Reactivity of a cobalt(III)-peroxo complex in oxidative nucleophilic reactions

A mononuclear cobalt(III)-peroxo complex bearing a macrocyclic tetradentate N₄ ligand, [Co^III(TMC)(O₂)]⁺ (TMC = 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane), was generated in the reaction of [Co^II(TMC)]²⁺ and H₂O₂ in the presence of triethylamine in CH₃CN. The reactivity of the cobalt(III)-peroxo complex was investigated in aldehyde deformylation with various aldehydes and compared with that of iron(III)- and manganese(III)-peroxo complexes, such as [Fe^III(TMC)(O₂)]⁺ and [Mn^III(TMC)(O₂)]⁺. In this reactivity comparison, the reactivities of metal-peroxo species were found to be in the order of [Mn^III(TMC)(O₂)]⁺ > [Co^III(TMC)(O₂)]⁺ > [Fe^III(TMC)(O₂)]⁺. A positive Hammett ρ value of 1.8, obtained in the reactions of [Co^III(TMC)(O₂)]⁺ and para-substituted benzaldehydes, demonstrates that the aldehyde deformylation by the cobalt(III)-peroxo species occurs via a nucleophilic reaction.     

The charge-transfer complex between protochlorophyllide and NADPH: an intermediate in protochlorophyllide photoreduction

A hypothesis describing the mechanism of photoactive protochlorophyllide (P) photoreduction in vivo, relating mainly to the molecular nature of the intermediates, is proposed. The hypothesis is compatible with currently published experimental data. After illumination of etiolated barley leaves at 143 to 153 K, the absorption of P remains essentially unchanged, but a new absorption band at 690 nm is observed. Appearance of this new intermediate enables to distinguish between light and dark stages of the photoconversion reaction. When returned to the higher temperature in the dark, the treated leaves begin accumulating chlorophyllide (Chlide), concomitant with the disappearance of the 690-nm band. The decay time of the excited P (P^*) is estimated at 300 ps, which approximates the time constant of photoinduced electron transfer (ET). It is suggested that the charge-transfer complex (CTC) in its ground state (GS) (ground state of CTC formed by the partial (δ) electron transfer), i.e. (P^δ−•••H–D^δ+), between P and NADPH – the electron and proton donor (H–D) – accumulates in the following sequence: P^* + H–D → (P^*•••H–D)→[(P^*•••H–D)←(P⁻•••H–D⁺)] → ¹(P⁻•••H–D⁺)] → ³(P⁻•••H–D⁺) → (P^δ−•••H–D ^δ+), where an equilibrium state (ES) – [(P^*•••H–D)←(P⁻•••H–D⁺)] – with a lifetime of about 1 to 2 ns, exists between the local excited (LE) and ET states. The existence of a triplet ET state – ³(P⁻•••H–D⁺) – is proposed because the time interval between recording of the ES and appearance of the CTC GS (35–250 ns) does not fit the lifetime of the singlet excited complex (exciplex). It is feasible that apart from NADPH, other intermediate proton carriers are contemporaneously involved in the dark reaction (P^δ−•••H–D^δ+) → Chlide, because proton binding to the C₇–C₈ bond in vivo takes place in the trans-configuration. The hydride ion may approach the C₇–C₈ bond from one side by heterolytic fission and an additional proton, donated by the protein group, may be simultaneously added to this bond from the opposite side of the porphyrin nucleus surface. This revised version was published online in June 2006 with corrections to the Cover Date.     

Conformational stabilities, infrared, and vibrational dichroism spectroscopy studies of tris(ethylenediamine) zinc(II) chloride

The conformational stabilities of the transition metal complex of Zn (en)₃Cl₂ were studied using density functional theory (DFT). Deformational potential energy profiles (PEPs), and pathways between the different isomeric conformational energies were calculated using DFT/B3LYP/6–31G. The relative conformational energies of Δ(λλλ), Δ(λλδ), Δ(λδδ) and Δ(δδδ) are 10.48, 7.08, 3.56, and 0.0 kcal/mol, respectively, which are small compared to the barrier heights for reversible phase transitions (49.56, 49.55, 49.52 kcal/mol, respectively). Frequency assignment was carried out by decomposing Fourier transform infrared (FTIR) spectra using Gaussian and Gaussview. The theoretical IR and vibrational dichroism spectroscopy (VCD) absorption spectra are presented for all conformations within the range of 400–3,500 cm^-1.     

Mechanism of peptide hydrolysis by co-catalytic metal centers containing leucine aminopeptidase enzyme: a DFT approach

In this density functional theory study, reaction mechanisms of a co-catalytic binuclear metal center (Zn1–Zn2) containing enzyme leucine aminopeptidase for two different metal bridging nucleophiles (H₂O and –OH) have been investigated. In addition, the effects of the substrate (l-leucine-p-nitroanilide → l-leucyl-p-anisidine) and metal (Zn1 → Mg and Zn2 → Co, i.e., Mg1–Zn2 and Mg1–Co2 variants) substitutions on the energetics of the mechanism have been investigated. The general acid/base mechanism utilizing a bicarbonate ion followed by this enzyme is divided into two steps: (1) the formation of the gem-diolate intermediate, and (2) the cleavage of the peptide bond. With the computed barrier of 17.8 kcal/mol, the mechanism utilizing a hydroxyl nucleophile was found to be in excellent agreement with the experimentally measured barrier of 18.7 kcal/mol. The rate-limiting step for reaction with l-leucine-p-nitroanilide is the cleavage of the peptide bond with a barrier of 17.8 kcal/mol. However, for l-leucyl-p-anisidine all steps of the mechanism were found to occur with similar barriers (18.0–19.0 kcal/mol). For the metallovariants, cleavage of the peptide bond occurs in the rate-limiting step with barriers of 17.8, 18.0, and 24.2 kcal/mol for the Zn1–Zn2, Mg1–Zn2, and Mg1–Co2 enzymes, respectively. The nature of the metal ion was found to affect only the creation of the gem-diolate intermediate, and after that all three enzymes follow essentially the same energetics. The results reported in this study have elucidated specific roles of both metal centers, the nucleophile, indirect ligands, and substrates in the catalytic functioning of this important class of binuclear metallopeptidases.     

Electronic structural investigations of ruthenium compounds and anticancer prodrugs

Several Ru^III compounds are propitious anticancer agents although the precise mechanisms of action remain unknown. With this paper we start to establish an experimental library of X-ray absorption spectroscopy (XAS) data for ten Ru compounds wherein the ligands [Cl⁻, dimethyl sulfoxide, imidazole, and indazole] were varied systematically to provide electronic structural information for future use in correlating spectroscopic signatures with chemical properties. Despite the considerable difference in the coordination environments of the complexes studied, the overall differences in spectral features and electronic structures calculated using density functional theory are unexpectedly small. However, the differences in the electronic structure of the Ru^III prodrugs KP1019 ([IndH][trans-RuCl₄(Ind)₂], Ind is indazole) and ICR ([ImH][trans-RuCl₄(Im)₂], Im is imidazole) observed in the XAS data show correlation with known chemical and biological activities in addition to the donor abilities of imidazole compared with indazole and reduction potentials of the complexes. These semiquantitative results lay the groundwork for future biochemical studies into the structure–function relationships of Ru-based anticancer drugs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Theoretical investigation of tautomerism in N-hydroxy amidines

A DFT study with QST3 approach method is used to calculate kinetic, thermodynamic, spectral and structural data of tautomers and transition state structures of some N-hydroxy amidines. All tautomers and transition states are optimized at the B3LYP/6-311++g** and B3LYP/aug-cc-pvtz level, with good agreement in energetic result with energies obtained from CBS-QB3, a complete basis set composite energy method. The result shows that the tautomer a (amide oxime) is more stable than the tautomer b (imino hydroxylamine) as is reported in the literature. In addition, our finding shows that, the energy difference between two tautomers is only in about 4–10 kcal/mol but the barrier energy found in traversing each tautomer to another one is in the range of 33–71 kcal/mol. Therefore, it is impossible to convert these two tautomers to each other at room temperature. Additionally, transition state theory is applied to estimate the barrier energy and reaction rate constants of the hydrogen exchange between tautomers in presence of 1–3 molecules of water. The computed activation barrier shows us that the barrier energy of solvent assisted tautomerism is about 9–20 kcal/mol and lower than simple tautomerism and this water-assisted tautomerism is much faster than simple tautomerism, especially with the assisting two molecules of water.     

Applications of the ETS-NOCV method in descriptions of chemical reactions

The present study characterizes changes in the electronic structure of reactants during chemical reactions based on the combined charge and energy decomposition scheme, ETS-NOCV (extended transition state–natural orbitals for chemical valence). Decomposition of the activation barrier, ΔE ^#, into stabilizing (orbital interaction, ΔE _orb, and electrostatic, ΔE _elstat) and destabilizing (Pauli repulsion, ΔE _Pauli, and geometry distortion energy, ΔE _dist) factors is discussed in detail for the following reactions: (I) hydrogen cyanide to hydrogen isocyanide, HCN → CNH isomerization; (II) Diels-Alder cycloaddition of ethene to 1,3-butadiene; and two catalytic processes, i.e., (III) insertion of ethylene into the metal-alkyl bond using half-titanocene with phenyl-phenoxy ligand catalyst; and (IV) B–H bond activation catalyzed by an Ir-containing catalyst. Various reference states for fragments were applied in ETS-NOCV analysis. We found that NOCV-based deformation densities (Δρ _i) and the corresponding energies ΔE _orb(i) obtained from the ETS-NOCV scheme provide a very useful picture, both qualitatively and quantitatively, of electronic density reorganization along the considered reaction pathways. Decomposition of the barrier ΔE^# into stabilizing and destabilizing contributions allowed us to conclude that the main factor responsible for the existence of positive values of ΔE ^# for all processes (I, II, III and IV) is Pauli interaction, which is the origin of steric repulsion. In addition, in the case of reactions II, III and IV, a significant degree of structural deformation of the reactants, as measured by the geometry distortion energy, plays an important role. Depending on the reaction type, stabilization of the transition state (relatively to the reactants) originating either from the orbital interaction term or from electrostatic attraction can be of vital importance. Finally, use of the ETS-NOCV method to describe catalytic reactions allows extraction of information on the role of catalysts in determination of ΔE ^#.     

Effect of nitrogen-donor ancillary ligand on oxidation behavior of Co(III) binuclear complexes with conjugated bis(catecholate) ligands

Two binuclear complexes of cobalt(III) have been prepared with 3,3′,4,4′-tetrahydroxy-5,5′-di-tert-butylbenzaldazine (H₄thBu) as bis(catecholate) ligand and two different ancillary ligands, 2,2′-bipyridine (bpy) or 2,2′-dipyridylamine (dpa). These compounds were characterized by ¹H NMR spectra, electrochemical measurements and UV–Vis spectra. In one case, [Co₂(dpa)₄(thBu)]²⁺, electrochemical oxidation of the complexes occurs at the bridges as two closely spaced one-electron couples (E_1/2 = 1 mV and 168 mV versus Fc/Fc⁺). Chemical oxidation of [Co₂(dpa)₄(thBu)]²⁺ using Ag⁺ is observed to occur as a stepwise two-electron process forming [Co₂(dpa)₄(thBu^Cat,SQ)]³⁺ or [Co₂(dpa)₄(thBu^SQ,SQ)]⁴⁺ by UV–Vis spectrum. However, [Co₂(bpy)₄(thBu)]²⁺ shows no change in electronic spectrum under the same conditions of oxidation. This illustrates the dependence of redox properties of the binuclear Co(III) complexes on the nature of the nitrogen-donor ancillary ligands. In this report we discuss the effect of two different nitrogen-donor ancillary ligands on the0 oxidation behavior of binuclear Co(III) complexes.     

Mechanistic insight into the initiation step of the reaction of Burkholderia pseudomallei catalase-peroxidase with peroxyacetic acid

The reaction of the catalase-peroxidase of Burkholderia pseudomallei with peroxyacetic acid has been analyzed using stopped-flow spectrophotometry. Two well-defined species were observed, the first defined by an increase in intensity and narrowing of the Soret band at 407 nm and a 10-nm shift of the charge transfer band from 635 to 625 nm. These features are consistent with a ferric spectrum with a greater proportion of sixth-coordination character and are assigned to an Fe^III–peroxyacetic acid complex. Complementary 9-GHz EPR characterization of the changes in the ferric signal of the resting enzyme induced by the binding of acetate in the heme pocket substantiates the proposal. Kinetic analysis of the spectral changes as a function of peroxyacetic acid concentration revealed two independent peroxyacetic acid binding events, one coincident with formation of the Fe^III–peroxyacetic acid complex and the other coincident with the heme oxidation to the subsequent ferryl intermediate. A model to explain the need for two peroxyacetic acid binding events is proposed. The reaction of the W330F variant followed similar kinetics, although the characteristic spectral features of the Fe^IV=O Por^•+ species were detected. The variant D141A lacking an aspartate at the entrance to the heme cavity as well as the R108A and D141A/R108A variants showed no evidence for the Fe^III–peroxyacetic acid complex, only the formation of ferryl species with absorbance maxima at 414, 545, and 585 nm. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Electrolyte effects on the intervalence transition within discrete binuclear cyano-bridged complexes. An estimation of activation free energy from static, optical and electrochemical data

A study of the metal-to-metal charge-transfer (MMCT) transition within the binuclear cyano-bridged complexes cis-[L¹³Co^III(μ-NC)Fe^II(CN)₅]⁻ (L¹³ = 12-methyl-1,4,7,10-tetraazacyclotridecan-12-amine), trans-[L¹⁴Co^III(μ-NC)Fe^II(CN)₅]⁻ (L¹⁴ = 6-methyl-1,4,8,11-tetraazacyclotetradecan-6-amine) and trans-[L¹⁵Co^III(μ-NC)Fe^II(CN)₅]⁻ (L¹⁵ = 10-methyl-1,4,8,12-tetraazacyclopentadecan-10-amine) has been carried out in electrolyte solutions at varying concentrations. Using these data, as well as the reaction free energies obtained from electrochemical measurements, the reorganisation and activation free energies for the forward and reverse thermal electron-transfer processes have been estimated. The changes of these parameters with the electrolyte concentration, as well as those of the energy of the maximum MMCT band and the reaction free energy, are mainly due to ion-pairing effects.     

Highly efficient photoactivation of Mn-depleted photosystem II by imidazole-liganded manganese complexes

The oxygen-evolving complex (OEC) of Mn-depleted photosystem II (PSII) can be reconstituted in the presence of exogenous Mn or a Mn complex under weak illumination, a process called photoactivation. Synthetic Mn complexes could provide a powerful system to analyze the assembly of the OEC. In this work, four mononuclear Mn complexes, [(terpy)₂Mn^II(OOCH₃)]·2H₂O (where terpy is 2,2′:6′,2″-terpyridine), Mn^II(bzimpy)₂, Mn^II(bp)₂(CH₃CH₂OH)₂ [where bzimpy is 2,6-bis(2-benzimidazol-2-yl)pyridine] and [Mn^III(HL)(L)(py)(CH₃OH)]CH₃OH (where py is pyridine) were used in photoactivation experiments. Measurements of the photoreduction of 2,6-dichorophenolindophenol and oxygen evolution demonstrate that photoactivation is more efficient when Mn complexes are used instead of MnCl₂ in reconstructed PSII preparations. The most efficient recoveries of oxygen evolution and electron transport activities are obtained from a complex, [Mn^III(HL)(L)(py)(CH₃OH)]CH₃OH, that contains both imidazole and phenol groups. Its recovery of the rate of oxygen evolution is as high as 79% even in the absence of the 33-kDa peptide. The imidazole ligands of the Mn complex probably accelerate P ₆₈₀ ^•+ reduction and consequently facilitate the process of photoactivation. Also, the strong intermolecular hydrogen bond probably facilitates interaction with the Mn-depleted PSII via reorganization of the hydrogen-bonding network, and therefore promotes the recovery of oxygen evolution and electron transport activities.     

Syntheses and characterization of vitamin B<Subscript>12</Subscript>–Pt(II) conjugates and their adenosylation in an enzymatic assay

Aiming at the use of vitamin B₁₂ as a drug delivery carrier for cytotoxic agents, we have reacted vitamin B₁₂ with trans-[PtCl(NH₃)₂(H₂O)]⁺, [PtCl₃(NH₃)]⁻ and [PtCl₄]²⁻. These Pt(II) precursors coordinated directly to the Co(III)-bound cyanide, giving the conjugates [{Co}–CN–{trans-PtCl(NH₃)₂}]⁺ (5), [{Co}–CN–{trans-PtCl₂(NH₃)}] (6), [{Co}–CN–{cis-PtCl₂(NH₃)}] (7) and [{Co}–CN–{PtCl₃}]⁻ (8) in good yields. Spectroscopic analyses for all compounds and X-ray structure elucidation for 5 and 7 confirmed their authenticity and the presence of the central “Co–CN–Pt” motif. Applicability of these heterodinuclear conjugates depends primarily on serum stability. Whereas 6 and 8 transmetallated rapidly to bovine serum albumin proteins, compounds 5 and 7 were reasonably stable. Around 20% of cyanocobalamin could be detected after 48 h, while the remaining 80% was still the respective vitamin B₁₂ conjugates. Release of the platinum complexes from vitamin B₁₂ is driven by intracellular reduction of Co(III) to Co(II) to Co(I) and subsequent adenosylation by the adenosyltransferase CobA. Despite bearing a rather large metal complex on the β-axial position, the cobamides in 5 and 7 are recognized by the corrinoid adenosyltransferase enzyme that catalyzes the formation of the organometallic C–Co bond present in adenosylcobalamin after release of the Pt(II) complexes. Thus, vitamin B₁₂ can potentially be used for delivering metal-containing compounds into cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Spectrophotometric kinetic study and analytical implications of the glucose oxidase-catalyzed reduction of [MIII(LL)2Cl2]+ complexes by d-glucose (M=Os and Ru, LL=2,2′-bipyridine and 1,10-phenanthroline type ligands)

 Glucose oxidase-catalyzed reduction of cis[M^III (LL)₂Cl₂]⁺ (M=Os and Ru) complexes to cis[M^II (LL)₂Cl₂] (LL=2,2′-bipyridine and 1,10-phenanthroline type ligands) by d-glucose is a first-order process in the complex and the enzyme in aqueous buffered solution. The reaction follows MichaelisMenten kinetics in d-glucose and the rate is independent of d-glucose concentration above 0.03 M. The reactivity decreases in the series [Ru(bpy)₂Cl₂]⁺ > [Os(phen)₂Cl₂]⁺ > [Os(4,4′-Me₂bpy)₂Cl₂]⁺ > [Os(4,7Me₂phen)₂Cl₂]⁺. The measured second-order rate constant for the oxidation of reduced glucose oxidase by [Os(phen)₂Cl₂]⁺ in air equals 1.2×10⁵ M^–1 s^–1 at pH 6.7, [d-glucose] 0.05 M, and 25  °C, which is ca. 20% less than that when the reaction solutions are purged with argon. In the case of [Ru(bpy)₂Cl₂]⁺ the rate constant equals 1.8×10⁵ M^–1 s^–1 under similar conditions in air, showing higher reactivity of Ru complexes compared with Os ones. The reduction is pH-dependent with a maximum around 7. Added for solubilization of poorly soluble metal complexes, surfactants decrease the rates of the enzymatic reaction. The retardation effect increases in the series: cetyltrimethylammonium bromide < Triton X-100 < sodium dodecyl sulfate, i.e. on going from positively charged to neutral and then to negatively charged surfactants. The behavior of the Os^III and Ru^III complexes toward reduced glucose oxidase contrasts to that of recently studied ferricenium cations. As opposed to the latter, the former do not show kinetically meaningful binding with the enzyme, and the Michaelis kinetics typical of the ferricenium case is not realized for the Os^III, and Ru^III species. The systems Os^III- or Ru^III-glucose oxidase are convenient for routine "one pot" spectrophotometric monitoring of the d-glucose content in samples, since the metal reduction to M^II is accompanied by a strong increase in absorbance in the visible spectral region. Received: 1 July 1998 / Accepted: 13 January 1999     

Density functional theory studies of model complexes for molybdenum-dependent nitrate reductase active sites

Molybdenum and tungsten complexes as models for the active sites of assimilatory or dissimilatory nitrate reductases (NR) were computed at the CPCM-B98/SDDp//B3LYP/Lanl2DZp* plus zero point energy level of density functional theory. The ligands were chosen on the basis of available experimental protein or small chemical model structures. A water molecule is found to bind to assimilatory NR models [(Me₂C₂S₂)MO(YMe)]⁻ (−11.5 kcal mol⁻¹ for M is Mo, Y is S) and may be replaced by nitrate (−4.5 kcal mol⁻¹) (but a hydroxy group may not). Nature’s choice of M is Mo and Y is S for NR has the largest activation energy for protein-free models (13.3 kcal mol⁻¹) and the least exothermic reaction energy for the nitrate reduction (−14.9 kcal mol⁻¹) compared with M is W and Y is O or Se alternatives. Water binding to dissimilatory NR model complexes [(Me₂C₂S₂)₂M(YR)]⁻ is considerably endothermic (10.3 kcal mol⁻¹); nitrate binding is only slightly so (1.5 kcal mol⁻¹ for RY⁻ is MeS⁻). The exchange of an oxo ligand (assimilatory NR) for a dithiolato ligand (dissimilatory NR model) reduces the exothermicity (−8.6 kcal mol⁻¹ relative to the fivefold-coordinate reduced complex) and raises the barrier for oxygen atom transfer (OAT) in the nitrate complex (19.2 kcal mol⁻¹). Not for the mono but only for the bisdithiolato complexes hydrogen bonding involving the coordinated substrate may significantly lower the OAT barrier as shown by explicitly adding water molecules. Substitution of tungsten for molybdenum generally lowers OAT activation energies and makes nitrate reduction reaction energies more negative. Bidentate carboxylato binding identified in Escherichia coli NarGHI is the preferred binding mode also for an acetato model. However, one dithiolato ligand folds when the Mo^VI center is bare of a good π-donor ligand, e.g., an oxo group. Computations on [(mnt)₂Mo^IV(YR)(PPh₃)]⁻ [mnt is (CN)₂C₂S₂ ²⁻] gave a smaller nitrate reduction activation energy for RY⁻ is Cl⁻, compared with RY⁻ is PhS⁻, although experimentally only the phenyl thiolato complex and not the chloro complex was found to be a functional NR model. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structure and magnetic properties of LnIII[Ru2(CO3)4] · 8H2O

Ln^III[Ru₂(CO₃)₄] · 8H₂O (Ln = Gd, Nd, Ho, Yb) is formed from the reaction of Ln^III and [Ru₂(CO₃)₄]^3? in water. These Ln^III materials have a 3D network structure composed of linked chains and μ_n-CO₃ linkages to both Ru and Ln^III sites, and are best described as Ln^III(OH₂)₄[Ru₂(CO₃)₄]_1/2[Ru₂(CO₃)₄(OH₂)₂]_1/2 · 3H₂O. Complete characterization of the Gd^III species is presented, as the other Ln^III are isostructural and exhibit large spin–orbit coupling leading to complex magnetic behavior. Magnetic ordering is not observed above 2 K.     

Purification and Characterisation of Rat Kidney Glutathione Reductase

Glutathione reductase [GR, E.C.1.8.1.7] catalyses NADPH dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH). Thus, it is the crucial enzyme to maintain high [GSH]/[GSSG] ratio and physiological redox status in cells. Kidney and liver tissues were considered as a rich source of GR. In this study, rat kidney GR was purified and some of its properties were investigated. The enzyme was purified 2,356 fold with a yield of 16% by using heat-denaturation and Sephadex G25 gel filtration, 2′,5′-ADP Agarose 4B, PBE94 column chromatographies. The purified enzyme had a specific activity (Vm) of 250 U/mg protein and the ratio of absorbances at wavelengths of A ₂₇₃/A _463, A ₂₈₀/A ₄₆₀, A ₃₆₅/A ₄₆₀, and A ₃₇₉/A ₄₆₃, were 7.1, 6.8, 1.2 and 1.0, respectively. Each mol of GR subunit bound 0.97 mol of FAD. NADH was used as a coenzyme by rat kidney GR but with a lower efficiency (32.7%) than NADPH. Its subunit molecular weight was estimated as 53 kDa. An optimum pH of 6.5 and optimum temperature of 65 °C were found for rat kidney GR. Its activation energy (Ea) and temperature coefficient (Q₁₀) were calculated as 7.02 kcal/mol and 1.42, respectively. The Km_(NADPH) and kcat/Km _(NADPH) values were found to be 15.3 ± 1.4 μM and 1.68 × 10⁷ M⁻¹ s⁻¹ for the concentration range of 10-200 μM NADPH and when GSSG is the variable substrate, the Km_(GSSG) and the kcat/Km_(GSSG) values of 53.1 ± 3.4 μM and 4.85 × 10⁶ M⁻¹ s⁻¹ were calculated for the concentration range of 20–1,200 μM GSSG.     

Decomposition Reaction of Dioxetanone in Firefly Bioluminescence by Computer Experiment

Firefly luciferin (Ln) reacts with molecular oxygen in the presence of the enzyme luciferase (E), the Mg⁺² ion and ATP to form a four-membered cyclic peroxide, so-called dioxetanone, which has not yet been observed by spectrophotometric techniques. Subsequently, dioxetanone decomposes into carbon dioxide (CO₂) and electronically excited oxyluciferin (Oxyln^−∗), emitting yellow-green light. In order to clarify the characteristics of the elementary reaction path from dioxetanone to Oxyln^−∗, the potential energy curve of the singlet ground-state (S₀-PEC) along the reaction coordinate was obtained by the intrinsic reaction coordinate (IRC) calculations using the AM1 Hamiltonian. Furthermore, the potential energy curve of the singlet excited-state (S₁-PEC) was calculated, because dioxetanone decomposes to Oxyln^−∗ along the reaction coordinate. The S₁-PEC relative to S₀-PEC was estimated at each point of the reaction coordinate using the INDO/S, where only the singly-excited configuration interactions (CI) constructed from 20 occupied and 20 unoccupied molecular orbitals (MOs) were considered. As a result of these calculations, it was concluded that (1) firefly dioxetanone might not be an intermediate but rather be in an unstable transition state; (2) the S₀-PEC has an activation barrier of 37.5 kcal/mol for dioxetanone formation and the reaction is exothermic along the S₀-PEC; (3) the S₁-PEC approaches the S₀-PEC in a concave manner where dioxetanone decomposes to efficiently produce Oxyln^−∗; and (4) rupturing of an O–O bond in dioxetanone can trigger the coming and going of electrons in a “cradle" motion mediated by S₀- and S₁-PECs in the chemiexcitation step toward Oxyln^−∗.     

Mechanism of dithionite reduction of the R2 protein of E. coli and mouse ribonucleotide reductase: evidence for reduction of FeIII 2 prior to the tyrosyl radical

 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 Fe^III ₂ (met-R2) component in this work at ∼40% levels, alongside the fully active enzyme Fe^III ₂ . . . 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 [S₂O₄ ^2–] and not [S₂O₄ ^2–]^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 S₂O₄ ^2– 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 Fe^III ₂ . . . Tyr* with S₂O₄ ^2– gives K=800 M^–1 and k _et=5.6×10^–5 s^–1. Bearing in mind the substantially smaller reduction potential for Fe^III ₂ 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 Fe^III ₂ is occurring in both cases, and since S₂O₄ ^2– is involved a two-equivalent change is proposed with the formation of Fe^II ₂ . . . Tyr* in the case of active R2. As a sequel to the second phase, intramolecular reduction of the strongly oxidising Tyr* by the Fe^II ₂ is rapid, and further decay of Fe^IIFe^III 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 Fe_A, are considered. Received: 25 February 1998 / Received: 20 August 1998     

Kinetics and mechanism of the reduction of (ImH)[<Emphasis Type="Italic">trans</Emphasis>-RuCl<Subscript>4</Subscript>(dmso)(Im)] by ascorbic acid in acidic aqueous solution

A systematic study of the reduction of (ImH)[trans-RuCl₄(dmso)(Im)] (NAMI-A; dmso is dimethyl sulfoxide, Im is imidazole), a promising antimetastasing agent entering phase II clinical trial, by l-ascorbic acid is reported. The rapid reduction of trans-[Ru^IIICl₄(dmso)(Im)]⁻ results in formation of trans-[Ru^IICl₄(dmso)(Im)]²⁻ in acidic medium (pH = 5.0) and is followed by successive dissociation of the chloride ligands, which cannot be suppressed even in the presence of a large excess of chloride ions. The reduction of NAMI-A strongly depends on pH and is accelerated on increasing the pH. Over the small pH range 4.9−5.1, the reaction is quite pH-independent and the influence of temperature and pressure on the reaction could be studied. On the basis of the reported activation parameters and other experimental data, it is suggested that the redox process follows an outer-sphere electron transfer mechanism. A small contribution from a parallel reaction ascribed to inner-sphere reduction of aqua derivatives of NAMI-A, was found to be favored by lower concentrations of the NAMI-A complex and higher temperature. In the absence of an excess of chloride ions, the reduction process is catalyzed by the Ru(II) products being formed. The reduction of NAMI-A is also catalyzed by Cu(II) ions and the apparent catalytic rate constant was found to be 1.5 × 10⁶ M⁻² s⁻¹ at 25 °C. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.