A possible crystal volume factor in the impact sensitivities of some energetic compounds

We have investigated the possibility of a link between the impact sensitivities of energetic compounds and the space available to their molecules in their crystal lattices. As a measure of this space, we use ΔV=V_eff−V(0.002), where V_eff is the effective molecular volume obtained from the crystal density and V(0.002) is that enclosed by the 0.002 au contour of the molecule’s gas phase electronic density, determined computationally. When experimental impact sensitivity was plotted against ΔV for a series of 20 compounds, the nitramines formed a separate group showing little dependence upon ΔV. Their impact sensitivities correlate well with an anomalous imbalance in the electrostatic potentials on their molecular surfaces, which is characteristic of energetic compounds in general. The imbalance is symptomatic of the weakness of the N–NO₂ bonds, caused by depletion of electronic charge. The impact sensitivities of non-nitramines, on the other hand, depend much more strongly upon ΔV, and can be quite effectively related to it if an electrostatically-based correction term is included.     

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     

An electrochemical method for the measurements of substrate-oxidizing activity of acetic acid bacteria using a carbon-paste electrode modified with immobilized bacteria

In order to measure the substrate-oxidizing activity of intact cells of Acetobacter pasteurianus no. 2, a given amount of the bacterial cells was immobilized on a carbon-paste electrode, and the current at the electrode was measured in a buffer solution. When Fe(CN)³⁻ ₆ was added to the buffer solution, an anodic current was observed at 0.5 V (against Ag/AgCl). Further, when ethanol was added to the solution, the current started to increase to reach a steady-state within 3 min. The electrode had a good response to acetaldehyde and lactic acid as well as ethanol. Culture conditions affected the current response to various substances; the response of the electrode modified with the cells grown in static culture was much higher than that of the electrode with the cells grown in shaking culture, and the electrode with ethanol-grown cells had a high response to ethanol and acetaldehyde compared with that of the electrode with glucose-grown cells. The increase in the amount of the current after the addition of ethanol (ΔI _EtOH) was linearly proportional to the total number of immobilized cells per electrode in the range 1.0 × 10⁴–1.0 × 10⁸ cells. The ΔI _EtOH values were measured with the electrode prepared with a fixed volume of the cell suspensions taken from the culture at 6-h intervals; the dependence of the ΔI _EtOH value on time agreed well with the cell growth measured by colony counting and turbidity in the lag and logarithmic phase. After the logarithmic phase, the value of ΔI _EtOH sharply decreased, resembling to the growth measured by colony counting, rather than by turbidity. Received: 30 October 1998 / Received revision: 2 February 1999 / Accepted: 5 February 1999     

A density functional theory study of phenyl formation initiated by ethynyl radical (C2H•) and ethyne (C2H2)

An ab initio computational density functional theory (DFT) was used to study the formation of the first cyclic molecule (phenyl) initiated by the ethynyl radical (C₂H•). The study covers a competition reaction between the addition reactions of C₂H• with ethyne (C₂H₂) and some molecular re-arrangement schemes. The minimum energy paths of the preferred cyclic formation route were characterized. A thorough thermochemical analysis was performed by evaluating the differences in the energy of activation (ΔE), enthalpy (ΔH), and Gibb's free energy (ΔG) of the optimized stable and transition state (TS) molecules. The reaction temperatures were set to normal (T = 298 K) and combustion (T = 1,200 K) conditions.

Respiratory and metabolic responses of the Australian Yabby Cherax destructor to progressive and sustained environmental hypoxia

The Australian Yabby, Cherax destructor, inhabits occasionally hypoxic water. The respiratory gas, acid-base, metabolite and energetic status of this crayfish was assessed during progressive hypoxia and during 3 h at a water PO₂ of 1.33 kPa. The O₂ affinity of haemocyanin from C. destructor was increased by lactate (Δlog P ₅₀/Δlog[lactate] = −0.111) and by Ca (Δlog P ₅₀/Δlog[Ca] = −0.62) but not by urate. While the non-bicarbonate buffering capacity was low (Δ[HCO₃ ⁻]/ ΔpH=−4.89) the haemocyanin had a low sensitivity to pH changes (ϕ = −0.33). The crayfish showed a compensatory hyperventilation, which induced a respiratory alkalosis, until the water O₂ partial pressure declined below 2.67 kPa, after which the O₂ uptake rate was approximately 10% of normoxic rates. The high haemocyanin-O₂ affinity maintained haemolymph O₂ content during progressive hypoxia despite the normally low arterial O₂ partial pressure of C. destructor. During severe hypoxia, pH decreased but increased lactate aided in maintaining haemocyanin-O₂ saturation. The importance of regulated haemocyanin-O₂ affinity in hypoxic C. destructor was reduced by lowered metabolism, including reduced cardiac output, and the consequent reduction in O₂ requirement. Anaerobiosis became important only at very low PO₂ but thereafter proceeded rapidly, supported by a marked hyperglycaemia. There was no depletion of adenylates, even after 3 h of severe hypoxia. The tail muscle of C. destructor held small amounts of glycogen which would sustain anaerobiosis for a only a few hours. Hypometabolism seems an important hypoxic response but severe hypoxia may encourage the crayfish to breathe air. Accepted: 26 February 1998     

Microbial fuel cells for energy self-sufficient domestic wastewater treatment—a review and discussion from energetic consideration

As the microbial fuel cell (MFC) technology is getting nearer to practical applications such as wastewater treatment, it is crucial to consider the different aspects that will make this technology viable in the future. In this paper, we provide information about the specifications of an energy self-sufficient MFC system as a basis to extrapolate on the potential benefits and limits of a future MFC-based wastewater treatment plant. We particularly emphasize on the importance of two crucial parameters that characterize an MFC: its electromotive force (E _emf) and its internal resistance (R _int). A numerical projection using state-of-art values (E _emf = 0.8 V and R _int = 5 Ω) emphasized on the difficulty at this moment to reach self-sufficiency using a reasonable number of MFCs at the laboratory scale. We found that a realistic number of MFCs to provide enough voltage (=5 V) at a sufficient current (=0.8 A) to power a pump requiring 4 W would be of 13 MFCs in series and 10 stacks of MFCs in parallel, resulting in a total number of 130 MFCs. That would result in a treatment capacity of 144 L of domestic wastewater (0.5 g-COD L⁻¹) per day. The total MFC system would be characterized by an internal resistance of 6.5 Ω.     

Changes in Morphology and Elemental Composition of <Emphasis Type="Italic">Vibrio splendidus</Emphasis> Along a Gradient from Carbon-limited to Phosphate-limited Growth

We examined morphology, elemental composition (C, N, P), and orthophosphate-uptake efficiency in the marine heterotrophic bacterium Vibrio splendidus grown in continuous cultures. Eight chemostats were arranged along a gradient of increasing glucose concentrations in the reservoirs, shifting the limiting factor from glucose to phosphate. The content of carbon, nitrogen, and phosphorus was measured in individual cells by x-ray microanalysis using a transmission electron microscope (TEM). Cell volumes (V) were estimated from length and width measurements of unfixed, air-dried cells in TEM. There was a transition from coccoid cells in C-limited cultures toward rod-shaped cells in P-limited cultures. Cells in P-limited cultures with free glucose in the media were significantly larger than cells in glucose-depleted cultures (P < 0.0001). We found functional allometry between cellular C-, N-, and P content (in femtograms) and V (in cubic micrometers) in V. splendidus (C = 224 × V ^0.89, N = 52.5 × V ^0.80, P = 2 × V ^0.65); i.e., larger bacteria had less elemental C, N, and P per V than smaller cells, and also less P relative to C. Biomass-specific affinity for orthophosphate uptake in large P-limited V. splendidus approached theoretical maxima predicted for uptake limited by molecular diffusion toward the cells. Comparing these theoretical values to respective values for the smaller, coccoid, C-limited V. splendidus indicated, contrary to the traditional view, that large size did not represent a trade-off when competing for the non-C-limiting nutrients.     

DFT and MP2 study on the electrophilic addition reaction of bromine to exo-tricyclo[3.2.1.02.4]oct-6-ene

The geometry and electronic structure of exo-tricyclo[3.2.1.0^2,4]oct-6-ene (exo-TCO) was investigated using DFT methods. The two faces of the endo-pyramidalised double bond of the molecule are not equivalent. The exo face of the double bond has regions with high electron density (q _i,HOMO) and greater negative potential. Molecular complexes of exo-TCO with bromine were investigated using the B3LYP/6-311++G(d,p) method; the exo-TCO . . . Br₂(exo) molecular complex was found to be relatively more stable than the exo-TCO . . . Br₂(endo) complex. The cationic intermediates of the reaction were studied by DFT and MP2 methods. The solvent effect was evaluated using the self-consistent isodensity polarised continuum model (SCI-PCM). The exo-bromonium cation was found to be more stable than the endo-bromonium cation. Exo-facial selectivity due to electronic and steric factors was observed upon addition of bromine to exo-TCO. The multicentre nonclassical delocalised bromocarbonium cation IV and the exo-bridged-bromonium cation I are more stable than the rearrangement cation V. The reaction products are formed via exo-bridged-bromonium I and nonclassical IV cations, which are the most stable intermediates and whose stabilities barely differ. The mechanism of the addition reaction is also discussed.     

Effects of phenolic compounds on the growth and the fatty acid composition of Lactobacillus plantarum

The effects of different phenolic compound concentrations on the fatty acid composition of Lactobacillus plantarum isolated from traditional home-made olive brines were determined. Increasing amounts of caffeic and ferulic acids induced a gradual increase in the amounts of myristic, palmitoleic, stearic and 9,10-methylenehexadecanoic (C17Δ, where Δ represents the cyclopropane group) acid with a concomitant decrease of lactobacillic acid (C₁₉Δ). On the other hand, the addition of tannins induced an increase in the C₁₉Δ level at the expense of vaccenic acid content. The presence of acidic phenols and tannins also affected bacterial growth, inducing the most obvious effect with tannin at 1 g l⁻¹. Received: 1 July 1997 / Received revision: 9 September 1997 / Accepted: 15 September 1997     

Decreased pulmonary diffusing capacity of divers over a 6-year period

Non-smoking, male, professional firemen divers (n = 15) underwent two pulmonary function tests (PFT) separated by 6 years. Measured data were compared to European Coal Steel Community recommended reference values to permit cross-sectional and then longitudinal study. Higher vital capacity (VC; P < 0.01) and forced expiratory volume in 1 s (FEV₁; P < 0.05), and lower maximal mid-expiratory flow (MMEF) coefficient with VC (MMEF/VC; P < 0.05) were observed in both PFT. Diver's pulmonary diffusing capacity (DL_CO) and the coefficient with alveolar volume (DL_CO/V _A) showed significantly (P < 0.001) different evolution profiles than those expected from predicted values. In divers, DL_CO and DL_CO/V _A decreased from 104.0% to 91.4% and from 106.4% to 91.5% of predicted values respectively. Changes in DL_CO and DL_CO/V _A correlated positively with the initial measurement of DL_CO (r = 0.67, P < 0.01) and DL_CO/V _A (r = 0.74, P < 0.01) respectively, whereas no correlation between changes in pulmonary gas transfer function and age or diving history parameters was found. Thus, it is suggested from our observations that hyperbaric atmosphere exposure increases the effects of aging on pulmonary diffusing capacity and that pulmonary gas transfer function should be regularly tested in professional and recreational divers. Accepted: 22 February 1997     

Swellographic Study of Peptide Resin Swelling Behavior during Solid Phase Peptide Synthesis

A representative array of peptide resin swelling data obtained using swellographic monitoring technique was analyzed to determine general tendencies in swelling volume dynamics in the course of Boc- and Fmoc-SPPS. Efficiency of the approach based on analyzing swelling volume changes (ΔV) as a function of nominal molecular weight change (ΔM) of the pendant peptide chain was demonstrated. The conclusion was made that a linear relationship between swelling volume and weight changes of peptide resin is a normal basic trend in line with results of early model studies carried out by Merrifield and coworkers. Excellent linear ΔV vs. ΔM correlations (r > 0.97) throughout SPPS for peptide resins swollen in DMF appeared to be typical of >60% of analyzed peptides and also are invariably observed for incorporation of the first four C-terminal amino acids and Boc-deprotected peptide resins swollen in 50% TFA-DCM. Formal theoretical interpretation of the observed regularities and anomalies of swelling volume dynamics and related solvation changes was made in terms of solvation numbers defined as a number of solvent molecules associated with a specific molecular segment of peptide-resin.     

CaATP inhibition of the MgATP-dependent proton pump (H+-ATPase) in bacterial photosynthetic membranes with a mechanism of alternative substrate inhibition

 The membrane-bound F1 sector of the H⁺–ATPase complex (F-type ATPase) in dark-adapted photosynthetic chromatophores is endowed with MgATP- and CaATP-dependent ATPase activities, both sensitive to inhibitors such as oligomycin and venturicidin. Because of contatamination of free Mg^{2 +} and Ca²⁺ ions in chromatophore preparations, kinetic characterization of the two hydrolitic reactions can be performed only in the presence of both substrates, using a model for two alternative substrates. The two activities are characterized by similar maximal rates and affinity constants [V_MgATP and V_CaATP: 13±1 and 10±1 nmol s^–1 ATP hydrolyzed (μmol BChl)^–1; K_MgATP and K_CaATP: 0.22±0.06 and 0.20±0.05 mm]. However, only the MgATP-dependent ATPase is coupled to Δ*_{H +} generation. In this process CaATP acts as an alternative substrate and a competitive inhibitor of the proton pump, with a K_I coincident with K_CaATP for the hydrolytic activity. This finding highlights the central role that the coordination chemistry of the ion-nucleotide complex plays in determining the proton gating mechanism at the catalytic site(s) of the enzyme complex. These results are discussed on the basis of the coordination properties of the ions and of the available information on the protein structure. Received: 5 December 1995 / Accepted: 7 March 1996     

Intramolecular and overall motion of proline: the influence of viscosity on carbon-13 spin-lattice relaxation times.

Nuclear magnetic resonance of ¹³C is used to probe the overall and internal motions of proline. Spin-lattice relaxation times (T₁) are reported for proline monomer dissolved in water/glycerol mixtures. Rates of overall molecular motion and internal motion depend on solvent composition but to different degrees. The effective correlation times (τ_eff) of the various proton-bearing carbon atoms in proline vary linearly as a function of solvent composition (%v/v) rather than of solution viscosity. The effective correlation time for molecular motion (τ_eff) is separated into contributions from overall molecular motion (τ_mol) and internal motion (τ_int). The γ-carbon of proline shows the smallest dependence of τ_int on solvent composition. The data indicate a high degree of intramolecular motion for the γ-carbon of proline. Inclusion of anisotropic molecular reorientation in the data analysis was found not to affect the above conclusions. The observed values of τ_eff indicate that the rotational diffusion model of molecular reorientations should apply to proline. The values of τ_eff calculated for proline using the Stokes-Einstein relation are larger than those observed; the discrepancy is discussed in terms of solvent-solute interactions.     

Heterologous expression and characterization of Choline Oxidase from the soil bacterium Arthrobacter nicotianae

In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15–70 °C). Specific activities were determined toward choline chloride (4.70 ± 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 ± 0.45 × 10^–2 U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 ± 0.12 × 10^–2 U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K _M = 1.51 ± 0.09 mM and V _max = 42.73 ± 0.42 mU/min for choline chloride and K _M = 4.77 ± 0.76 mM and V _max = 48.40 ± 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.     

A physical model for PDZ-domain/peptide interactions

The PDZ domain is an interaction motif that recognizes and binds the C-terminal peptides of target proteins. PDZ domains are ubiquitous in nature and help assemble multiprotein complexes that control cellular organization and signaling cascades. We present an optimized energy function to predict the binding free energy (ΔΔG) of PDZ domain/peptide interactions computationally. Geometry-optimized models of PDZ domain/peptide interfaces were built using Rosetta, and protein and peptide side chain and backbone degrees of freedom are minimized simultaneously. Using leave-one-out cross-validation, Rosetta’s energy function is adjusted to reproduce experimentally determined ΔΔG values with a correlation coefficient of 0.66 and a standard deviation of 0.79 kcal mol⁻¹. The energy function places an increased weight on hydrogen bonding interactions when compared to a previously developed method to analyze protein/protein interactions. Binding free enthalpies (ΔΔH) and entropies (ΔS) are predicted with reduced accuracies of R = 0.60 and R = 0.17, respectively. The computational method improves prediction of PDZ domain specificity from sequence and allows design of novel PDZ domain/peptide interactions.     

Sweat distribution before and after repeated heat exposure

We investigated the impact of short-term, moderate humidity heat acclimation upon sweat distribution. Eight males completed six daily heat exposures [cycling: ambient temperature 39.5 (0.2)°C, relative humidity 59.2 (0.8)%], during which auditory canal temperature (T _ac) was maintained 1.4°C above pre-exposure levels for 70 min by manipulating the work rate. On days 1 and 6, T _ac and local sweat rates (m˙ _sw: eight sites) were monitored. The pre-exposure, resting T _ac and the T _ac sweat threshold decreased from day 1 to day 6 [36.83 (0.05)°C vs 36.62 (0.05)°C, and 36.90 (0.05)°C vs 36.75 (0.05)°C, respectively; both P<0.05]. However, the sweat-onset time, sweat sensitivity (Δm˙ _sw/ΔT _ac) and established m˙ _sw were unaltered (P > 0.05). There was also no evidence of a post-acclimation redistribution in established m˙ _sw between the eight skin regions, though both the sweat sensitivity and established m˙ _sw for the forehead and hand were significantly greater than at the remaining sites (P<0.05). It is concluded that the 5-day heat acclimation regimen provided only a minimal stimulus for sudomotor adaptation. Accepted: 3 March 1997     

Hydrogen bonding interactions in PN···HX complexes: DFT and ab initio studies of structure, properties and topology

Spin-restricted DFT (X3LYP and B3LYP) and ab initio (MP2(fc) and CCSD(fc)) calculations in conjunction with the Aug-CC-pVDZ and Aug-CC-pVTZ basis sets were performed on a series of hydrogen bonded complexes PN···HX (X = F, Cl, Br) to examine the variations of their equilibrium gas phase structures, energetic stabilities, electronic properties, and vibrational characteristics in their electronic ground states. In all cases the complexes were predicted to be stable with respect to the constituent monomers. The interaction energy (ΔE) calculated using a super-molecular model is found to be in this order: PN···HF > PN···HCl > PN···HBr in the series examined. Analysis of various physically meaningful contributions arising from the Kitaura-Morokuma (KM) and reduced variational space self-consistent-field (RVS-SCF) energy decomposition procedures shows that the electrostatic energy has significant contribution to the over-all interaction energy. Dipole moment enhancement (Δμ) was observed in these complexes expected of predominant dipole-dipole electrostatic interaction and was found to follow the trend PN···HF > PN···HCl > PN···HBr at the CCSD level. However, the DFT (X3LYP and B3LYP) and MP2 levels less accurately determined these values (in this order HF < HCl < HBr). Examination of the harmonic vibrational modes reveals that the PN and HX bands exhibit characteristic blue- and red shifts with concomitant bond contraction and elongation, respectively, on hydrogen bond formation. The topological or critical point (CP) analysis using the static quantum theory of atoms in molecules (QTAIM) of Bader was considered to classify and to gain further insight into the nature of interaction existing in the monomers PN and HX, and between them on H-bond formation. It is found from the analysis of the electron density ρ _c , the Laplacian of electron charge density ∇²ρ_c, and the total energy density (H _c ) at the critical points between the interatomic regions that the interaction N···H is indeed electrostatic in origin (ρ_c > 0, ∇²ρ_c > 0 and H_c > 0 at the BCP) whilst the bonds in PN (ρ_c > 0, ∇²ρ_c > 0 and H_c < 0) and HX ((ρ_c > 0, ∇²ρ_c < 0 and H_c < 0)) are predominantly covalent. A natural bond orbital (NBO) analysis of the second order perturbation energy lowering, E⁽²⁾, caused by charge transfer mechanism shows that the interaction N···H is n(N) → BD*(HX) delocalization.     

A Series of Novel Rare Earth Molybdotungstosilicate Heteropolyoxometalates Binding to Bovine Serum Albumin: Spectroscopic Approach

Heteropolyoxometalate complexes have been widely applied in many fields. In this paper, the interaction between a series of novel rare earth molybdotungstosilicate heteropolyoxometalates, K₁₀H₃[Ln(SiMo₆W₅O₃₉)₂]·xH₂O (abbr. LnW₅, Ln = Pr (x = 30), Gd (x = 29), Dy (x = 28), and Yb (x = 31)), and bovine serum albumin (BSA) was investigated by spectroscopic approach under the physiological conditions. In the mechanism discussion, it was proved that the fluorescence quenching of BSA by LnW₅ is a result of the formation of LnW₅–BSA complex. Fluorescence quenching constants were determined using the Stern–Volmer equation to provide a measure of the binding affinity between LnW₅ and BSA. The binding affinity ranked in the order GdW₅ > DyW₅ > PrW₅ > YbW₅. The results of thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures indicate that van der Waals interactions and hydrogen bonds play a major role for LnW₅–BSA association. Furthermore, the distance r between donor (BSA) and acceptor (LnW₅) was obtained according to fluorescence resonance energy transfer.     

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     

Spectroscopic studies of the tungsten-containing formaldehyde ferredoxin oxidoreductase from the hyperthermophilic archaeon Thermococcus litoralis

Thermococcus litoralis (Tl) have been investigated by using the combination of EPR and variable-temperature magnetic circular dichroism (VTMCD) spectroscopies. The results reveal a [Fe₄S₄]^2+,+ cluster (E _m=−368 mV) that undergoes redox cycling between an oxidized form with an S=0 ground state and a reduced form that exists as a pH- and medium-dependent mixture of S=3/2 (g=5.4; E/D=0.33) and S=1/2 (g=2.03, 1.93, 1.86) ground states, with the former dominating in the presence of 50% (v/v) glycerol. Three distinct types of W(V) EPR signals have been observed during dye-mediated redox titration of as-isolated Tl FOR. The initial resonance observed upon oxidation, termed the “low-potential” W(V) species (g=1.977, 1.898, 1.843), corresponds to approximately 25–30% of the total W and undergoes redox cycling between W(IV)/W(V) and W(V)/W(VI) states at physiologically relevant potentials (E _m=−335 and −280 mV, respectively). At higher potentials a minor “mid-potential” W(V) species, g=1.983, 1.956, 1.932, accounting for less than 5% of the total W, appears with a midpoint potential of −34 mV and persists up to at least +300 mV. At potentials above 0 mV, a major “high-potential” W(V) signal, g=1.981, 1.956, 1.883, accounting for 30–40% of the total W, appears at a midpoint potential of +184 mV. As-isolated samples of Tl FOR were found to undergo an approximately 8-fold enhancement in activity on incubation with excess Na₂S under reducing conditions and the sulfide-activated Tl FOR was partially inactivated by cyanide. The spectroscopic and redox properties of the sulfide-activated Tl FOR are quite distinct from those of the as-isolated enzyme, with loss of the low-potential species and changes in both the mid-potential W(V) species (g=1.981, 1.950, 1.931; E _m=−265 mV) and high-potential W(V) species (g=1.981, 1.952, 1.895; E _m=+65 mV). Taken together, the W(V) species in sulfide-activated samples of Tl FOR maximally account for only 15% of the total W. Both types of high-potential W(V) species were lost upon incubation with cyanide and the sulfide-activated high-potential species is converted into the as-isolated high-potential species upon exposure to air. Structural models are proposed for each of the observed W(V) species and both types of mid-potential and high-potential species are proposed to be artifacts of ligand-based oxidation of W(VI) species. A W(VI) species with terminal sulfido or thiol ligands is proposed to be responsible for the catalytic activity in sulfide-activated samples of Tl FOR. Received: 9 September 1999 / Accepted: 17 February 2000