the social and ecological responsibility. In this context, the article based on an ecological point of view, analyzed the shortcomings of ecological imbalance caused by a single mode of agricultural production and the negative impact on the quality of agricultural products, and discussed the core values of ecological agriculture. On this basis, we put forward the concept of sustainable security of agricultural products. Based on this concept, an agricultural platform was established under the healthy ecosysphere environment, and from this agricultural platform, agricultural products could be safely and sustainably obtained. Around the central value of the concept, we designed the agricultural sustainable and security production model. Finally, we compared the responsibility, benefiting groups, agronomic practices selection and other aspects of sustainable agriculture with organic agriculture, and proved the advancement of sustainable agricultural model in agricultural production quality and safety. ©, 2015, Editorial Board of Chinese Journal of Applied Ecology. All right reserved.
Ferrocenylamides, Fc-(CH2)n-CONH2 with n = 0 (3a), 1 (3b), 2 (3c), and 3 (3d) and Fc = ferrocenyl = FeII(C5H5)(C5H4), were synthesised by reacting aqueous ammonia with the appropriate acid chlorides, Fc-(CH2)n-COCl, 2a-2d, under interfacial conditions. The acid chlorides were obtained from Fc-(CH2)n-COOH, 1a-1d, by treatment with SOCl2, (COCl)2 or PCl3. The effectiveness of the different chlorination reagents for these ferrocene acid derivatives is compared. The amides were subsequently treated with LiAlH4 to generate the amines Fc-(CH2)m-NH2 with m = 1 (4a), 2 (4b), 3 (4c), and 4 (4d); they may be stored as the hydrochloride salts but cold storage is preferable. A cyclic voltammetric study of the amides 3a-3d and amines 4a-4d in CH3CN/0.1 M [N(nBu)4][PF6] and the hydrochlorides 4a.HCl-4d.HCl in water containing 10 mM HCl and 1 M KCl showed the formal oxidation potential, Eo′ versus FcH/FcH+, of the ferrocenyl group decreased non-linearly with increasing values of n. Formal redox potentials of the ferrocenylamides, -amines, -hydrochlorides, the precursor acids and of the related ferrocene-containing alcohols Fc-(CH2)n+1-OH, 5a-5d, are compared. The cytoxicity of Fc-CH2-CONH2, expressed as the concentration causing 50 % HeLa cell growth inhibition (the IC50 value) was 39.7 μM, is almost the same as that of the alcohol Fc-CH2-CH2-OH (IC50 = 35.0 μM). This Fc-CH2-CONH2 cytotoxic result implies that the largest ferrocenyl group redox potential a radical-active cytotoxic ferrocene-containing compound can have to still possess cytotoxic activity against HeLa cancer cells is ca. −0.003 V versus FcH/FcH+.
Chalcones of the type Mc-CO–CHCH-Ph, Ph-CO–CHCH-Mc and Mc-CO–CHCH-Mc with Mc=Fc=ferrocenyl or Rc=ruthenocenyl, and Ph=phenyl were synthesized. Synthesis was achieved by base catalyzed Claisen–Schmidt condensation of the appropriated acetyl and aldehyde in ethanol. Cyclic voltammetry in CH3CN in the presence of 0.1moldm−3 [N(Bu)4][PF6] revealed chemical and electrochemical reversible behaviour for the Fc/Fc+ couple and irreversible electrochemistry for the two electron Rc/Rc2+ couple. The potential ranges for the Fc/Fc+ couple varied in the range 138⩽E°′⩽302mV while Epa for Rc/Rc2+ couple was between 358 and 510mV vs. FcH/FcH+, the internal standard. Chalcones with the carbonyl group adjacent to the metallocene, are more difficult to oxidize.
The purpose of this chapter is to highlight a progress in the transition metal chemistry of some enzymes that catalyze oxidative and reductive reactions. These enzymes are referred to as oxidoreductases (1,2) and transition metals are usually found in their active sites. It is essential to understand basic principles of structural organization and mechanisms of action of the key enzymes used in amperometric biosensors. These are glucose oxidase from Aspergillus niger, horseradish peroxidase (HRP), and a group of pyrroloquinoline quinine (PQQ)-dependent enzymes such as alcohol and glucose dehydrogenases. Natural substrates of HRP are aromatic amines, phenols, indoles, sulfonates, which are usually oxidized into oligomers or polymers. Mediators as electron donors act instead of H2A in the peroxidase catalysis. Their role is to rapidly reduce Compounds I and II into the resting state. Glucose oxidase from A. niger and horseradish peroxidase are the enzymes that catalyze absolutely different reactions.
Organometallic chemistry is an interdisciplinary science which continues to grow at a rapid pace. Although there is continued interest in synthetic and structural studies the last decade has seen a growing interest in the potential of organometallic chemistry to provide answers to problems in catalysis, synthetic organic chemistry and also in the development of new materials. This Specialist Periodical Report aims to reflect these current interests, reviewing progress in theoretical organometallic chemistry, main group chemistry, the lanthanides and all aspects of transition metal chemistry. Volume 31 covers literature published during 2001. Specialist Periodical Reports provide systematic and detailed review coverage in major areas of chemical research. Compiled by teams of leading authorities in the relevant subject areas, the series creates a unique service for the active research chemist, with regular, in-depth accounts of progress in particular fields of chemistry. Subject coverage within different volumes of a given title is similar and publication is on an annual or biennial basis.
The series of ferrocene-containing tris-β-diketonato aluminum(III) complexes [Al(FcCOCHCOR)(3)] (R = CF(3), 1; CH(3), 2; C(6)H(5), 3; and Fc = ferrocenyl = Fe(η(5)-C(5)H(5))(η(5)-C(5)H(4)), 4) were synthesized and investigated structurally and electrochemically; complex 1 was subjected to cytotoxicity tests. (1)H NMR-spectroscopy distinguished between the mer and fac isomers of 2 and 3. Complex 1 existed only as the mer isomer. A single crystal X-ray crystallographic determination of the structure of a mer-isomer of Al(FcCOCHCOCF(3))(3), 1, (Z = 4, space group P2(1)2(1)2(1)) demonstrated extensive delocalization of all bonds which explained the pronounced electrochemically observed intramolecular communication between molecular fragments. In contrast to electrochemical studies in CH(2)Cl(2)/[N((n)Bu)(4)][PF(6)], the use of the supporting electrolyte [N((n)Bu)(4)][B(C(6)F(5))(4)] allowed identification of all Fc/Fc(+) electrochemical couples by cyclic and square wave voltammetry for 1-4. For R = Fc, formal reduction potentials of the six ferrocenyl groups were found to be E°' = 33, 123, 304, 432, 583, and 741 mV versus free ferrocene respectively. Complex 1 (IC(50) = 10.6 μmol dm(-3)) was less cytotoxic than the free FcCOCH(2)COCF(3) ligand having IC(50) = 6.8 μmol dm(-3) and approximately 2 orders of magnitude less toxic to human HeLa neoplastic cells than cisplatin (IC(50) = 0.19 μmol dm(-3)).
Ferrocene derivatives may possess antineoplastic activity. Those with low ferrocenyl reduction potentials often have the highest anticancer activity, as cell components have to oxidise them to the active ferrocenium species before cytotoxicity can be recorded. Some gold(I) complexes also possess anticancer activity. This study examined the cytotoxicity of ferrocenyl-ethynyl and ruthenocenyl-ethynyl complexes of gold and platinum. The results were related to the ease of iron oxidation in the ferrocenyl fragment and compared with the cytotoxicity of cisplatin, [(H(3)N)(2)PtCl(2)] and [Au(PPh(2)CH(2)CH(2)PPh(2))(2)]Cl.
Ferrocene-containing gold and platinum complexes of the type Fc-C≡C-PPh(2), 1, and Fc-C≡C-PPh(2)→M with Fc=ferrocenyl (Fe(II)(η(5)-C(5)H(5)) (η(5)-C(5)H(4))), Ph=phenyl (C(6)H(5)) and M=Au-Cl, 2, Au-C≡C-Fc, 3, or Au-C≡C-Rc, 4 (Rc=ruthenocenyl, (Ru(II)(η(5)-C(5)H(5)) (η(5)-C(5)H(4))) and the complex [(Fc-C≡C-PPh(2))(2)PtCl(2)], 5, were investigated. Cytotoxicity tests were determined on the HeLa (human cervix epitheloid) cancer cell line, ATCC CCL-2. Cell survival was measured by means of the colorometric 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide assay.
The IC(50) values of compounds 1-4 from four experiments causing 50% cell growth inhibition, ranged between 4.6 and 27 μmol dm(-3). Drug activity was inversely proportional to the sum of all formal reduction potentials, E(o'), of the ferrocenyl groups of the Fc-C≡C-PPh(2) and Fc-C≡C-ligands coordinated to the gold centre. The Fc-C≡C-PPh(2)→Au-Cl complex, compound 2, was most cytotoxic with IC(50)=4.6 μmol dm(-3) , demonstrating the beneficial effect the Cl(-) ion has on the cytotoxicity of these neutral gold complexes. The platinum complex [(Fc-C≡C-PPh(2))(2)PtCl(2)], compound 5, resembling the structure of cisplatin, in principle should exhibit good cytotoxicity, but was not tested due to its total insolubility in any biocompatible medium.
A series of ferrocene-containing rhodium complexes of the type [Rh(FcCOCHCOR)(cod)] (cod = 1,5-cyclooctadiene) with R = CF(3), 1, (E(pa)(Rh) = 269; E(o)'(Fc) = 329 mV vs. Fc/Fc(+)), CCl(3), 2, (E(pa) = 256; E(o)' = 312 mV), CH(3), 3, (E(pa) = 177; E(o)' = 232 mV), Ph = C(6)H(5), 4, (E(pa) = 184; E(o)' = 237 mV), and Fc = ferrocenyl = (C(5)H(5))Fe(C(5)H(4)), 5, (E(pa) = 135; E(o)'(Fc1) = 203; E(o)'(Fc2) = 312 mV), have been studied electrochemically in CH(3)CN. Results indicated that the rhodium(I) centre is irreversibly oxidised to Rh(III) in a two-electron transfer process before the ferrocenyl fragment is reversibly oxidized in a one-electron transfer process. The peak anodic (oxidation) potential, E(pa), (in V vs. Fc/Fc(+)) of the rhodium core in 1-5 relates to k(2), the second-order rate constant for the substitution of (FcCOCHCOR)(-) with 1,10-phenanthroline in [Rh(FcCOCHCOR)(cod)] to form [Rh(phen)(cod)](+) in methanol at 25 °C with the equation lnk(2) = 39.5 E(pa)(Rh) - 3.69, while the formal oxidation potential of the ferrocenyl groups in 1-5 relates to k(2) by lnk(2) = 40.8 E(o)'(Fc)-6.34. Complex 4 (IC(50) = 28.2 μmol dm(-3)) was twice as cytotoxic as the free FcCOCH(2)COPh ligand having IC(50) = 54.2 μmol dm(-3), but approximately one order of magnitude less toxic to human HeLa neoplastic cells than cisplatin (IC(50) = 2.3 μmol dm(-3)).
For Abstract see ChemInform Abstract in Full Text.
The large subunit of ribonucleotide reductase from Escherichia coli contains redox-active cysteine residues. In separate experiments, five conserved and 2 nonconserved cysteine residues were substituted with alanines by oligonucleotide-directed mutagenesis. The activities of the mutant proteins were determined in the presence of three different reductants: thioredoxin, glutaredoxin, or dithiothreitol. The results indicate two different classes of redox-active cysteines in ribonucleotide reductase: 1) C-terminal Cys-754 and Cys-759 responsible for the interaction with thioredoxin and glutaredoxin; and 2) Cys-225 and Cys-439 located at the nucleotide-binding site. Our classification of redox-active cysteines differs from the location of the active site cysteines in E. coli ribonucleotide reductase suggested previously (Lin, A.-N. I., Ashley, G. W., and Stubbe, J. (1987) Biochemistry 26, 6905–6909).
The large subunit of ribonucleotide reductase from Escherichia coli contains redox-active cysteine residues. In separate experiments, five conserved and 2 nonconserved cysteine residues were substituted with alanines by oligonucleotide-directed mutagenesis. The activities of the mutant proteins were determined in the presence of three different reductants: thioredoxin, glutaredoxin, or dithiothreitol. The results indicate two different classes of redox-active cysteines in ribonucleotide reductase: 1) C-terminal Cys-754 and Cys-759 responsible for the interaction with thioredoxin and glutaredoxin; and 2) Cys-225 and Cys-439 located at the nucleotide-binding site. Our classification of redox-active cysteines differs from the location of the active site cysteines in E. coli ribonucleotide reductase suggested previously (Lin, A.-N. I., Ashley, G. W., and Stubbe, J. (1987) Biochemistry 26, 6905-6909).
The nrdB gene of Escherichia coli, coding for the B2 protein of ribonucleotide reductase, has been cloned in a runaway-replication vector. The runaway derivative pBEU17 carries the promoter-proximal portion of the E. coli alanyl-tRNA synthetase gene and proved useful for expressing cloned genes lacking their native transcription initiation signals. The alaS promoter is located approximately 500 base pairs upstream of a single BamHI restriction endonuclease cleavage site utilized in the construction of an expression recombinant plasmid, pBS1, for the nrdB product. After 5-h thermal induction of cells carrying the runaway recombinant pBS1, protein B2 constituted 40% of the soluble protein fraction of the cells. The high concentration of protein B2 in crude extracts of induced cells has enabled a simplified purification scheme to be developed for production of homogeneous and concentrated B2 preparations. Protein B2 produced from pBS1 is identical to the chromosomally encoded nrdB product of E. coli as regards molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme activity, tyrosine radical content, and structure of the binuclear iron center. Amino acid sequence analysis showed that the two polypeptide chains of protein B2 are identical. They start with an alanine residue, and the first 30 residues confirmed the amino acid sequence predicted from the nucleotide sequence of the nrdB gene, apart from an NH2-terminal processing removal of the initiator methionine.
Protein B2, a subunit of ribonucleotide reductase from Escherichia coli, contains in its active form a tyrosyl free radical as part of the polypeptide chain and a dimeric iron center that stabilizes the radical. The enzyme depends on this radical for its catalytic activity. Treatment with hydroxyurea scavenges the radical without disturbing the iron center and, thereby, results in an inactive form of the subunit, B2/HU. A second inactive form, apoB2, lacking both the radical and the iron center, is obtained by treatment of B2 with 8-hydroxyquinoline. Here we describe an enzyme activity in extracts from E. coli that transforms the catalytically inactive B2/HU form into the active B2 subunit by regeneration of the tyrosyl radical. This reaction requires the presence of oxygen, dithiothreitol, and Mg2+ and does not proceed through apoB2. Under anaerobic conditions, we obtained evidence for a second activity in the bacterial extract that destroys the free radical and transforms B2 into B2/HU. We suggest that this novel type of protein modification is functionally related to the synthesis of deoxyribonucleotides and DNA.
Reconstitution of the tyrosyl radical in ribonucleotide reductase protein R2 requires oxidation of a diferrous site by oxygen. The reaction involves one externally supplied electron in addition to the three electrons provided by oxidation of the Tyr-122 side chain and formation of the mu-oxo-bridged diferric site. Reconstitution of R2 protein Y122F, lacking the internal pathway involving Tyr-122, earlier identified two radical intermediates at Trp-107 and Trp-111 in the vicinity of the di-iron site, suggesting a novel internal transfer pathway (Sahlin, M., Lassmann, G., Pötsch, S., Sjöberg, B. -M., and Gräslund, A. (1995) J. Biol. Chem. 270, 12361-12372). Here, we report the construction of the double mutant W107Y/Y122F and its three-dimensional structure and demonstrate that the tyrosine Tyr-107 can harbor a transient, neutral radical (Tyr-107(.)). The Tyr-107(.) signal exhibits the hyperfine structure of a quintet with coupling constants of 1.3 mT for one beta-methylene proton and 0.75 mT for each of the 3 and 5 hydrogens of the phenyl ring. Rapid freeze quench kinetics of EPR-visible intermediates reveal a preferred radical transfer pathway via Trp-111, Glu-204, and Fe-2, followed by a proton coupled electron transfer through the pi-interaction of the aromatic rings of Trp-(Tyr-)107 and Trp-111. The kinetic pattern observed in W107Y/Y122F is considerably changed as compared with Y122F: the Trp-111(.) EPR signal has vanished, and the Tyr-107(.) has the same formation rate as does Trp-111(.) in Y122F. According to the proposed consecutive reaction, Trp-111(.) becomes very short lived and is no longer detectable because of the faster formation of Tyr-107(.). We conclude that the phenyl rings of Trp-111 and Tyr-107 form a better stacking complex so that the proton-coupled electron transfer is facilitated compared with the single mutant. Comparison with the formation kinetics of the stable tyrosyl radical in wild type R2 suggests that these protein-linked radicals are substitutes for the missing Tyr-122. However, in contrast to Tyr-122(.) these radicals lack a direct connection to the radical transfer pathway utilized during catalysis.
Syntheses of DL-ferrocenylalanine from ferrocenecarboxyaldehyde by the azlactone method, and, more efficiently, from (ferrocenylmethyl) trimethylammonium iodide via (ferrocenylmethyl)formamidomalonic ester are described. The latter starting material has also been converted into ferrocenyl-acetic and -propionic acid.
Ferrocene is much more reactive towards Friedel-Crafts acylation than either benzene or anisole. The first acyl substituent deactivates even the unsubstituted ring. The high reactivity of ferrocene is utilised in its conversion into its mono-aldehyde by the N-methylformanilide method. This aldehyde shows the typical properties of an aromatic aldehyde; it undergoes the Cannizzaro reaction, has been converted via its oxime into ferrocenyl cyanide, and has also been condensed with malonic acid, with α-picoline methiodide, and, in a mixed benzoin condensation, with benzaldehyde. An alternative preparation of the aldehyde is also described.
Riassunto Si riferisce sulla applicazione delle reazioni di Reformatskii e di Wittig al formil- e all'acetil-ferrocene per preparare i corrispondenti acidi ferrocenilacrilici. Viene anche descritta la condensazione del formilferrocene col nitrometano.
Electron exchange between ferrocene and the ferricinium cation proceeds with half-times of a few milliseconds between -65° and -75°C. The second-order rate constants are best reproduced by calculations based on the Marcus tunnelling theory. Electron exchange between a series of aqua-ammines of CoII and CoIII proceeds through bridged transition states. The energy of activation remains constant in this series but the entropy of activation becomes less negative as ammine ligands are replaced by hydroxo-ligands. Bridging mechanisms may operate in other systems in which substantial reorganization of the primary co-ordination sphere is involved.
1-Ferrocenyl-4,4,4-trifluorobutane-1,3-dione (ferrocenoyltrifluoroacetone, Hfctfa, pKa1 = 6.53 ± 0.03), 4,4,4-trichloro-1-ferrocenylbutane-1,3-dione (trichloroferrocenoylacetone, Hfctca, pKa1 = 7.15 ± 0.02), 1-ferrocenylbutane-1,3-dione (ferrocenoylacetone, Hfca, pKa1 = 10.01 ± 0.02), 1-ferrocenyl-3-phenylpropane-1,3-dione (benzoylferrocenoylmethane, Hbfcm, pKa1 = 10.41 ± 0.02) and 1,3-diferrocenylpropane-1,3-dione (diferrocenoylmethane, Hdfcm, pKa1 = 13.1 ± 0.1) were prepared by Claisen condensation of acetylferrocene with an appropriate ester under the influence of sodium amide, sodium ethoxide or lithium diisopropylamide. The group electronegativity of the ferrocenyl group is 1.87 (Gordy scale) as inferred from a linear β-diketone pKa1–group electronegativity relationship as well as from a linear methyl ester IR carbonyl stretching frequency–group electronegativity relationship. Complexes [Rh(β-diketone)(cod)] were obtained in yields approaching 80% by treating the β-diketones with [Rh2Cl2(cod)2], while the copper(II) chelates form just as readily. Treatment of all [Rh(β-diketone)(cod)] complexes with 1,10-phenanthroline (phen) and some of its derivatives resulted in substitution of the β-diketone ligand to form [Rh(cod)(phen)]+. The uncomplexed β-diketones are increasingly stable towards the OH– nucleophile in the order Hdfcm (apparent most unstable) < Hfctfa < Hbfcm < Hfctca < Hfca (most stable). Asymmetric enolisation in the direction furthest from the ferrocenyl group was observed for all β-diketones. This finding is considered to be the result of resonance driving forces rather than inductive electronic effects of substituents on the pseudo-aromatic β-diketone core.
Dithionite has been found to reduce directly (without mediators) the Escherichia coli R2 subunit of ribonucleotide reductase. With dithionite (∼10 mM) in large excess, the reaction at 25 °C is complete in ∼10 h. Preparations of E. coli R2 have an FeIII2 (met-R2) component in this work at ∼40% levels, alongside the fully active enzyme FeIII2 . . . Tyr*, which has a tyrosyl radical at Tyr-122. In the pH range studied (7–8) the kinetics are biphasic. Rate laws for both phases give [S2O42–] and not [S2O42–]1/2 dependencies, and saturation kinetics are observed for the first time in R2 studies. No dependence on pH was detected. The kinetics (25 °C) of the first phase are reproduced in separate experiments using only met-R2, with association of S2O42– to met-R2, K=330 M–1, occurring prior to electron transfer, k
et=4.8×10–4 s–1, I=0.100 M (NaCl). The second phase assigned to the reaction of FeIII2 . . . Tyr* with S2O42– gives K=800 M–1 and k
et=5.6×10–5 s–1. Bearing in mind the substantially smaller reduction potential for FeIII2 compared to Tyr*, this is a quite remarkable finding, with implications similar to those already reported for the reaction of R2 with hydrazine, but with additional information provided by the saturation kinetics. The similarity in rates for the two phases (∼fourfold difference) suggests that reduction of FeIII2 is occurring in both cases, and since S2O42– is involved a two-equivalent change is proposed with the formation of FeII2 . . . Tyr* in the case of active R2. As a sequel to the second phase, intramolecular reduction of the strongly oxidising Tyr* by the FeII2 is rapid, and further decay of FeIIFeIII is also fast. There is no stable mouse met-R2 form, and the single-phase reaction with dithionite gives saturation kinetics with K=208 M–1 and k
et=1.7±10–3 s–1. Mechanistic implications, including the applicability of a pathway for electron transfer via FeA, are considered.
In light of the cytostatic properties of the ferricenium radical cation against certain murine tumors, it has been of interest to combine the biooxidizable ferrocenyl group with the structural framework of the anticancer agent cisplatin (cis-diamminedichloroplatinum(II)). Dichloro(1,6-diferrocenyl-2,5-diazahexane)platinum(II) (2), and cis-dichlorobis(1-ferrocenylethylamine)platinum(II) (4) are presented as examples of such combination. The synthesis of 2 is brought about by treatment of the 1,2-diamine 1, 1,6-diferrocenyl-2,5-diazahexane, with the PtCl42- anion in aqueous EtOH at pH ≥ 7. The analogous reaction of 1-ferrocenylethylamine (3) with PtCl42- leads to 4, the cis configuration of the amine ligands in this complex being accepted on a tentative basis. At pH < 7 the formation of (substituted) ammonium tetrachloroplatinate(II) salts (such as 5 and 6) is favored as a consequence of the inductive donor properties of the metallocene substituents, which tend to stabilize the protonated amine sites. Spectroscopic data are presented for 1-6 in support of the assigned structures.
The methiodide of N,N-dimethylaminomethylferrocene was treated with potassium cyanide to afford ferrocylacetonitrile. This nitrile was converted to various derivatives. Evidence is presented for the structures of these compounds. An anomalous Leuckart reaction is reported.
Cross-reaction rate constants k12 (22 °C) at pH 7.0 have been determined for the reduction of FeIII2 and tyrosyl-radical-containing active-R2 from E. coli ribonucleotide reductase with eight organic radicals (OR), e.g., MV•+ from methyl viologen. The more reactive OR's were generated in situ using pulse radiolysis (PR) techniques, and other OR's were generated by prior reduction of the parent with dithionite, followed by stopped-flow (SF) studies. In both procedures it was necessary to include consideration of doubly-reduced parent forms. Values of k12 are in the range 109 to 104 M-1 s-1 and reduction potentials Eo1 for the OR vary from −0.446 to +0.194 V. Samples of E. coli active-R2 also have an FeIII2 met-R2 component (with no Tyr•), which in the present work was close to 40%. From separate experiments met-R2 gave similar k12 rate constants (on average 66% bigger) to those for active-R2, suggesting that reduction of the FeIII2 center is the common rate-limiting step. A single Marcus free-energy plot of log k12 − 0.5 log f vs −Eo1/0.059 describes all the data, and the slope of 0.54 is in satisfactory agreement with the theoretical value of 0.50. It is concluded that the rate-limiting step involves electron transfer. In addition, the intercept at −Eo1/0.059 = 0 is 5.94, where values of the reduction potential and self-exchange rate constant for met-R2 contribute to this value. To maintain electroneutrality at the 10 Å buried active site H+ uptake is also required. For both e- and H+ transfer the conserved pathway Trp-48, Asp-237, His-118 to FeA is a possible candidate requiring further examination.
Five ω-ferrocenyl aliphatic acids have been synthesized; these are ferrocenylacetic acid, β-ferrocenylpropionic acid, γ-ferrocenylbutyric acid, δ-ferrocenylvaleric acid and ε-ferrocenylcaproic acid. Synthetic methods employed include the Willgerodt reaction of ferrocenyl ketones, carbethoxylation of acetylferrocene and Friedel-Crafts acylations of ferrocene.
The effect of aliphatic chain length on cyclization has been studied in the series β-ferrocenylpropionic acid (Ib), aγ-ferrocenylbutryic acid (Ic), δ-ferrocenylvaleric acid (Id) and ε-ferrocenylcaproic acid (Ie). The first member gives the heteroannularly bridged 1,1′-(α-ketotrimethylene)-ferrocene (IIb) while the second and third members give the homoannularly cyclized products IIIc and IIId and ε-ferrocenylcaproic acid gives only a polymer (XVII) of low molecular weight. Synthetic methods employed and some of the reactions of the mono-bridged compound are discussed, as are a pair of geometrical isomers (XIVa and XIVb) obtained from ferrocene-1,1′-dibutyric acid (XIII).
Samples of the Escherichia coli R2 protein of ribonucleotide reductase (RNR) normally have two components, the fully active tyrosyl radical (Tyr•) and FeIII2-containing protein (60%) and the FeIII2-only met-R2 form (40%). Reaction with the 1- or multi- (maximum 4-) equiv reagent hydrazine under anaerobic conditions gives biphasic kinetics. From UV−vis absorbance changes, the first stage of reaction corresponds unexpectedly to reduction of the FeIII2 met-R2 component, rate constant 7.4 × 10-3 M-1 s-1 (25 °C) at pH 7.5. The slower second stage is assigned as net reduction of Tyr• and one FeIII of the FeIII2 center, rate constant 1.7 × 10-3 M-1 s-1. Separate experiments with met-R2 protein, and previous evidence from EPR spectroscopy for the formation of an FeIIFeIII intermediate, support such a mechanism. Reduction of the second FeIII is then rapid. The corresponding reduction of the Tyr122Phe R2 variant gives a rate constant of 7.7 × 10-4 M-1 s-1, which is substantially (×10) less than that for met-R2. This is in part explained by the decreased reduction potential of the variant. From pH variations in the range pH 6.6−8.5, N2H4 is the prime reactant with little or no contribution from N2H5+ (pKa 8.2). Phenylhydrazine (250 mV) is unable to reduce the FeIII2 center, and reacts only with the tyrosyl radical (a 1-equiv process) of the active R2 protein (0.184 M-1 s-1). The reaction is >102 times faster than the 2-equiv N2H4 reduction of Tyr• and FeIII2. The pKa for C6H5N2H4+ is 5.27, C6H5N2H3 is the dominant species present under the pH conditions (6.5−8.5) investigated, and no pH dependence is observed. Contrary to a previous report, we conclude that the stability of the diimide (N2H2) does not allow separate studies of the reduction of the R2 protein with this reagent.
The experiment described is a five-period exercise in experimental design and optimization performed in a junior laboratory course that integrates synthesis and analysis. Keywords (Audience): Upper-Division Undergraduate
It is the purpose of this article is to provide a description of cyclic voltammetry and its capabilities. This article is accompanied by an experiment which has been developed to demonstrate important features of CV. Keywords (Audience): Upper-Division Undergraduate
This article details the procedural steps and their theoretical implications for a laboratory in cyclic voltammetry. Keywords (Audience): Upper-Division Undergraduate
A simple experiment is descried here that has become extremely popular in chemical research because it can provide useful information about redox reactions in a form that is easily obtained and interpreted. In this paper the authors present the principles of the method and illustrate its use in the study of four electrode reactions. From State-of-the-Art Symposium: Electrochemistry, ACS meeting, Kansas City, 1982. Keywords (Audience): Graduate Education / Research
Ferriciniumsalze lassen sich auch in saurem Medium nur mit geringer Ausbeute cyanieren, besser mit flüss. Blausäure in Tetrahydrofuran. Das Ferrocen-carbon-säurenitril entsteht aber in 80-proz. Ausb., wenn man das Ferriciniumsalz durch Ferrocen und FeCl3 ersetzt. Bei der gleichen Reaktion mit Alkylferrocen tritt die CN-Gruppe in den substituierten Cyclopentadienyl-Ring; sie sucht den unsubstituierten Ring auf, wenn man vom Monochlorferrocen oder Ferrocen-carbonsäurenitril ausgeht.
Rate constants have been determined for the biphasic reduction of the tyrosyl radical Tyr (κl) and Fe2III (κ2) centres of the R2 protein from E. coli ribonucleotide reductase with two cationic CoII complexes. At 25°C and pH 7.6 the reduction with [Co(sep)]2+ (−300 mV) gives rate constants 0.95 M−1 s−l and 0.0160 M−1 s−1, and the reduction with [Co(9-aneN3)2]2+ (−400 mV) gives rate constants 0.085 M−1 s−1 and 0.0076 M−1 s−1 respectively, where formal reduction potentials (versus nhe) are given in parentheses. The rate constants for the latter reaction are less, in part due to the smaller electron-self-exchange rate constant for the [Co(9-aneN3)2]2+/3+ reaction. In spite of the favourable thermodynamic driving forces the rates applying are extremely slow. There are for example substantial differences in κl values (∼108) compared to reactions with the methyl viologen MV+ (−446 mV) and phenosafranin Pf (−252 mV) radicals. The results indicate the difficulty that cationic complexes have in reducing R2, and unfavourable work terms for the reactions are indicated. The possible involvement of a site previously proposed for electron transfer via the surface Trp-48 is discussed.
Several carboxy-substituted ferrocene compounds are prepared and investigated by cyclic voltammetry in acetonitrile solution. The half-wave potentials of most of the acids studied (E
1/2=0.34–0.58 V versus s.c.e.) are more positive than that of ferrocene (0.33 V), reflecting a diminished susceptibility to oxidation of these compounds relative to the parent metallocene. Only -ferrocenylpropionic acid (0.325 V) is effectively identical with the latter in its oxidation behaviour, and -ferrocenylbutyric acid (0.31 V) tends to be more readily oxidized. The results are of interest for subsequent chemical oxidation studies of ferrocenylcarboxylic acids.
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 FeIII 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 FeIIFeIII 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 FeIIFeIII occurs at a comparable rate to account for the transient behaviour of FeIIFeIII. For mouse R2 the combined FeIII 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
FeIII. 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.
Ribonucleotide reductase from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides. Multiple cysteins have been postulated to play a key role in this process. To test the role of various cysteines in nucleotide reduction, a variety of single and double mutants of the R1 subunit were prepared: C754S, C759S, C754-759S, C462S, C462A, C230S, and C292S. Due to the expression system, each mutant contains small amounts of contaminating wt-R1 (estimated to be 1.5-3% based on activity). An epitope tagging method in conjunction with anion exchange chromatography was used to partially resolve the mutant R1 from the wt-R1. The interaction of these mutants with the normal substrate was studied, which allowed a model to be proposed in which five cysteines of the R1 subunit of RDPR play a role in catalysis. C754S and C759S R1s catalyze CDP formation at rates similar to wt-R1 when DTT is used as a reductant. However, when thioredoxin (TR)/thioredoxin reductase (TRR)/NADPH is used as reductant, the rates of dNDP production are similar to those expected for contaminating wt-R1 present as a heterodimer with the mutant. The impaired nature of these mutants with respect to reduction by TR suggests that their function is to transfer reducing equivalents from TR to the active site disulfide of R1 produced during NDP reduction. Single-turnover experiments, designed to avoid the problem of contaminating wt-R1, also support this role for C754 and C759. The double serine mutant of 754 and 759 has catalytic activity with DTT that is one-third the rate of wt-R1 with thioredoxin. C225 and C462 are thought to be the active site cysteines oxidized concomitantly with NDP reduction. Conversion of these cysteines to serines results in R1 mutants which convert the normal substrate into a mechanism-based inhibitor. C462SR1 upon incubation with R2 and [3'-3H,U-14C]UDP results in uracil release, 3H2O production, 3H,14C-labeled protein which has an absorbance change at 320 nm, and slow loss of the tyrosyl radical on R2. The isotope effect (kH/k3H) on 3' carbon-hydrogen bond cleavage is 1.7. This sequence of events is independent of the reductant, consistent with the postulate that C462 is an active site thiol. The C462AR1 has properties similar to C462SR1. Several additional mutant R1s, C230SR1, and C292SR1 were shown to have activities similar to wt-R1 with both TR/TRR/NADPH and DTT.
Ribonucleotide reductase from Escherichia coli consists of two dissociable, nonidentical homodimeric proteins called R1 and R2. The role of the C-terminal region of R2 in forming the R1R2 active complex has been studied. A heterodimeric R2 form with a full-length polypeptide chain and a truncated one missing the last 30 carboxyl-terminal residues was engineered by site-directed mutagenesis. Kinetic analysis of the binding of this protein to R1, compared with full-length or truncated homodimer, revealed that the C-terminal end of R2 accounts for all of its interactions with R1. The intrinsic dissociation constant of the heterodimeric R2 form, with only one contact to R1, 13 microM, is of the same magnitude as that obtained previously [Climent, I., Sjöberg, B.-M., & Huang, C. Y. (1991) Biochemistry 30, 5164-5171] for synthetic C-terminal peptides, 15-18 microM. We have also mutagenized the only two invariant residues localized at the C-terminal region of R2, glutamic acid-350 and tyrosine-356, to alanine. The binding of these mutant proteins to R1 remains tight, but their catalytic activity is severely affected. While E350A protein exhibits a low (240 times less active than the wild-type) but definitive activity, Y356A is completely inactive. A catalytic rather than structural role for these residues is discussed.
Ribonucleotide reductase is the only enzyme that catalyses de novo formation of deoxyribonucleotides and is thus a key enzyme in DNA synthesis. The radical-based reaction involves five cysteins. Two redox-active cysteines are located at adjacent antiparallel strands in a new type of ten-stranded alpha/beta-barrel, and two others at the carboxyl end in a flexible arm. The fifth cysteine, in a loop in the centre of the barrel, is positioned to initiate the radical reaction.
The crystal structure of the ribonucleotide reductase free radical protein R2 from Escherichia coli has been determined by multiple isomorphous replacement and twofold molecular averaging. The structure has been refined at 2.2 A resolution to R = 0.175. The subunit structure of the R2 protein has a novel fold where the basic motif is a bundle of eight long helices. The R2 dimer has two equivalent dinuclear iron centers. Each iron center is well buried in the subunit. The iron atoms have both histidine and carboxyl acid ligands and are bridged by an oxide ion and the carboxylate group of Glu115. One iron atom is octahedrally coordinated with small deviations from ideal values, while the coordination of the other iron ion is more distorted, mainly due to the fact that Asp84 is a bidental ligand to this iron atom. The oxidation of the enzymatically essential tyrosine residue (Tyr122) and the dinuclear iron center by molecular oxygen is suggested to take part in a suitable conserved oxygen-binding pocket between the iron center and the tyrosine zeta-oxygen 5.3 A away from the closest iron ion. The tyrosine proton can be abstracted by the dioxygen and the deprotonated tyrosine residue is then more easily oxidized to a radical species. Tyr122 is buried inside the protein about 10 A from the surface. This has the consequence that the tyrosyl radical cannot participate directly in hydrogen abstraction from the substrate ribose at the active site of the holoenzyme located on the R1 subunit. The radical must then be indirectly involved in the mechanism of the enzyme and an electron transfer reaction between the active site and the tyrosine must take place. Based on the analysis of the available ribonucleotide reductase sequences, the binding surface for the large ribonucleotide reductase protein R1, and a possible route for an electron transport between the buried radical and this surface is described.
Ribonucleotide reductases (RNRs) catalyze the formation of the deoxyribonucleotides that are essential for DNA synthesis. The R2 subunit of Escherichia coli RNR is a homodimer containing one dinuclear iron centre per monomer. A tyrosyl radical is essential for catalysis, and is formed via a reaction in which the reduced, diferrous form of the iron centre activates dioxygen. To help understand the mechanism of oxygen activation, we examined the structure of the diferrous form of R2.
The crystal structures of reduced forms of both wild type R2 and a mutant of R2 (Ser211--> Ala) have been determined at 1.7 A and 2.2 A resolution, respectively. The diferrous iron centre was compared to the previously determined structure of the oxidized, diferric form of R2. In both forms of R2 the iron centre is coordinated by the same carboxylate dominated ligand sphere, but in the reduced form there are clear conformational changes in three of the carboxylate ligands and the bridging mu-oxo group and two water molecules are lost. In the reduced form of R2 the coordination number decreases from six to four for both ferrous ions, explaining their high reactivity towards dioxygen. The structure of the mutant Ser211--> Ala, known to have impaired reduction kinetics, shows a large conformational change in one of the neighbouring helices although the iron coordination is very similar to the wild type protein.
Carboxylate shifts are often important for carboxylate coordinated metal clusters; they allow the metals to achieve different coordination modes in redox reactions. In the case of reduced R2 these carboxylate shifts allow the formation of accessible reaction sites for dioxygen. The Ser211--> Ala mutant displays a conformational change in the helix containing the mutation, explaining its altered reduction kinetics.
In plants and algae, photosystem II uses light energy to oxidize water to oxygen at a metalloradical site that comprises a
tetranuclear manganese cluster and a tyrosyl radical. A model is proposed whereby the tyrosyl radical functions by abstracting
hydrogen atoms from substrate water bound as terminal ligands to two of the four manganese ions. Molecular oxygen is produced
in the final step in which hydrogen atom transfer and oxygen-oxygen bond formation occur together in a concerted reaction.
This mechanism establishes clear analogies between photosynthetic water oxidation and amino acid radical function in other
enzymatic reactions.
Further to a linear free-energy correlation of cross-reaction rate constants k12 for the reaction of eight organic radicals (OR), e.g. MV*+, from methyl viologen, with cytochrome c(III), we consider here similar studies for the reduction of the R2 protein of Escherichia coli ribonucleotide reductase, which has FeIII2 and Tyr* redox components. The same two techniques of pulse radiolysis and stopped-flow were used. Cross-reaction rate constants (22 degrees C) at pH 7.0, I=0.100 M (NaCl), were determined for the reduction of active-R2 with the eight ORs, reduction potentials E0(1) from -0.446 to +0.194 V. Samples of active-R2 have an FeIII2 met-R2 component, which in the present studies was close to 40%. Concurrent reactions have to be taken into account for the five most reactive ORs, corresponding to reduction of the FeIII2 of met-R2 and then of active-R2. Separate experiments on met-R2 reproduced the first of these rate constants, which on average is approximately 66% larger than the second rate constant. A single Marcus free-energy plot of log k12-0.5 log10f versus -E0(1)/0.059 describes all the data and the slope of 0.54 is in satisfactory agreement with the theoretical value of 0.50. Such behaviour is unexpected since the Tyr* is a much stronger oxidant (E0 approximately 1.0 V versus NHE) as compared to FeIII2 (E0 close to zero). X-ray structures of the met- and red-R2 states have indicated that electroneutrality of the approximately 10 A buried active site is maintained. Proton transfer is therefore proposed as a rapid sequel to electron transfer. Other reactions considered are the much slower conventional time-range reductions of active-R2 with hydrazine and dithionite. For these reactions one and/or two-equivalent changes are possible. With both reductants, met-R2 reacts about four-fold faster than active-R2, and as with the ORs the less strongly oxidising FeIII2 component is reduced before the Tyr*.
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