Probing the ground state of the purple mixed valence CuA center in nitrous oxide reductase: a CW ENDOR (X-band) study of the 65Cu, 15N-histidine labeled enzyme and interpretation of hyperfine couplings by molecular orbital calculations

 CW ENDOR (X-band) spectra for the purple mixed-valence [Cu(1.5+)...Cu(1.5+)], S = 1/2, Cu_A site in nitrous oxide reductase were obtained after insertion of ⁶⁵Cu or both ⁶⁵Cu and ¹⁵N-histidine. The ¹⁴N/¹⁵N isotopic substitution allowed for an unambiguous deconvolution of proton and nitrogen hyperfine couplings in the spectra. A single nitrogen coupling with a value of 12.9 ± 0.4 MHz for ¹⁴N was detected. Its anisotropy was characteristic for imidazole bound to copper. A spin density of 3–5% was estimated for the nitrogen donors to Cu_A, indicating that the ground state is ²B_3u. Proton hyperfine structure was detected from four C_β protons of coordinating cysteine residues. Their isotropic and anisotropic parts were deconvoluted by spectral simulation. From the anisotropic couplings a spin density of 16–24% was estimated for each of the cysteine thiolate donors of Cu_A. The [N_HisCu(RS)₂CuN_His]⁺ core structure of Cu_A in nitrous oxide reductase from Pseudomonas stutzeri is predicted to be similar to the crystallographically determined Cu_A* structure (Wilmanns M, Lappalainen P, Kelly M, Sauer-Eriksson E, Saraste M (1995) Proc Natl Acad Sci USA 92 : 11955–11959), but distinct from the Cu_A structure of Paracoccus denitrificans cytochrome c oxidase (Iwata S, Ostermeier C, Ludwig B, Michel H (1995) Nature 376 : 660–669). The angular dependence of the isotropic couplings as a function of the electronic ground state was calculated by the INDO/S method. The Mulliken atomic-spin populations calculated by a gradient-corrected density functional method and the semiempirical INDO/S method were compared with experimentally derived spin populations, and good agreement between theory and experiment was found for both calculations. The ground state of Cu_A is best represented by the resonance structures of the form [Cu^IS^–S^–Cu^II↔ Cu^IS^•S^–Cu^I↔ Cu^IS^–S^•Cu^I↔ Cu^IIS^–S^–Cu^I]. It is proposed that the Cu 4s,p as well as sulfur 3d orbitals play a role in the stabilization of this novel type of cluster. Received: 17 September 1997 / Accepted: 28 October 1997     

Modulation of Bacillus pasteurii cytochrome c 553 reduction potential by structural and solution parameters

 Direct cyclic voltammetry and ¹H NMR spectroscopy have been combined to investigate the electrochemical and spectroscopic properties of cytochrome c ₅₅₃ isolated from the alkaliphilic soil bacterium Bacillus pasteurii. A quasi-reversible diffusion-controlled redox process is exhibited by cytochrome c ₅₅₃ at a pyrolitic graphite edge microelectrode. The temperature dependence of the reduction potential, measured using a non-isothermal electrochemical cell, revealed a discontinuity at 308 K. The thermodynamic parameters determined in the low-temperature range (275–308 K;ΔS°′=–162.7±1.2 J mol^–1 K^–1, ΔH°′=–53.0±0.5 kJ mol^–1, ΔG°′=–4.5±0.1 kJ mol^–1, E°′=+47.0±0.6 mV) indicate the presence of large enthalpic and entropic effects, leading, respectively, to stabilization and destabilization of the reduced form of cytochrome c ₅₅₃. Both effects are more accentuated in the high-temperature range (308–323 K;ΔS°′=–294.1±8.4 J mol^–1 K^–1, ΔH°′=–93.4±3.1 kJ mol^–1, ΔG°′=–5.8±0.6 kJ mol^–1, E°′=+60.3±5.8 mV), with the net result being a slight increase of the standard reduction potential. These thermodynamic parameters are interpreted using the compensation theory of hydration of biopolymers as indicating the extrusion, upon reduction, of water molecules from the hydration sphere of the cytochrome. The low-T and high-T conformers differ by the number of water molecules in the solvation sphere: in the high-T conformer, the number of water molecules extruded upon reduction increases, as compared to the low-T conformer. The ionic strength dependence of the reduction potential at 298 K, treated within the frame of extended Debye-Hückel theory, yields values of E ^°′ _(I=0) =–25.4±1.4 mV, z _red=–11.3, and z _ox=–10.3. The pH dependence of the reduction potential at 298 K shows a plateau in the pH range 7–10 and an increase at more acidic pH, allowing the calculation of pK _O=5.5 and pK _R=5.7, together with the estimate of the reduction potentials of completely protonated (+71 mV) and deprotonated (+58 mV) forms of cytochrome c ₅₅₃. ¹H NMR spectra of the oxidized paramagnetic cytochrome c ₅₅₃ indicate the presence of a His-Met axial coordination of the low-spin (S=1/2) heme iron, which is maintained in the temperature interval 288–340 K at pH 7 and in the pH range 4.8–10.0 at 298 K. The temperature dependence of the hyperfine-shifted signals shows both Curie-type and anti-Curie-type behavior, with marked deviations from linearity, interpreted as indicating the presence of a fast equilibrium between the low-T and high-T conformers, having slightly different heme electronic structures resulting from the T-induced conformational change. Increasing the NaCl concentration in the range 0–0.2 M causes a slight change of the ¹H NMR chemical shifts of the hyperfine-shifted signals, with no influence on their linewidth. The calculated lower limit value of the apparent affinity constant for specific ion binding is estimated as 5.2±1.1 M^–1. The pH dependence of the isotropically shifted ¹H NMR signals of the oxidized cytochrome displays at least one ionization step with pK _O=5.7. The thermodynamic and spectroscopic data indicate a large solvent-derived entropic effect as the main cause for the observed low reduction potential of B. pasteurii cytochrome c ₅₅₃. Received: 9 January 1998 / Accepted: 8 April 1998     

Effects of trans-pyridine ligands on the interactions of RuII and RuIII ammine complexes N7-coordinated to purine nucleosides and DNA

 DNA binding by trans-[(H₂O)(Pyr)(NH₃)₄Ru^II]²⁺ (Pyr=py, 3-phpy, 4-phpy, 3-bnpy, 4-bnpy) is highly selective for G⁷ with K _G=1.1×10⁴ to 2.8×10⁴, with the more hydrophobic Pyr ligands exhibiting slightly higher binding. A strong dependence on ionic strength indicates that ion-pairing with DNA occurs prior to binding. At μ=0.05, d[Ru^II-DNA]/dt=k[Ru^II][DNA], where k=0.17–0.21 M^–1s^–1 with the various Pyr ligands. The air oxidation of [(py)(NH₃)₄Ru^II] _n -DNA to [(py)(NH₃)₄Ru^III] _n -DNA at pH 6 occurs with a pseudo-first-order rate constant of k _obs=5.6×10^–4s^–1 at μ=0.1, T=25  °C. Strand cleavage of plasmid DNA appears to occur by both Fenton/Haber-Weiss chemistry and by base-catalyzed routes, some of which are independent of oxygen. Base-catalyzed cleavage is more efficient than O₂ activation at neutral pH and involves the disproportionation of covalently bound Ru^III and, in the presence of O₂, Ru-facilitated autoxidation to 8-oxoguanine. Disproportionation of [py(NH₃)₄Ru^III] _n -DNA occurs according to the rate law: d[Ru^II–G_DNA]/dt=k ₀[Ru^III–G_DNA]+k ₁[Ru^III–G_DNA][OH^–], where k ₀=5.4×10^–4s^–1 and k ₁=8.8 M^–1s^–1 at 25  °C, μ=0.1. The appearance of [(Gua)(py)(NH₃)₄Ru^III] under argon, which occurs according to the rate law: d[Ru^III–G]/dt=k ₀[Ru^III–G_DNA]+k ₁[OH^–][Ru^III–G_DNA] (k ₀=5.74×10^–5 s^–1, k ₁=1.93×10^–2M^–1s^–1 at T=25  °C, μ=0.1), is consistent with lysis of the N-glycosidic bond by Ru^IV-induced general acid hydrolysis. In air, the ratio of [Ru-8-OG]/[Ru-G] and their net rates of appearance are 1.7 at pH 11, 25  °C. Small amounts of phosphate glycolate indicate a minor oxidative pathway involving C4′ of the sugar. In air, a dynamic steady-state system arises in which reduction of Ru^IV produces additional Ru^II. Received: 11 November 1998 / Accepted: 3 March 1999     

The metal-binding properties of the blue crab copper specific CuMT-2: a crustacean metallothionein with two cysteine triplets

Most crustacean metallothioneins (MTs) contain 18 Cys residues and bind six divalent metal ions. The copper-specific CuMT-2 (MTC) of the blue crab Callinectes sapidus with 21 Cys residues, of which six are organized in two uncommon Cys-Cys-Cys sequences, represents an exception. However, its metal-binding properties are unknown. By spectroscopic and spectrometric techniques we show that all 21 Cys residues of recombinant MTC participate in the binding of Cu(I), Zn(II), and Cd(II) ions, indicating that both Cys triplets act as ligands. The fully metallated M₈ ^II–MTC (M is Zn, Cd) form possesses high- and low-affinity metal binding sites, as evidenced by the formation of Zn₆–MTC and Cd₇–MTC species from M₈ ^II–MTC after treatment with Chelex 100. The NMR characterization of Cd₇–MTC suggests the presence of a two-domain structure, each domain containing one Cys triplet and encompassing either the three-metal or the four-metal thiolate cluster. Whereas the metal–Cys connectivities in the three-metal cluster located in the N-terminal domain (residues 1–31) reveal a Cd₃Cys₉ cyclohexane-like structure, the presence of dynamic processes in the C-terminal domain (residues 32–64) precluded the determination of the organization of the four-metal cluster. Absorption and circular dichroism features accompanying the stepwise binding of Cu(I) to MTC suggest that all 21 Cys are involved in the binding of eight to nine Cu(I) ions (Cu_8–9–MTC). The subsequent generation of Cu₁₂–MTC involves structural changes consistent with a decrease in the Cu(I) coordination number. Overall, the metal-binding properties of MTC reported here contribute to a better understanding of the role of Cys triplets in MTs.     

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     

Effects of temperature on photosynthesis of two morphologically contrasting plant species along an altitudinal gradient in the tropical high Andes

The effects of temperature on photosynthesis of a rosette plant growing at ground level, Acaena cylindrostachya R. et P., and an herb that grows 20–50 cm above ground level, Senecio formosus H.B.K., were studied along an altitudinal gradient in the Venezuelan Andes. These species were chosen in order to determine – in the field and in the laboratory – how differences in leaf temperature, determined by plant form and microenvironmental conditions, affect their photosynthetic capacity. CO₂ assimilation rates (A) for both species decreased with increasing altitude. For Acaena leaves at 2900 m, A reached maximum values above 9 μmol m⁻² s⁻¹, nearly twice as high as maximum A found at 3550 m (5.2) or at 4200 m (3.9). For Senecio leaves, maximum rates of CO₂ uptake were 7.5, 5.8 and 3.6 μmol m⁻² s⁻¹ for plants at 2900, 3550 and 4200 m, respectively. Net photosynthesis-leaf temperature relations showed differences in optimum temperature for photosynthesis (A _o.t.) for both species along the altitudinal gradient. Acaena showed similar A _o.t. for the two lower altitudes, with 19.1°C at 2900 m and 19.6°C at 3550 m, while it increased to 21.7°C at 4200 m. Maximum A for this species at each altitude was similar, between 5.5 and 6.0 μmol m⁻² s⁻¹. For the taller Senecio, A _o.t. was more closely related to air temperatures and decreased from 21.7°C at 2900 m, to 19.7°C at 3550 m and 15.5°C at 4200 m. In this species, maximum A was lower with increasing altitude (from 6.0 at 2900 m to 3.5 μmol m⁻² s⁻¹ at 4200 m). High temperature compensation points for Acaena were similar at the three altitudes, c. 35°C, but varied in Senecio from 37°C at 2900 m, to 39°C at 3550 m and 28°C at 4200 m. Our results show how photosynthetic characteristics change along the altitudinal gradient for two morphologically contrasting species influenced by soil or air temperatures. Received: 5 July 1997 / Accepted: 25 October 1997     

The reaction mechanism of nitrosothiols with copper(I)

Copper and other transition metal ions and their complexes are catalysts for the decomposition of nitrosothiols. In this way they catalyze the biological functions of nitrosothiols. The kinetics and mechanism of the reaction of two nitrosothiols, S-nitrosothiolactic acid and S-nitrosoglutathione (GSNO), with copper(I) are reported. The kinetics of the reaction of Cu(MeCN) _n ⁺ (n=0–3) with the nitrosothiols were studied. The results indicate that Cu⁺ _aq is the active species in the GSNO system, with k(Cu⁺ _aq+GSNO)=(9.4 ±2.0)×10⁷ dm³ mol⁻¹ s⁻¹. The results also indicate that the Cu(MeCN) _n ⁺ (n=0–3) complexes react with S-nitrosothiolactic acid. Transient species are formed in these processes. The results suggest that these species contain copper(I) and thiol. The results shed light on the catalytic role of copper complexes in the decomposition of S-nitrosothiols. Received 10 April 1999 / Accepted 17 December 1999     

The relationship between preferred and optimal positioning during submaximal cycle ergometry

This study was designed to determine how changes in oxygen uptake (O₂) and heart rate (HR) during submaximal cycle ergometry were determined by changes in cycle geometry and/or lower-limb kinematics. Fourteen trained cyclists [Mean (SD): age, 25.5 (6.4) years; body mass 74.4 (8.8) kg; peak O₂, 4.76 (0.79) l. min⁻¹ peak] were tested at three seat-tube angles (70°, 80°, 90°) at each of three trunk angles (10°, 20°, 30°) using a modified Monark cycle ergometer. All conditions were tested at a power output corresponding to 95% of the O₂ at each subject's ventilatory threshold while pedalling at 90 rpm and using aerodynamic handlebars. Sagittal-view kinematics for the hip, knee, and ankle joints were also recorded for all conditions and for the subjects' preferred positioning on their own bicycles. No combination of seat-tube and trunk angle could be considered optimal since many of the nine conditions elicited statistically similar mean O₂ and HR values. Mean hip angle (HA) was the only kinematic variable that changed consistently across conditions. A regression relationship was not observed between mean O₂ or HR and mean hip angle values (P > 0.45). Significant curvilinear relationships were observed, however, between ΔO₂ (O₂ − minimum O₂) and ΔHA (mean HA − preferred HA) using the data from all subjects (R = 0.45, SEE = 0.13 l . min⁻¹) and using group mean values (R = 0.93, SEE = 0.03 l . min⁻¹). In both cases ΔO₂ minimized at ΔHA = 0, which corresponded to the subjects' preferred HA from their own bicycles. Thus, subjects optimized their O₂ cost at cycle geometries that elicited similar lower-limb kinematics as the preferred geometries from their own bicycles. Accepted: 3 July 1996     

Phenoxyl-copper(II) complexes: models for the active site of galactose oxidase

 The reaction of the macrocycles 1,4,7-tris (3,5-di-tert-butyl-2-hydroxy-benzyl)-1,4,7-triazacyclononane, L¹H₃, or 1,4,7-tris(3-tert-butyl-5-methoxy-2-hydroxy-benzyl)-1,4,7-triazacyclononane, L²H₃, with Cu(ClO₄)₂·6H₂O in methanol (in the presence of Et₃N) affords the green complexes [Cu^II(L¹H)] (1), [Cu^II(L²H)]·CH₃OH (2) and (in the presence of HClO₄) [Cu^II(L¹H₂)](ClO₄) (3) and [Cu^II(L²H₂)] (ClO₄) (4). The Cu^II ions in these complexes are five-coordinate (square-base pyramidal), and each contains a dangling, uncoordinated pendent arm (phenol). Complexes 1 and 2 contain two equatorially coordinated phenolato ligands, whereas in 3 and 4 one of these is protonated, affording a coordinated phenol. Electrochemically, these complexes can be oxidized by one electron, generating the phenoxyl-copper(II) species [Cu^II(L¹H)]^+ ·, [Cu(L²H)]^+ ·, [Cu^II(L¹H₂)]^2+ ·, and [Cu^II(L²H₂)]^2+ ·, all of which are EPR-silent. These species are excellent models for the active form of the enzyme galactose oxidase (GO). Their spectroscopic features (UV-VIS, resonance Raman) are very similar to those reported for GO and unambiguously show that the complexes are phenoxyl-copper(II) rather than phenolato-copper(III) species. Received: 10 February 1997 / Accepted: 7 April 1997     

Haemoglobin components and oxygen transport in relation to habitat distribution in triplefin fishes (Tripterygiidae)

Haemoglobin components were analysed for nine species of New Zealand triplefins and their isoelectric points (pI) ranged from 5.1 to 7.0. The number of well-expressed isohaemoglobins was larger in shallow-water and tidal pool species, ranging from four in Grahamina signata to eight in Grahamina capito, and were relatively cathodal. Two strongly anodal isohaemoglobins were expressed in the mid-depth species Ruanoho decemdigitatus and Ruanoho whero, and one in the deeper water species Karalepis stewarti and Forsterygion malcolmi. The red blood cell oxygen-binding properties were determined at 15 °C and 25 °C in the pH range 6.7–7.9 for the shallow-water species G. capito, the shallow to mid-depth species Forsterygion varium, and the deep-water species F. malcolmi. Oxygen affinity was highest for G. capito and the magnitude of the Bohr effect lower (Δlog P ₅₀/ΔpH = −0.37 at 25 °C, where P ₅₀ is the half-saturation coefficient) compared to the two Forsterygion species (Δlog P ₅₀/ΔpH = −0.52 to −0.59). Further, the cooperativity factor, n ₅₀, was lower in G. capito thus maintaining oxygen transport over a wide range of environmental oxygen pressures. Oxygen binding was similarly influenced by temperature in both G. capito and F. malcolmi (maximum heat of oxygenation ΔH_max = −27 kJ mol⁻¹ and −37 kJ mol⁻¹, respectively). Thus, triplefin fishes living in shallow, thermally unstable habitats possess a greater number of cathodally migrating isohaemoglobins, and their red blood cells have a higher oxygen affinity and reduced cooperativity which is less sensitive to changes in pH than do species occurring in more stable, deeper water habitats. Our analysis of an assemblage of closely related species circumvents some of the difficulties inherent in studies where interpretation of experimental results is confounded by phylogeny. Accepted: 18 March 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     

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     

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     

Gd(III) complexes as contrast agents for magnetic resonance imaging: a proton relaxation enhancement study of the interaction with human serum albumin

 The non-covalent interaction between human serum albumin (HSA) and DOTA-like Gd(III) complexes containing hydrophobic benzyloxymethyl (BOM) substituents has been thoroughly investigated by measuring the solvent proton relaxation rates of their aqueous solutions. The binding association constants (K _A) to HSA are directly related to the number of hydrophobic substituents present on the surface of the complexes. Furthermore, an estimation of ΔH° and ΔS° has been obtained by the temperature dependence of K _A. Assays performed with the competitor probes warfarin and ibuprofen established that the complexes interact with HSA through two nearly equivalent binding sites located in the subdomains IIA and IIIA of the protein. Strong relaxation enhancements, promoted by the formation of slowly tumbling paramagnetic adducts, have been measured at 20 MHz for complexes containing two and three hydrophobic substituents. The macromolecular adduct with the latter species has a relaxivity of 53.2±0.7 mM^–1 s^–1, which represents the highest value so far reported for a Gd(III) complex. The temperature dependence of the relaxivity for the paramagnetic adducts with HSA indicates long exchange lifetimes for the water molecules dipolarly interacting with the paramagnetic centre. This is likely to be related to the formation, upon hydrophobic interaction of the complexes with HSA, of a clathrate-like, second-coordination-sphere arrangement of water molecules. Besides affecting the dissociative pathway of the coordinated water molecule, this water arrangement may itself significantly contribute to enhancement of the bulk solvent relaxation rate. Received: 6 November 1995 / Accepted: 17 April 1996     

Ethidium bromide binding to native and denatured poly(dA)poly(dT)

Isotherms of the EtBr adsorption on native and denatured poly(dA)poly(dT) in the temperature interval 20–70°C were obtained. The EtBr binding constants and the number of binding sites were determined. The thermodynamic parameters of the EtBr intercalation complex upon changes of solution temperature 20–48°C were calculated: 1.0·10⁶ M⁻¹≤K≤1.4·10⁶ M⁻¹, free energy ΔG ^o=−8.7±0.3 kcal/mol, enthalpy ΔH ^o≅0, and entropy ΔS ^o=28±0.5 cal/(mol deg). UV melting has shown that the melting temperature (T _m) of EtBr-poly(dA)poly(dT) complexes (μ=0.022,4.16·10⁻⁵ M EtBr) increased by 17°C as compared with the ΔT _m of free homopolymer, whereas the half-width of the transition (T _m) is not changed. It was shown for the first time that EtBr forms complexes of two types on single-stranded regions of poly(dA)poly(dT) denatured at 70°C: strong (K ₁=1.7·10⁵ M⁻¹; ΔG ^o=−8.10±0.03 kcal/mol) and weak (K ₂=2.9·10³ M⁻¹; ΔG ^o=−6.0±0.3 kcal/mol).The ΔG ^o of the strong and weak complexes was independent of the solution ionic strength, 0.0022≤μ≤0.022. A model of EtBr binding with single-stranded regions of poly(dA)poly(dT) is discussed.     

Effects of nonspecific ion-protein interactions on the redox chemistry of cytochrome c

 The effects of the ionic atmosphere on the enthalpic and entropic contributions to the reduction potential of native (state III) beef heart cytochrome c have been determined through variable-temperature direct electrochemistry experiments. At neutral or slightly alkaline pH values, from 5 to 50  °C, the reduction enthalpy and entropy become less negative with decreasing ionic strength. The reduction entropy extrapolated at null ionic strength is approximately zero, indicating that, in the absence of the screening effects of the salt ions on the network of the electrostatic interactions at the protein-solvent interface, the solvation properties and the conformational flexibility of the two redox states are comparable. The moderate decrease in E°′ observed with increasing ionic strength [ΔE°′_IS =(E°′) _{I =0.1 M}–(E°′) _{I =0 M}=–0.035 V at 25  °C], once the compensating enthalpic and entropic effects of the salt-induced changes in the hydrogen bonding within the hydration sphere of the molecule in the two redox states are factorized out, results in being ultimately determined by the stabilizing enthalpic effect of the negatively charged ionic atmosphere on the ferri form. At pH 9, the ionic strength dependence of the reduction termodynamics of cytochrome c follows distinctive patterns, possibly as a result of specific binding of the hydroxide ion to the protein. A decrease in ionic strength at constant pH, as well as a pH increase at constant ionic strength, induces a depression of the temperature of the transition from the low-T to high-T conformer of cytochrome c, which suggests that a temperature-induced decrease in the pK _a for a residue deprotonation is the key event of this conformational change. Received: 7 April 1999 / Accepted: 19 July 1999     

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     

Metabolic rates in an anadromous clupeid, the American shad (Alosa sapidissima)

To assess the energetics of migration in an anadromous fish, adult American shad (Alosa sapidissima) were swum in a large respirometer at a range of speeds (1.0–2.3 body lengths (BL) s⁻¹, 13–24 °C). Metabolic rate (M_O2) was logarithmically related to swimming speed (Bl s⁻¹; r ² = 0.41, slope = 0.23 ± 0.037) and tailbeat frequency (beats × min⁻¹; r ² = 0.52, slope = 0.003 ± 0.0003). Temperature had a significant effect on metabolic rate (r ² = 0.41) with a Q₁₀ of 2.2. Standard metabolic rate (SMR), determined directly after immobilization with the neuroblocker gallamine triethiodide, ranged from 2.2–6.2 mmolO₂ kg⁻¹ h⁻¹ and scaled with mass (W) such that SMR = 4.0 (±0.03)W^{0.695(±0.15)}. Comparison of directly determined and extrapolated SMR suggests that swimming respirometry provides a good estimate of SMR in this species, given the differences in basal activity monitored by the two methods. Overall, American shad metabolic rates (M_O2 and SMR) were intermediate between salmonids and fast-swimming perciforms, including tunas, and may be a result of evolutionary adaptation to their active pelagic, schooling life history. This study demonstrates variability in metabolic strategy among anadromous fishes that may be important to understanding the relative success of different migratory species under varying environmental conditions. Accepted: 3 March 1999     

Facultative hypothermia as a thermoregulatory strategy in the phyllostomid bats, Carollia perspicillata and Sturnira lilium

The present study questions whether hypothermia is an artifact due to captivity-induced stress or a thermoregulatory strategy for bats of the neotropical family Phyllostomidae. In Guanacaste, Costa Rica, Carollia perspicillata and Sturnira lilium exhibited a bimodal distribution of body temperatures when submitted to an ambient temperature of 21 °C. Body temperature was highly correlated with body mass in both species. C. perspicillata of mass ≥20 g and S. lilium of mass ≥17 g remained normothermic (body temperature >37 °C), whereas at masses below 18 g and 13 g, respectively, >80% of individuals were hypothermic (body temperature ≤32 °C). In two treatment groups for each species, we restricted food intake to ca. 20% of body mass on either night 1 or night 4 following capture. Hypothermia was significantly related to food-restriction, but not time in captivity. Metabolic rate (ml O₂ ·  g⁻¹ h⁻¹) at ambient temperature = 21 °C was MR = e ^{(–2.11 + 0.101 Tb)} (r ² = 0.7, P < 0.001) for C. perspicillata and MR = e ^{(−2.62 + 0.115 Tb)} (r ² = 0.89) for S. lilium. Free-ranging, radio tagged C. perspicillata exhibited daily depression of body temperature to 33–34 °C. We conclude that hypothermia is an thermoregulatory strategy that allows phyllostomid bats to adjust metabolic rate to feeding success and the level of fat stores. Accepted: 20 August 1996     

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