Trans-pyridine tetraammine complexes of RuII and RuIII with N7-coordninated purine nucleosides

 The synthesis, spectroscopic, and electrochemical properties of trans-[L(Pyr)(NH₃)₄Ru^II/III] (Pyr=py, 3-phpy, 4-phpy, 3-bnpy, or 4-bnpy; L=H₂O, Guo, dGuo, 1MeGuo, Gua, Ino, or G⁷-DNA) are reported. As expected, the Pyr ligand slows DNA binding by trans-[(H₂O)(Pyr)(NH₃)₄Ru^II]²⁺ relative to [(H₂O)(NH₃)₅Ru^II]²⁺ and favors reduction of Ru^III by about 150 mV. The pyridine ligand also promotes the disproportionation of Ru^III to afford the corresponding complexes of Ru^II and, presumably, Ru^IV. For L=Ino, disproportionation follows the rate law: d[Ru^II]/dt=k ₀[Ru^III]+k ₁[OH^–][Ru^III], k ₀=(2.7±0.7)×10^–4s^–1 and k ₁=70±1 M^–1s^–1. Received: 11 May 1998 / Accepted: 3 March 1999     

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     

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     

Kinetic studies on the reactions of Fusarium galactose oxidase with five different substrates in the presence of dioxygen

 Reactions (25  °C) of galactose oxidase, GOase_ox from Fusarium NRRL 2903 with five different primary-alcohol-containing substrates RCH₂OH:- D-galactose (I) and 2-deoxy-d-galactose (II) (monosaccharides); methyl-β-d-galactopyranoside (III) (glycoside);d-raffinose (IV) (trisaccharide); and dihydroxyacetone (V) have been studied in the presence of O₂. The GOase_ox state has a tyrosyl radical coordinated at a square-pyramidal Cu^II active site, and is a two-equivalent oxidant. Reactant concentrations were [GOase_ox] (0.8–10 μM), RCH₂OH (1.0–6.0 mM), and O₂ (0.14–0.29 mM), with I=0.100 M (NaCl). The reactions, monitored at 450 nm by stopped-flow spectrophotometry, terminated with depletion of the O₂. Each trace was fitted to the competing reactions GOase_ox+RCH₂ OH → GOase_redH₂+RCHO (k ₁), and GOase_redH₂+O₂→ GOase_ox+H₂O₂ (k ₂), with GOase_redH₂ written as the doubly protonated two-electron-reduced Cu^I product. It was necessary to avoid auto-redox interconversion of GOase_ox and GOase_semi . Information obtained at pH 7.5 indicates a 5 : 95 (ox : semi) "native" mix equilibration complete in ∼3 h. At pH >7.5, rate constants 10^–4 k ₁ / M^–1 s^–1 for the reactions of GOase_ox with (I) (1.19), (II) (1.07), (III) (1.29), (IV) (1.81), (V) (2.94) were determined. On decreasing the pH to 5.5, k ₁ values decreased by factors of up to a half, and acid dissociation pK _as in the range 6.6–6.9 were obtained. UV-Vis spectrophotometric studies on GOase_ox gave an independently determined pK _a of 6.7. No corresponding reactions of the Tyr495Phe variant were observed, and there are no similar UV-Vis absorbance changes for this variant. The pK _a is therefore assigned to protonation of Tyr-495 which is a ligand to the Cu. The rate constant k ₂ (1.01×10⁷ M^–1 s^–1) is independent of pH in the range 5.5–9.0 investigated, suggesting that H⁺ (or H-atoms) for the O₂ → H₂O₂ change are provided by the active site of GOase_red . The Cu^I of GOase_red is less extensively complexed, and a coordination number of three is likely. Received: 4 February 1997 / Accepted: 16 May 1997     

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

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

Kinetic studies of electron transfer in the cyt b 5 : metHb system

 The kinetics of methemoglobin reduction by cytochrome b ₅ has been studied by stopped-flow and saturation transfer NMR. A forward rate constant k _f = 2.44×10⁴ M^–1 s^–1 and a reverse rate constant k _b = 540 M^–1s^–1 have been observed at 10 mm, pH 6.20, 25  °C. The ratio k _f/k _b = k _eq = 43.6 is in good agreement with the equilibrium constant calculated from the electrochemical potential between cyt b ₅ and methemoglobin. A bimolecular collisional mechanism is proposed for the electron transfer from cyt b ₅ to methemoglobin based on the kinetic data analysis. The dependence of the rate constants on ionic strengths supports such collisional mechanism. It is also found that the reaction rate strongly depends on the conformations of methemoglobin. Received: 20 February 1996 / Accepted: 4 June 1996     

Electrochemistry of the CuA domain of Thermus thermophilus cytochrome ba 3

 The electrochemistry of a water-soluble fragment from the Cu_A domain of Thermus thermophilus cytochrome ba ₃ has been investigated. At 25  °C, Cu_A exhibits a reversible reduction at a pyridine-4-aldehydesemicarbazone-modified gold electrode (0.1 M Tris, pH 8) with E° = 0.24 V vs NHE. Thermodynamic parameters for the [Cu(Cys)₂Cu]^+/0 electrode reaction were determined by variable-temperature electrochemistry (ΔS°_rc = –5.4(12) eu, ΔS° = –21.0(12) eu, ΔH° = –11.9(4) kcal/mol;ΔG° = –5.6 (11) kcal/mol). The relatively small reaction entropy is consistent with a low reorganization energy for [Cu(Cys)₂Cu]^+/0 electron transfer. An irreversible oxidation of [Cu(Cys)₂Cu]⁺ at 1 V vs NHE confirms that the Cu^II:Cu^II state of Cu_A is significantly destabilized relative to the Cu^II state of analogous blue-copper proteins. Received: 3 June 1996 / Accepted: 26 August 1996     

Steady-state kinetics, micellar effects, and the mechanism of peroxidase-catalyzed oxidation of n -alkylferrocenes by hydrogen peroxide

 Kinetics of the steady-state oxidation of n–alkylferrocenes (alkyl = H, Me, Et, Bu and C₅H₁₁) by H₂O₂ to form the corresponding ferricenium cations catalyzed by horseradish peroxidase has been studied in micellar systems of Triton X-100, CTAB, and SDS, mostly at pH 6.0 and 25  °C. The rate of oxidation of ferrocenes with longer alkyl radicals is too slow to be measured. The reaction obeying the [RFc]:[H₂O₂] = 2 : 1 stoichiometry is strictly first-order in both HRP and RFc in a wide concentration range. The corresponding observed second-order rate constants k, which refer to the interaction of the peroxidase compound II (HRP-II) with RFc, decrease with the elongation of the alkyl substituent R, and this in turn is accompanied by an increase in the formal redox potentials E°′ in the same medium. Increasing the surfactant concentration lowers the rate constants k, the effect being due to the nonproductive binding of RFc to micelles rather than to enzyme inactivation. The micellar effects are accounted for in terms of the Berezin pseudo-phase model of micellar catalysis applied to the interaction of enzyme with organometallic substrates. The oxidation was found to occur primarily in the aqueous pseudo-phase and the calculated intrinsic second-order rate constants k _w are (1.9 ± 0.5)×10⁵, (2.7 ± 0.1)×10⁴, and (5.9 ± 0.6)×10³ M^–1 s^–1 for HFc, EtFc, and n–BuFc, respectively. The data obtained were used for estimating the self-exchange rate constants for the HRP-II/HRP couple in terms of the Marcus formalism. Received: 15 July 1996 / Accepted: 15 November 1996     

Intramolecular electron transfer in the oxidation of amines by methylamine oxidase from Arthrobacter P1

 The intramolecular electron-transfer rate constant for the Cu(II)–topa_NH2⇌ Cu(I)–topa_SQ equilibrium in methylamine oxidase has been measured by temperature-jump relaxation techniques. At pH 7.0 the estimated k_obs = 150±30 s^–1 for both methylamine and benzylamine; assuming the equilibrium constant is ≈0.7–1 at pH 7.0 and 296 K, this would correspond to a forward electron-transfer rate constant k_ET≈ 60–75 s^–1. Although substantially slower than the previously determined k_ET≈ 20 000 s^–1 for pea seedling amine oxidase [5] steady-state kinetics measurements established that k_ET > k_cat≈ 4–10 s^–1. Thus the Cu(I)-semiquinone state is a viable intermediate in methylamine oxidase turnover. Received: 16 August 1995 / Accepted: 21 December 1995     

Kinetic study of DNA binding of cisplatin and of a bicycloalkyl-substituted (ethylenediamine)dichloroplatinum(II) complex

 Salmon sperm DNA platination has been conducted under strictly pseudo-first-order conditions with cisplatin (1) and rac-{(1S,2S,4S)-exo-2-(aminomethyl)-2-amino-7-bicyclo[2.2.1]heptane}dichloroplatinum(II) (2). An aquation step first occurs for both complexes, with the rate constants k ₁ = 1.12(0.02)×10^–4 s^–1 and 1.47(0.02)×10^–4 s^–1 respectively for 1 and 2 at 37  °C, values in agreement with those previously reported. It is followed by the actual platination step whose second-order rate constant has been determined for the first time by physicochemical techniques. The values for 1 and 2 respectively are: k ₂ = 2.08(0.07) M^–1 s^–1 and 3.9(0.4) M^–1 s^–1. These kinetic data are discussed in the context of a comparison of several biological properties of the two complexes. Received: 15 May 1998 / Accepted: 26 June 1998     

Biochemical effects of binding [(H2O)(NH3)5RuII]2+ to DNA and oxidation to[(NH3)5RuIII]n—DNA.

The reaction of [(H₂O)(NH₃)₅Ru^II]²⁺ with calf thymus and salmon sperm DNA has been studied over a wide fange of transition metal ion concentrations. Kinetic studies revealcd a biphasic reaction with an initial fairly rapid coordination of the metal ion being followed by slower reactions. Binding studies were done under pseudo-equilibrium conditions following completion of the initial rapid reaction. Spectra and HPLC of acid-hydrolyzed samples of [(NH3)₅Ru^II]n-DNA prepared by incubation of [(H₂O)(NH₃)₅Ru^II]²⁺ with DNA (where [P_DNA] = 1.5 mM and reactant [Ru^II]/[P_DNA] ratios were in the fange 0.1 to 0.3) followed by air oxidation showed the predominant binding site on helical DNA to be in the major groove at the N-7 of guanine. The equilibrium constant for [(H₂O)(NH₃)₅Ru^II]²⁺ binding to the G⁷ site in helical CT DNA is 5.1 x 103. Differential pulse voltammetry exhibited a single peak at 48 mV, which is attributed to the reduction of Rum on the G⁷ sites.At [Run]/[P_DNA] <0.5, Tm values for the DNA decreased linearly with increasing ruthenium concentration and an increase in the intensity of the 565 nm dG→ Ru(III) charge transfer band was noted upon melting. The UV and CD spectra of these samples indicated no extensive destacking or alteration in geometry (B family) compared to unsubstituted DNA. At [Run]/[P_DNA]〉 0.5 or when single-stranded DNA was used, increased absorbance at 530 nm and 480 nm suggested additional binding to the exocyclic amine sites of adenine and cytosine residues. HPLC and individual spectrophotometric identification of the products derived from hydrolysis of these spec~es yielded both [(Gua)(NH₃)₅Ru^III] and [(Ade)(NH₃)₅Ru^III]. Earlier studies have established the cytidine and adenosine binding sites of [(NH₃)₅Ru^III] to be at their exocyclic amines (C4 and A6). Coordination to these positions indicates disruption of the double helix since these amines are involved in hydrogen bonding on the interior of B-DNA.Agrose gel electrophoresis of superhelical pBR322 plasmid DNA after exposure to various complexes of [(nh₃)₅Ruⁱⁱⁱ] in the presence of a reductant and air generally revealed moderately efficient cleavage of the DNA, presumably due to the generation of hydroxyl radical via Fenton's chemistry. However, similar studies involving [(NH₃)₅Ru^III] directly coordinated to the DNA showed no strand cutting above background. Polyacrylamide gel electrophoresis of a 381 bp, 3′-³²P-labeled fragment of pBR322 plasmid DNA containing low levels of bound [(NH₃)₅ Ru^III] further indicated negligible DNA cutting by the coordinated metal ion.     

Characterization of the cadmium complex of peptide 49–61: a putative nucleation center for cadmium-induced folding in rabbit liver metallothionein IIA

 The synthetic peptide fragment containing residues 49–61 of rabbit liver metallothionein II (MT-II) (Ac-Ile-Cys-Lys-Gly-Ala-Ser-Asp-Lys-Cys-Ser-Cys-Cys-Ala-COOH), which includes the only sequential four cysteines bound to the same metal ion in Cd₇MT, forms a stable, monomeric Cd-peptide complex with 1 : 1 stoichiometry (Cd:peptide) via Cd-thiolate interactions. This represents the first synthesis of a single metal-binding site of MT independent of the domains. The ¹¹¹Cd NMR chemical shift at 716 ppm indicates that the ¹¹¹Cd²⁺ in the metal site is terminally coordinated to four side-chain thiolates of the cysteine residues. The pH of half dissociation for this Cd-peptide derivative, ∼3.3, demonstrates an affinity similar to that for Cd₇MT. Molecular mechanics calculations show that the thermodynamically most stable folding for this isolated Cd²⁺ center has the same counterclockwise chirality (Λ or S) observed in the native holo-protein. These properties are consistent with its proposed role as a nucleation center for cadmium-induced protein folding. However, the kinetic reactivity of the CdS₄ structure toward 5,5′-dithiobis(5-nitrobenzoate) (DTNB) and EDTA is greatly increased compared to the complete cluster (α-domain or holo-protein). The rate law for the reaction with DTNB is rate=(k _uf +k _1,f +k _2,f [DTNB])[peptide], where k _uf=0.15 s^–1, k _1,f=2.59×10^–3s^–1, and k _2,f=0.88 M^–1s^–1. The ultrafast step (uf), observable only by stopped-flow measurement, is unprecedented for mammalian (M₇MT) and crustacean (M₆MT) holo-proteins or the isolated domains. The accommodation of other metal ions by the peptide indicates a rich coordination chemistry, including stoichiometries of M-peptide for Hg²⁺, Cd²⁺, and Zn²⁺, M₂-peptide for Hg²⁺ and Au⁺, and (Et₃PAu)₂-peptide. Received: 9 December 1998 / Accepted: 20 May 1999     

Metal-metal bonding and charge localisation in [Ru2X9], X=Cl or Br; n=1, 2, 3, 4. A spectroelectrochemical study

A spectroelectrochemical study of [Ru₂X₉]ⁿ⁻, X=Cl, Br; n=1, 2, 3, 4 has been undertaken. Stable solutions of n=4, 2, 1 can be formed by electrolysis at low temperatures. Analysis of the Vis-NIR spectra of the complexes indicate that the Ru^II---R^III dimers (n=4) have delocalised mixed valence and that the Ru^III---R^III (n=3) dimers have a strong Ru---Ru bond. The more oxidised materials do not form a Ru---Ru bond; the spectroscopic data indicates the Ru^III---R^IV dimers have localised valency.     

Direct assessment of water exchange on a Gd(III) chelate bound to a protein

 The Gd(III) complex of 4-pentylbicyclo[2.2.2]octane-1-carboxyl-di-l-aspartyl-lysine-derived DTPA, [GdL(H₂O)]^2–, binds to serum albumin in vivo, through hydrophobic interaction. A variable temperature ¹⁷O NMR, EPR, and Nuclear Magnetic Relaxation Dispersion (NMRD) study resulted in a water exchange rate of k ²⁹⁸ _ex=4.2×10⁶ s^–1, and let us conclude that the GdL complex is identical to [Gd(DTPA)(H₂O)]^2– in respect to water exchange and electronic relaxation. The effect of albumin binding on the water exchange rate has been directly evaluated by ¹⁷O NMR. Contrary to expectations, the water exchange rate on GdL does not decrease considerably when bound to bovine serum albumin (BSA); the lowest limit can be given as k _{ex, GdL-BSA}=k _ex, GdL / 2. In the knowledge of the water exchange rate for the BSA-bound GdL complex, the analysis of its NMRD profile at 35  °C yielded a rotational correlation time of 1.0 ns, one order of magnitude shorter than that of the whole protein. This value is supported by the longitudinal ¹⁷O relaxation rates. This indicates a remarkable internal flexibility, probably due to the relatively large distance between the protein- and metal-binding moieties of the ligand. Received: 25 June 1998 / Accepted: 11 August 1998     

Recombinant horseradish peroxidase isoenzyme C: the effect of distal haem cavity mutations (His42→Leu and Arg38→Leu) on compound I formation and substrate binding

 Horseradish peroxidase isoenzyme C (HRPC) mutants were constructed in order to understand the role of two key distal haem cavity residues, histidine 42 and arginine 38, in the formation of compound I and in substrate binding. The role of these residues as general acid-base catalysts, originally proposed for cytochrome c peroxidase by Poulos and Kraut in 1980 was assessed for HRPC. Replacement of histidine 42 by leucine [(H42L)HRPC*] decreased the apparent bimolecular rate constant for the reaction with hydrogen peroxide by five orders of magnitude (k ₁ = 1.4×10² M^–1s^–1) compared with both native-glycosylated and recombinant forms of HRPC (k ₁ = 1.7×10⁷ M^–1s^–1). The first-order rate constant for the heterolytic cleavage of the oxygen-oxygen bond to form compound I was estimated to be four orders of magnitude slower for this variant. Replacement of arginine 38 by leucine [(R38L)HRPC*] decreased the observed pseudo-first-order rate constant for the reaction with hydrogen peroxide by three orders of magnitude (k ₁ = 1.1×10⁴ M^–1s^–1), while the observed rate constant of oxygen bond scission was decreased sixfold (k ₂ = 142 s^–1). These rate constants are consistent with arginine 38 having two roles in catalysing compound I formation: firstly, promotion of proton transfer to the imidazole group of histidine 42 to facilitate peroxide anion binding to the haem, and secondly, stabilisation of the transition state for the heterolytic cleavage of the oxygen-oxygen bond. These roles for arginine 38 explain, in part, why dioxygen-binding globins, which do not have an arginine in the distal cavity, are poor peroxidases. Binding studies of benzhydroxamic acid to (H42L)HRPC* and (R38L)HRPC* indicate that both histidine 42 and arginine 38 are involved in the modulation of substrate affinity. Received: 21 July 1995 / Accepted: 27 November 1995     

Type I blue copper proteins as enantioselective quenchers of the photoluminescence of Δ,Λ-Eu(pyridine-2,6-dicarboxylate)3 3–: azurin from Pseudomonas aeruginosa and its Met44→Lys mutant, amicyanin from Paracoccus versutus and parsley plastocyanin

 The dynamic quenching of the luminescence of racemic Eu(III)(pyridine-2,6-dicarboxylate=dpa)₃ ^3– by the title proteins is investigated and the enantioselectivity of the proteins in the quenching of the Δ and Λ enantiomers of Eu(dpa)₃ ^3– is determined. The two diastereomeric quenching rate constants pertaining to azurin (k _q ^Δ=3.3×10⁶, k _q ^Λ=2.7×10⁶ M^–1s^–1, pH 7.2, ionic strength I=22 mM) are lower than for its Met→44Lys mutant (k _q ^Δ=1.9×10⁷, k _q ^Λ=1.4×10⁷ M^–1 s^–1, same pH and I), indicating that energy transfer occurs from Eu(dpa)₃ ^3– to the Cu(II) centre when the luminophore is bound to the hydrophobic patch of the protein near residue 44. The enantioselectivity remains unaltered by the mutation: k _q ^Δ/k _q ^Λ=1.27±0.04, so Lys44 is probably not in direct contact with the Eu chelate. The I and pH dependence of k _q indicate that the lysine residue interacts electrostatically with Eu(dpa)₃ ^3–. For plastocyanin the quenching rates are of the order of 10⁶ M^–1 s^–1; for amicyanin they are two orders of magnitude larger (k _q ^Δ=12×10⁷, k _q ^Λ=11×10⁷ M^–1 s^–1, pH 7.2, I=22 mM). The variation of k _q is attributed to differences in the charge distribution on the proteins, which influences the binding of the luminophore to the protein surface. For amicyanin the anion binding site near Lys59 and Lys60 may be involved in the energy transfer. Received: 16 June 1998 / Accepted: 18 September 1998     

Water influx and efflux in free-flying pigeons

We used tritium-labeled water to measure total body water, water influx (which approximated oxidative water production) and water efflux in free-flying tippler pigeons (Columba livia) during flights that lasted on average 4.2 h. At experimental air temperatures ranging from 18 to 27 °C, mean water efflux by evaporation and excretion [6.3 ± 1.3 (SD) ml · h⁻¹, n = 14] exceeded water influx from oxidative water and inspired air (1.4 ± 0.7 ml · h⁻¹, n = 14), and the birds dehydrated at 4.9 ± 0.9 ml · h⁻¹. This was not significantly different from gravimetrically measured mass loss of 6.2 ± 2.1 g · h⁻¹ (t = 1.902, n = 14, P>0.05). This flight-induced dehydration resulted in an increase in plasma osmolality of 4.3 ± 3.0 mosmol · kg⁻¹ · h⁻¹ during flights of 3–4 h. At 27 °C, the increase in plasma osmolality above pre-flight levels (ΔP _osm = 7.6±4.29 mosmol · kg⁻¹ · h⁻¹, n = 6) was significantly higher than that at 18 °C (ΔP _osm = 0.83±2.23 mosmol · kg⁻¹ · h⁻¹, (t = 3.43, n = 6, P < 0.05). Post-flight haematocrit values were on average 1.1% lower than pre-flight levels, suggesting plasma expansion. Water efflux values during free flight were within 9% of those in the one published field study (Gessaman et al. 1991), and within the range of values for net water loss determined from mass balance during wind tunnel experiments (Biesel and Nachtigall 1987). Our net water loss rates were substantially higher than those estimated by a simulation model (Carmi et al. 1992) suggesting some re-evaluation of the model assumptions is required. Accepted: 8 April 1997     

Walking performance and economy in chronic heart failure patients pre and post exercise training

The effect of a 3-week exercise programme on performance and economy of walking was analysed in 16 male patients with chronic heart failure [mean age 51.8 (SD 6.9) years, height 174.9 (SD 6.3) cm, body mass 75.3 (SD 11.5) kg, ejection fraction 20.8 (SD 5.0)%]. They were submitted to a cardiopulmonary exercise test on a cycle ergometer and a 6-min walking test on a treadmill before and after the period of exercise training. The training programme consisted of interval cycle (five times a week for 15 min), and treadmill ergometer training (three times a week for 10 min) at approximately 70% cycling peak oxygen uptake (O_2peak) and supplementary exercises (three times a week for 20 min). Compared to the pre values cycling O_2peak [11.9 (SD 2.9) vs 14.0 (SD 2.3) ml ·  kg^–1 · min^–1], maximal self paced walking speed [0.68 (SD 0.33) vs 1.16 (SD 0.30) m · s^–1], and net walking power [2.16 (SD 0.89) vs 2.73 (SD 0.91) W · kg^–1] had increased (P < 0.01) while net energy cost [3.31 (SD 0.66) vs 2.33 (SD 0.38) J · kg^–1 ·  m^–1] had decreased (P < 0.001) after the training period. Approximately 42% of the increase of walking speed resulted from a higher walking power output, whereas approximately 58% corresponded to a positive effect on walking economy. The improvement in walking economy was a function of an increase in walking velocity itself and a result of a more efficient walking technique. These results would indicate that in patients with marked exercise intolerance, adequate exercise training programmes could contribute to favourable metabolic changes with positive effects on the economy of motion. Accepted: 29 August 1996     

Disproportionation of pentaammineruthenium(III)–nucleoside complexes leads to two-electron oxidation of nucleosides without involving oxygen molecules

Pentaammineruthenium(III) complexes of deoxyinosine (dIno) and xanthosine (Xao) ([Ru^III(NH₃)₅(L)], L?is?dIno, Xao) in basic solution were studied by UV?Cvis spectroscopy, liquid chromatography/electrospray ionization mass spectrometry, and high-performance liquid chromatography. Both Ru^III complexes disproportionate to Ru^II and Ru^IV. Disproportionation followed the rate law d[Ru^II]/dt?=?(k _o?+?k ₁[OH^?])[Ru^III]. k _o and k ₁ of disproportionation at 25?°C were 2.1 (±0.1)?×?10^?3?s^?1 and 21.4?±?3.2?M^?1 s^?1, respectively, for [Ru^III(NH₃)₅(dIno)], and 3.5 (±0.7)?×?10^?4?s^?1 and 59.7?±?3.6?M^?1?s^?1, respectively, for [Ru^III(NH₃)₅(Xao)]. The [Ru^III(NH₃)₅(Xao)] complex disproportionates at a faster rate than [Ru^III(NH₃)₅(dIno)] owing to the stronger electron-withdrawing effect of exocyclic oxygen in Xao. The activation parameters ??H ^? and ??S ^? for k ₁ of [Ru^III(NH₃)₅(dIno)] were 80.2?±?15.2?kJ?mol^?1 and 47.6?±?9.8?J?K^?1 mol^?1, respectively, indicating that the disproportionation of Ru^III to Ru^II and Ru^IV is favored owing to the positive entropy of activation. The final products of both complexes in basic solution under Ar were compared with those under O₂. Under both conditions [Ru(NH₃)₅(8-oxo-L)] was produced, but via different mechanisms. In both aerobic and anaerobic conditions, the deprotonation of highly positively polarized C8-H of Ru-L by OH^? initiates a two-electron redox reaction. For the next step, we propose a one-step two-electron redox reaction between L and Ru^IV under anaerobic conditions, which differentiates from Clarke??s mechanism of two consecutive one-electron redox reactions between L, Ru^III, and O₂.     

Uptake of 3-O-methyl-D-[U-14C]glucose into brain of rainbow trout: possible effects of melatonin

The influx of glucose into the brain and plasma glucose disappearance were estimated in rainbow trout (Oncorhynchus mykiss) intravenously injected (1 ml · kg⁻¹ body weight) with a single dose (15 μCi · kg⁻¹ body weight) of 3-O-methyl-D-[U-¹⁴C]glucose ([U-¹⁴C]-3-OMG) at different times (2–160 min), and after intravenous injection at 15 min of increased doses (10–60 μCi · kg⁻¹ body weight) of [U-¹⁴C]-3-OMG. Brain and plasma radiotracer concentrations were measured, and several kinetic parameters were calculated. The apparent brain glucose influx showed a maximum after 15–20 min of injection then decreased to a plateau after 80 min. Brain distribution space of 3-OMG increased from 2 min to 20 min reaching equilibrium from that time onwards at a value of 0.14 ml · g⁻¹. The unidirectional clearance of glucose from blood to brain (k₁) and the fractional clearance of glucose from brain to blood (k₂) were estimated to be 0.093 ml · min⁻¹ · g⁻¹, and 0.867 min⁻¹, respectively. A linear increase was observed in brain and plasma radiotracer concentrations when increased doses of [U-¹⁴C]-3-OMG were used. All these findings support a facilitative transport of glucose through the blood-brain barrier of rainbow trout with characteristics similar to those observed in mammals. The injection of different doses of melatonin (0.25–1.0 mg · kg⁻¹) significantly increased brain glucose influx suggesting a possible role for melatonin in the regulation of glucose transport into the brain. Accepted: 26 January 2000