Experimental data and calculated parameters in FeS polymetallic centers in proteins

 Covariance between J and B parameters and indetermination of the actual symmetry suggest caution in making general statements about their values in Fe_nS_n centers. The Fe₄S₄ ³⁺ case is further complicated by electronic isomerism and lack of information on the actual symmetry. Electronic ground states and relative J and B values are discussed in the light of hyperfine coupling values. Received: 12 January 1996 / Accepted: 26 January 1996     

1H NMR studies of the Fe7S8 ferredoxin from Bacillus schlegelii: a further attempt to understand Fe3S4 clusters

 The oxidized Fe₇S₈ ferredoxin from Bacillus schlegelii, containing both [Fe₃S₄]⁺ and [Fe₄S₄]²⁺ clusters, has been investigated by ¹H NMR spectroscopy. An extensive sequence-specific assignment of the hyperfine-shifted resonances has been obtained by making use of a computer-generated structural model. The pattern and the temperature dependence of the hyperfine shifts of the β-CH₂ protons of the cysteines coordinating the [Fe₃S₄]⁺ cluster are rationalized in terms of magnetic interactions between the iron ions. The same approach holds for the hyperfine coupling with ⁵⁷Fe. It is shown that the magnetic interactions are more asymmetric in Fe₇S₈ ferredoxins than in Fe₃S₄ ferredoxins. The NMR non-observability of the β-CH₂ protons of coordinated cysteines in the one-electron-reduced form has been discussed. Received: 19 June 1996 / Accepted: 2 August 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     

Identification of random amplified polymorphic DNA (RAPD) markers for self-incompatibility alleles in Corylus avellana L.

 Random amplified polymorphic DNA (RAPD) markers were identified for self-incompatibility (SI) alleles that will allow marker-assisted selection of desired S-alleles in hazelnut (Corylus avellana L.). DNA was extracted from young leaves collected from field-planted parents and 26 progeny of the cross OSU 23.017 (S₁S₁₂)×VR6-28 (S₂S₂₆) (OSU23×VR6). Screening of 10-base oligonucleotide RAPD primers was performed using bulked segregant analysis. DNA samples from 6 trees each were pooled into four ‘bulks’, one for each of the following: S₁ S₂, S₁ S₂₆ ^, S₂ S₁₂, and S₁₂ S₂₆. ‘Super bulks’ of 12 trees each for S₁, S₂, S₁₂, and S₂₆ were then created for each allele by combining the appropriate bulks. The DNA from these four super bulks and from the parents was used as a template in the PCR assays. A total of 250 primers were screened, and one RAPD marker each was identified for alleles S₂ (OPI07₇₅₀) and S₁ (OPJ14₁₇₀₀). OPJ14₁₇₀₀ was identified in 13 of 14 S₁ individuals of the cross OSU23×VR6 used in bulking and yielded a false positive in 1 non-S₁ individual. This same marker was not effective outside the original cross, identifying 4 of 5 S₁ progeny in another cross, ‘Willamette’×VR6-28 (‘Will’×VR6), but yielded false positives in 4 of 9 non-S₁ individuals from the cross ‘Casina’×VR6-28 (‘Cas’×VR6). OPI07₇₅₀ served as an excellent marker for the S₂ allele and was linked closely to this allele, identifying 12 of 13 S₂ individuals in the OSU23×VR6 population with no false positives. OPI07₇₅₀ was found in 4 of 4 S₂ individuals from ‘Will’×VR and 7 of 7 S₂ individuals of ‘Cas’×VR6 with no false positives, as well as 10 of 10 S₂ individuals of the cross OSU 296.082 (S₁S₈)×VR8-32 (S₂S₂₆), with only 1 false positive individual out of 21 progeny. OPI07₇₅₀ was also present in 5 of 5 cultivars carrying the S₂ allele, with no false-positive bands in non-S₂ cultivars, and correctly identified all but 2 S₂ individuals in 57 additional selections in the breeding program. In the OSU23×VR6 population, the recombination rate between the marker OPJ14₁₇₀₀ and the S₁ allele was 7.6% and between the OPI07₇₅₀ marker and the S₂ allele was 3.8%. RAPD marker bands were excised from gels, cloned, and sequenced to enable the production of longer primers (18 or 24 bp) that were used to obtain sequence characterized amplified regions (SCARs). Both the S₁ and S₂ markers were successfully cloned and 18 bp primers yielded the sole OPJ14₁₇₀₀ product, while 24-bp primers yielded OPI07₇₅₀ as well as an additional smaller product (700 bp) that was not polymorphic but was present in all of the S-genotypes examined. Received: 10 January 1998 / Accepted: 26 January 1998     

Ferroglobus placidus gen. nov., sp. nov., a novel hyperthermophilic archaeum that oxidizes Fe2+ at neutral pH under anoxic conditions

A novel coccoid, anaerobic, Fe²⁺-oxidizing archaeum was isolated from a shallow submarine hydrothermal system at Vulcano, Italy. In addition to ferrous iron, H₂ and sulfide served as electron donors. NO₃ ^– was used as electron acceptor. In the presence of H₂, also S₂O₃ ^2– could serve as electron acceptor. The isolate was a neutrophilic hyperthermophile that grew between 65° C and 95° C. It represents a novel genus among the Archaeoglobales that we name Ferroglobus. The type species is Ferroglobus placidus (DSM 10642). Received: 7 March 1996 / Accepted: 4 September 1996     

Cometabolic degradation of 4-chlorophenol by Alcaligenes eutrophus

 Alcaligenes eutrophus was grown in batch cultures using either phenol as a sole substrate or mixtures of phenol and 4-chlorophenol. Phenol was found to be the sole source for carbon and energy while 4-chlorophenol was utilized only as a cometabolite. Maximum growth rates on phenol reached only 0.26 h^-1, significantly below the growth rates reported earlier with Pseudomonas putida. The cometabolite was found to decrease biomass yield and increase lag time before logarithmic growth occurred. Both phenol and 4-chlorophenol were found to inhibit the growth rate linearly with maximum concentrations of 1080 ppm and 69 ppm respectively, beyond which no growth occurred. The best-fit parameters are incorporated into a simple, dynamic (i.e. time-varying) model capable of predicting all the batch growth conditions presented here. It is shown that P. putida is capable of faster bioremediation when phenol is the sole carbon source or for mixed substrates with low concentrations of the cometabolite, but for high concentrations of 4-chlorophenol, A. eutrophus becomes superior because of the long lag times that occur in the Pseudomonas species. Received: 25 January 1996/Received revision: 13 March 1996/Accepted: 15 April 1996     

A new approach to the investigation on the tonogenic groups of root cell walls

Acid-base properties of wheat, lupin, pea root cell walls were investigated. The roots of etiolated and green plants of different age were analysed by the potentiometric method. The ion exchange capacity of root cell walls (S_i) was estimated at various pH values (pH_i 2 to pH_i 12) and constant ion strength of the solution (10 mM). To analyse polysigmoid curves pH_i =f (S_i), Gregor's equation was used. It was shown that Gregor's model fits fairly well the experimental data. The total quantities of cation-exchange (S_t ^cat) and anion-exchange (S_t ^an) groups were determined in the root cell walls. It was shown that the quantity of anion exchange groups is varied through a small range (60–185 μmol/g dry wt.) in plant species tested, and that the S_t ^cat differs widely from 550 to 1300 μmol/g dry wt. For leguininous plants the quantity of acidic groups (fixed anions) is nearly twice as large as that for cereals. It was found that in seedlings as well as in plants, there are 3 cation-exchange groups and one anion-exchange group in root cell walls. The quantity of functional groups of each type (S^j) was estimated, and the corresponding values of n^j and pK_a ^j were calculated. It can be assumed that the groups with the pK_a ¹ ≈ 3.2 are amine groups, the ones with PK_a ² ≈ 5 are groups of galacturonic acid, the ones with pK_a ≈ 7.5 are the carboxyl groups of the second species, and the ones with pK_a ⁴ ≈ 40 are the phenolic groups. The values of dissociation constants (pK_a ^j) and S^j indicate that the root cell walls of wheat, lupin and pea are identical in qualitative structure of ionogenic groups but vary in the quantity of each ionogenic group. It was demonstrated that the summarized quantity of carboxyl groups (S² + S³) should be connected directly with the pH gradient in the extracellar space at the membrane surface. The gradient arises from ion-exchange reactions between cations of an outer medium and protons of the ionized carboxyl groups of the cell walls. The results suggest that, S_t ^cat and S_t ^an allow the quantitative estimation of ion exchange properties of the cell walls. The resulting parameters (S^j, pK_a ^j and n^j) allow prediction of changes in an ionic composition of a medium that bathes the cell membrane, during the first step of mineral nutrition uptake. This revised version was published online in June 2006 with corrections to the Cover Date.     

Thermal resistance parameters of the air environment at various altitudes

 The thermal properties of atmospheric air surrounding the human body at various altitudes are characterized with a system of parameters. This system comprises resistance of the air to convective heat transfer h _c ^–1, °C (W/m²)⁻¹ and to water vapour transfer h _D ^–1, s/m. The concept of ’evaporative resistance’h _e ^–1, hPa (W/m²)⁻¹) following the similarity of the processes is introduced. In obtaining the altitude dependencies of investigated paramters, a respective heat transfer equation expressing the rate of heat exchange at the boundary body surface – ambient air is applied. The use of the body thermal state of the established altitude dependencies is discussed. The concept of ’thermal stability’ related to the evaporative resistance parameter h _e ^–1 is introduced. This parameter is assumed as: (1) an indicator of the human body thermal stability and (2) distributor and predictor of environmental influence on the body thermal state. Received: 5 January 1996 / Accepted 5 November 1996     

Reconstruction of the carbon balance for microsites in a boreal oligotrophic pine fen, Finland

 Carbon dioxide (CO₂) exchange was studied at flark (minerotrophic hollow), lawn and hummock microsites in an oligotrophic boreal pine fen. Statistical response functions were constructed for the microsites in order to reconstruct the annual CO₂ exchange balance from climate data. Carbon accumulation was estimated from the annual net CO₂ exchange, methane (CH₄) emissions and leaching of carbon. Due to high water tables in the year 1993, the average carbon accumulation at the flark, Eriophorum lawn, Carex lawn and hummock microsites was high, 2.91, 6.08, 2.83 and 2.66 mol C m^–2, respectively, and for the whole peatland it was 5.66 mol m^–2 year^–1. During the maximum primary production period in midsummer, hummocks with low water tables emitted less methane than predicted from the average net ecosystem exchange (NEE), while the Carex lawns emitted slightly more. CH₄ release during that period corresponded to 16% of the contemporary NEE. Annual C accumulation rate did not correlate with annual CH₄ release in the microsites studied, but the total community CO₂ release seemed to be related to CH₄ emissions in the wet microsites, again excluding the hummocks. The dependence of CO₂ exchange dynamics on weather events suggests that daily balances in C accumulation are labile and can change from net carbon uptake to net release, primarily in high hummocks on fens under warmer, drier climatic conditions. Received: 16 August 1996 / Accepted: 30 November 1996     

Uptake of lactose and continuous lactic acid fermentation by entrapped non-growing Lactobacillus helveticus in whey permeate

 Continuous production of lactic acid from lactose has been carried out in a stirred-tank reactor with non-growing Lactobacillus helveticus entrapped in calcium alginate beads. A considerably longer operation half-life was obtained in a continuously operated reactor than in a batch-operated reactor. It is possible to simulate the action of entrapped non-growing cells on the basis of information from diffusion and kinetic experiments with suspended free cells. The simulation fit the experimental data over a broad range of substrate concentrations if the specific lactic acid production rate, q _P, was used as a variable parameter in the model. The dynamic mathematical model used is divided into three parts: the reactor model, which describes the mass balance in a continuously operated stirred-tank reactor with immobilized biomass, the mass-transfer model including both external diffusion and internal mass transfer, and the kinetic model for uptake of substrate on the basis of a Michaelis-Menten-type mechanism. From kinetic data obtained for free biomass experiments it was found, with the use of non-linear parameter estimation techniques, that the conversion rate of lactose by L. helveticus followed a Michaelis-Menten-type mechanism with K _S at half-saturation=0.22±0.01 g/l. The maximum specific lactose uptake rate for growing cells, q _S,max, varied between 4.32±0.02 g lactose g cells^-1 h^-1 and 4.89 ±0.02 g lactose g cells^-1 h^-1. The initial specific lactose uptake rate for non-growing cells, q _S,0, was found to be approximately 40% of the maximum specific lactose uptake rate for growing cells. Received: 4 October 1995/Received last revision: 23 April 1996/Accepted: 29 April 1996     

Investigation of exchange couplings in [Fe3S4]+ clusters by electron spin-lattice relaxation

3 S₄]⁺, S=1/2, composed of three, antiferromagnetically coupled high-spin ferric ions) by continuous wave (CW) and pulsed EPR techniques: Azotobacter vinelandii ferredoxin I, Desulfovibrio gigas ferredoxin II, and the 3Fe forms of Pyrococcus furiosus ferredoxin and aconitase. The 35 GHz (Q-band) CW EPR signals are simulated to yield experimental g tensors, which either had not been reported, or had been reported only at X-band microwave frequency. Pulsed X- and Q-band EPR techniques are used to determine electron spin-lattice (T ₁, longitudinal) relaxation times at several positions on the samples' EPR envelope over the temperature range 2–4.2 K. The T ₁ values vary sharply across the EPR envelope, a reflection of the fact that the envelope results from a distribution in cluster properties, as seen earlier as a distribution in g ₃ values and in ⁵⁷ Fe hyperfine interactions, as detected by electron nuclear double resonance spectroscopy. The temperature dependence of 1/T ₁ is analyzed in terms of the Orbach mechanism, with relaxation dominated by resonant two-phonon transitions to a doublet excited state at ∼20 cm⁻¹ above the doublet ground state for all four of these 3Fe proteins. The experimental EPR data are combined with previously reported ⁵⁷Fe hyperfine data to determine electronic spin exchange-coupling within the clusters, following the model of Kent et al. Their model defines the coupling parameters as follows: J ₁₃=J, J ₁₂=J(1+ε′), J ₂₃=J(1+ε), where J _ij is the isotropic exchange coupling between ferric ions i and j, and ε and ε′ are measures of coupling inequivalence. We have extended their theory to include the effects of ε′≠0 and thus derived an exact expression for the energy of the doublet excited state for any ε, ε′. This excited state energy corresponds roughly to ε J and is in the range 5–10 cm⁻¹ for each of these four 3Fe proteins. This magnitude of the product ε J, determined by our time-domain relaxation studies in the temperature range 2–4 K, is the same as that obtained from three other distinct types of study: CW EPR studies of spin relaxation in the range 5.5–50 K, NMR studies in the range 293–303 K, and static susceptibility measurements in the range 1.8–200 K. We suggest that an apparent disagreement as to the individual values of J and ε be resolved in favor of the values obtained by susceptibility and NMR (J≳200 cm⁻¹ and ε≳0.02 cm⁻¹ ), as opposed to a smaller J and larger ε as suggested in CW EPR studies. However, we note that this resolution casts doubt on the accepted theoretical model for describing the distribution in magnetic properties of 3Fe clusters. Received: 23 December 1999 / Accepted: 8 March 2000     

Isotope exchange reactions with radiolabeled sulfur compounds in anoxic seawater

The isotope exchange between³⁵S-labeled sulfur compounds of sulfate (SO₄ ^2–), elemental sulfur (S⁰), polysulfide (S_n ^2–), hydrogen sulfide (HS^–: H₂S + HS^– + S^2–), iron sulfide (FeS), and pyrite (FeS₂) was studied at pH 7.6 and 20 °C in anoxic, sterile seawater. Isotope exchange was observed between S⁰, S₂ ^2– HS^–, and FeS, but not between³⁵S labeled SO₄ ^2– or FeS₂ and the other sulfur compounds. Polysulfide mediated the isotope exchange between S⁰ and bisulfide (HS^–). The isotope exchange between S⁰ and S_n ^2–) reached 50% of equilibrium within < 2 min while exchange between S₂ ^2– and HS^– approached equilibrium within 0.5-1 h. In all the experiments HS^–, revealed a fraction exchange from 0.79 to 1.00. Isotope exchange between S^2– and FeS took place only via S₂ ^2– and/or HS^–. The isotope exchange between iron sulfide and the other sulfur compounds was not complete within 24 h as shown by a fraction exchange of 0.07–0.83. This lack of equilibrium (fraction exchange < 1) was due to the isotope exchange between dissolved compounds and surfaces of sulfur particles. The isotopic exchange reactions limit the usefulness of radiotracers in process studies of the inorganic sulfur species. Exchange reactions will also affect the stable isotope distribution among the sulfur species. The kinetics of the isotopic exchange reactions, however, depend on both pH and temperature.     

Crossover of a high-spin (S=7/2, 3/2) to a low-spin (S=1/2) iron-selenium cluster in FA and FB of photosystem I on rebinding of PsaC onto P700-FX cores

 PsaC is a tightly bound ferredoxin in the Photosystem I (PS I) reaction center which contains two [4Fe-4S] clusters named F_A and F_B. We recently proposed that the mixed-ligand F_B cluster in C14D_PsaC and the mixed-ligand F_A cluster in C51D_PsaC exist in a spin state of S=3/2, and that a spin state crossover to S=1/2 occurs when the PsaC mutants are rebound onto P700-F_X cores. Since EPR signals from a highly rhombic S=3/2 spin state can be difficult to study, wild-type PsaC was reconstituted with iron and selenium to introduce an easily detected S=7/2 spin state similar to that shown for Clostridial ferredoxin. When the unbound [4Fe-4Se] PsaC was chemically reduced, a sharp derivative resonance was found at g=5.171 attributed to the excited ±3/2 doublet from an S=7/2 spin multiplet. An additional peak was found at g=5.616 attributed to the superimposed ±1/2 and ±3/2 doublets from a highly rhombic S=3/2 spin multiplet, and an axial set of resonances found around g=2.0 attributed, in part, to a classical S=1/2 spin state. When the [4Fe-4Se] PsaC was rebound onto P700-F_X cores, the spin population derived from the S=7/2 and 3/2 spin states was negligible. Illumination of the rebuilt PS I complex at 15 K resulted in two rhombic sets of resonances, one with g values of 2.043, 1.941 and 1.854, diagnostic of F_A, and the other with g values of 2.067, 1.941 and 1.878, diagnostic of F_B. Chemical reduction with sodium dithionite at pH 10.5 or photoaccumulation by freezing during illumination resulted in a set of resonances with g values of 2.046, 1.938, 1.920 and 1.883, characteristic of a spin-coupled F_A ^–/F_B ^– pair. The spin state crossover in this iron chalcogenide cluster is the first known to be induced by protein-protein association and reinforces the hypothesis that an S=3/2 to 1/2 crossover occurs in the PS I-rebound mutants C14D_PsaC and C51D_PsaC. Received: 6 August 1996 / Accepted: 28 December 1996     

Value of the β transfer integral in Fe-S clusters

 The purpose of this commentary is to stress that in mixed-valence systems, electron localization and electron delocalization are competitive, the first being most often the stronger. In iron-sulfur clusters where a delocalized Fe(II)Fe(III) pair of maximal spin exists, a minimum value of 1200 cm^–1 is proposed for the resonance integral β parametrizing the electron delocalization. Received: 12 January 1996 / Accepted: 23 January 1996     

Mono-manganese mechanism of the photosytem II water splitting reaction by a unique Mn4Ca cluster

The molecular mechanism of the water oxidation reaction in photosystem II (PSII) of green plants remains a great mystery in life science. This reaction is known to take place in the oxygen evolving complex (OEC) incorporating four manganese, one calcium and one chloride cofactors, that is light-driven to cycle four intermediates, designated S₀ through S₄, to produce four protons, five electrons and lastly one molecular oxygen, for indispensable resources in biosphere. Recent advancements of X-ray crystallography models established the existence of a catalytic Mn₄Ca cluster ligated by seven protein amino acids, but its functional structure is not yet resolved. The ¹⁸O exchange rates of two substrate water molecules were recently measured for four S_i-state samples (i = 0-3) leading to ³⁴O₂ and ³⁶O₂ formations, revealing asymmetric substrate binding sites significantly depending on the S_i-state. In this paper, we present a chemically complete model for the Mn₄Ca cluster and its surrounding enzyme field, which we found out from some possible models by using the hybrid density functional theoretic geometry optimization method to confirm good agreements with the 3.0 Å resolution PSII model [B. Loll, J. Kern, W. Saenger, A. Zouni , J. Biesiadka, Nature 438 (2005) 1040-1044] and the S-state dependence of ¹⁸O exchange rates [W. Hillier and T. Wydrzynski, Phys. Chem. Chem. Phys. 6 (2004) 4882-4889]. Furthermore, we have verified that two substrate water molecules are bound to asymmetric cis-positions on the terminal Mn ion being triply bridged (μ-oxo, μ-carboxylato, and a hydrogen bond) to the Mn₃CaO₃(OH) core, by developing a generalized theory of ¹⁸O exchange kinetics in OEC to obtain an experimental evidence for the cross exchange pathway from the slow to the fast exchange process. Some important experimental data will be discussed in terms of this model and its possible tautomers, to suggest that a cofactor, Cl⁻ ion, may be bound to CP43-Arg357 nearby Ca²⁺ ion and that D1-His337 may be used to trap a released proton only in the S₂-state.     

Redox chemistry of biological tungsten: an EPR study of the aldehyde oxidoreductase from Pyrococcus furiosus

 Aldehyde:ferredoxin oxidoreductase (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus is a homodimeric protein. Each subunit carries one [4Fe-4S] cubane and a novel tungsten cofactor containing two pterins. A single iron atom bridges between the subunits. AOR has previously been studied with EPR spectroscopy in an inactive form known as the red tungsten protein (RTP): reduced RTP exhibits complex EPR interaction signals. We have now investigated the active enzyme AOR with EPR, and we have found an S = 1/2 plus S = 3/2 spin mixture from a non-interacting [4Fe-4S]¹⁺ cluster in the reduced enzyme. Oxidized AOR affords EPR signals typical for W(V) with g–values of 1.982, 1.953, and 1.885. The W(V) signals disappear at a reduction potential E _m,7.5 of +180 mV. This unexpectedly high value indicates that the active-site redox chemistry is based on the pterin part of the cofactor. Received: 18 December 1995 / Accepted: 26 March 1996     

The Relative Rates of Thiol–Thioester Exchange and Hydrolysis for Alkyl and Aryl Thioalkanoates in Water

This article reports rate constants for thiol–thioester exchange (k _ex), and for acid-mediated (k _a), base-mediated (k _b), and pH-independent (k _w) hydrolysis of S-methyl thioacetate and S-phenyl 5-dimethylamino-5-oxo-thiopentanoate—model alkyl and aryl thioalkanoates, respectively—in water. Reactions such as thiol–thioester exchange or aminolysis could have generated molecular complexity on early Earth, but for thioesters to have played important roles in the origin of life, constructive reactions would have needed to compete effectively with hydrolysis under prebiotic conditions. Knowledge of the kinetics of competition between exchange and hydrolysis is also useful in the optimization of systems where exchange is used in applications such as self-assembly or reversible binding. For the alkyl thioester S-methyl thioacetate, which has been synthesized in simulated prebiotic hydrothermal vents, k _a = 1.5 × 10⁻⁵ M⁻¹ s⁻¹, k _b = 1.6 × 10⁻¹ M⁻¹ s⁻¹, and k _w = 3.6 × 10⁻⁸ s⁻¹. At pH 7 and 23°C, the half-life for hydrolysis is 155 days. The second-order rate constant for thiol–thioester exchange between S-methyl thioacetate and 2-sulfonatoethanethiolate is k _ex = 1.7 M⁻¹ s⁻¹. At pH 7 and 23°C, with [R″S(H)] = 1 mM, the half-life of the exchange reaction is 38 h. These results confirm that conditions (pH, temperature, pK _a of the thiol) exist where prebiotically relevant thioesters can survive hydrolysis in water for long periods of time and rates of thiol–thioester exchange exceed those of hydrolysis by several orders of magnitude.     

Effects of physical factors on photosynthesis by the Antarctic liverwort Marchantia berteroana

 Effects of irradiance, temperature and water availability on respiration and photosynthesis in a maritime Antarctic liverwort, Marchantia berteroana, were investigated. Carbon dioxide exchange was measured using an infra-red gas analysis system under controlled conditions. The relationships between respiration, photosynthesis, irradiance and temperature were modelled. Application of these models to year-round micro-climate data provided an estimate of yearly net productivity of 823 (SE=75) mg C⋅g^-1 ash-free dry weight. year^-1; this is somewhat higher than figures obtained for other Antarctic cryptogams. Desiccation had a highly adverse affect on Marchantia. Photosynthetic capacity was reduced below a water content of 12 g⋅g^-1 afdw, and there was only a limited recovery (ca. 10%) after dehydration. Freezing also caused a great reduction in photosynthesis, although the model suggested that photosynthesis at sub-zero temperatures is likely. It is suggested that seasonality in the photosynthetic capacity and the survival of sub-zero temperatures might be important. It is concluded that Marchantia is a relatively productive Antarctic cryptogam that may dominate favourable areas, but that its low tolerance of environmental stress, particularly desiccation, limits its distribution to relatively mild habitats. Received: 8 January 1996/Accepted: 12 May 1996     

Microbial sensors for naphthalene using Sphingomonas sp. B1 or Pseudomonas fluorescens WW4

 Amperometric biosensors for naphthalene were developed using either immobilized Sphingomonas sp. B1 or Pseudomonas fluorescens WW4 cells. The microorganisms were immobilized within a polyurethane-based hydrogel, which was used for a microbial biosensor for the first time. Both strains were shown to be equally suited for the quantification of naphthalene in aqueous solutions. The biosensors were tested in a flow-through system and a stirred cell (batch method). In both systems a linear response down to the detection limit was obtained. Measurements in the flow-through system gave sensitivities of up to 1.2 nA mg⁻¹ l⁻¹ and a linear range from 0.03 mg/l to 2.0 mg/l. The response time (t ₉₅) was 2 min and the sample throughput six per hour; the repeatability was within ±5 %. With the batch method, sensitivities of between 3 nA mg⁻¹ l⁻¹ and 5 nA mg⁻¹l⁻¹ and a linear range of 0.01–3.0 mg/l were obtained; the response time was between 3 min and 5 min. The sensors reached an operational lifetime of up to 20 days. The sensitivity of both sensors for naphthalene was, in most cases, more than four times higher than for various other substrates. Received: 18 October 1995/Received revision: 22 December 1995/Accepted: 22 January 1996     

Biodegradation kinetics of monoterpenes in liquid and soil-slurry systems

Batch experiments were conducted to evaluate the biodegradation rates of limonene, α-pinene, γ-terpinene, terpinolene and α-terpineol at 23 °C under aerobic conditions. Biodegradation was demonstrated by the depletion of monoterpene mass, CO₂ production and a corresponding increase in biomass. Monoterpene degradation in liquid cultures devoid of soil followed Monod kinetics. The maximum specific growth rate (μ_max) was 0.02 h⁻¹ and 0.06 h⁻¹ and the half-velocity constant (K _s ) varied from 32 mg/l to 3 mg/l for the limonene and α-terpineol respectively. The recovery of monoterpenes by solvent extraction from autoclaved and azide-amended soil-slurry samples decreased over time and ranged from 69% to 73% for 120 h of incubation period. Although a significant fraction of monoterpene hydrocarbon could not be extracted, mineralization of these compounds in the soil-slurry systems took place, as shown by CO₂ production. The soil-normalized degradation rates for the hydrocarbon monoterpenes ranged from 0.6 μg g⁻¹ h⁻¹ to 2.1 μg g⁻¹ h⁻¹. A kinetic model – which combined monoterpene biodegradation in the liquid phase and net desorption – was developed and applied to data obtained from soil-slurry assays. Received: 10 September 1996 / Received revision: 16 December 1996 / Accepted: 10 January 1997