The influence of temperature on the function of renal cortical slices frozen in various cryoprotectants

Renal cortical slices were frozen to various subzero temperatures after treatment with 2.1 M of one of three cryoprotectants, dimethyl sulfoxide (Me2SO), ethylene glycol, or glycerol. The effects on tissue [K+]/[Na+] of cooling to these temperatures were tested (using identical procedure times, cooling rates, and warming rates) by holding the slices at each experimental temperature for appropriate periods of time prior to rewarming. The effects of the holding time were assessed by comparison with slices which were cooled and rewarmed with no intermediate holding time. Slices treated with ethylene glycol or glycerol were found to exhibit a continuous decrease in [K+]/[Na+] with lowered temperatures, in contrast to those treated with Me2SO. Slices treated with Me2SO actually experienced a continuous increase in [K+]/[Na+] with lowered temperature (-12 to -33 degrees C). Me2SO does exhibit toxic effects at subzero temperatures. Adverse effects of holding time on viability are seen for Me2SO-treated slices at higher subzero temperatures. These effects were alleviated as the temperature is reduced, suggesting that temperature has a greater effect on survival of renal cortical tissue than Me2SO concentration. However, the toxicity observed at higher subzero temperatures is expected to be of importance, particularly for slowly cooled tissues which are exposed to these temperatures for relatively long periods of time.     

Damage in Escherichia coli cells treated with a combination of high hydrostatic pressure and subzero temperature

The relationship between membrane permeability, changes in ultrastructure, and inactivation in Escherichia coli strain K-12TG1 cells subjected to high hydrostatic pressure treatment at room and subzero temperatures was studied. Propidium iodide staining performed before and after pressure treatment made it possible to distinguish between reversible and irreversible pressure-mediated cell membrane permeabilization. Changes in cell ultrastructure were studied using transmission electron microscopy (TEM), which showed noticeable condensation of nucleoids and aggregation of cytosolic proteins in cells fixed after decompression. A novel technique used to mix fixation reagents with the cell suspension in situ under high hydrostatic pressure (HHP) and subzero-temperature conditions made it possible to show the partial reversibility of pressure-induced nucleoid condensation. However, based on visual examination of TEM micrographs, protein aggregation did not seem to be reversible. Reversible cell membrane permeabilization was noticeable, particularly for HHP treatments at subzero temperature. A correlation between membrane permeabilization and cell inactivation was established, suggesting different mechanisms at room and subzero temperatures. We propose that the inactivation of E. coli cells under combined HHP and subzero temperature occurs mainly during their transiently permeabilized state, whereas HHP inactivation at room temperature is related to a balance of transient and permanent permeabilization. The correlation between TEM results and cell inactivation was not absolute. Further work is required to elucidate the effects of pressure-induced damage on nucleoids and proteins during cell inactivation.     

Fast photochemical reactions of cytochrome P450 at subzero temperatures.

Several reactions of the cytochrome P450 multi-step cycle have been studied by fast light activation combined with subzero temperatures. A flash device was adapted to an Aminco-Chance DW 2 spectrophotometer equipped for subzero temperature thermostatisation. The first electron can be introduced into the cycle by non specific reducing agents such as reduced flavin mononucleotide (FMNH2) or methylviologen radical (MV.). This first reduction remains a fast process even at subzero temperatures. The oxy-compound Fe2+-O2 can thus be formed either directly from Fe2+ or via the photodissociation of the carboxy-ferro adduct. Fe2+-O2 is stable at subzero temperatures towards spontaneous autoxidation as well as further reduction by FMNH2 or MW.. In addition, the recombination of CO after flash photodissociation of Fe2+-CO was used to study in more details the specific behaviors of the purified microsomal cytochrome.     

A simple method for determining the boundary layer resistance in leaf cuvettes

Abstract. A new method is described for determining the boundary layer resistance over wet filter paper exposed within a leaf cuvette, based on the energy balance of the filler paper. The boundary layer resistance is calculated by an iterative procedure from measurements of the relative humidity and temperature of the air in the cuvette. Comparisons between the new and the conventional method, involving measurement of the filter paper temperature, show close agreement.
To simplify the method further, a graph has been constructed for the relationship between boundary layer resistance and cuvette relative humidity at temperatures from 15 to 35°C, determined at one value of the ratio of the flow rate through the cuvette to the filter paper area.
An analysis of errors suggests that the new method is less sensitive than the conventional to errors in temperature and humidity.     

Damage in Escherichia coli Cells Treated with a Combination of High Hydrostatic Pressure and Subzero Temperature

The relationship between membrane permeability, changes in ultrastructure, and inactivation in Escherichia coli strain K-12TG1 cells subjected to high hydrostatic pressure treatment at room and subzero temperatures was studied. Propidium iodide staining performed before and after pressure treatment made it possible to distinguish between reversible and irreversible pressure-mediated cell membrane permeabilization. Changes in cell ultrastructure were studied using transmission electron microscopy (TEM), which showed noticeable condensation of nucleoids and aggregation of cytosolic proteins in cells fixed after decompression. A novel technique used to mix fixation reagents with the cell suspension in situ under high hydrostatic pressure (HHP) and subzero-temperature conditions made it possible to show the partial reversibility of pressure-induced nucleoid condensation. However, based on visual examination of TEM micrographs, protein aggregation did not seem to be reversible. Reversible cell membrane permeabilization was noticeable, particularly for HHP treatments at subzero temperature. A correlation between membrane permeabilization and cell inactivation was established, suggesting different mechanisms at room and subzero temperatures. We propose that the inactivation of E. coli cells under combined HHP and subzero temperature occurs mainly during their transiently permeabilized state, whereas HHP inactivation at room temperature is related to a balance of transient and permanent permeabilization. The correlation between TEM results and cell inactivation was not absolute. Further work is required to elucidate the effects of pressure-induced damage on nucleoids and proteins during cell inactivation.     

Influence of soil moisture on egg cold hardiness in the migratory locust Locusta migratoria (Orthoptera: Acridiidae)

Abstract.  The present study investigates the influence of environmental moisture on cold hardiness of the migratory locust, Locusta migratoria . The water content of locust eggs kept in soil at 30 °C varies according to the moisture content of the substrate. In turn, it can significantly affect the supercooling point of locust eggs (range from −26 to −14.8 °C) and the mortality when exposed to subzero temperatures. Environmental moisture influences the supercooling capacity of eggs and their survival at low temperature. When locust eggs of the same water content are exposed to subzero temperatures under different soil moistures, their mortality varies between short-time exposure and long-time exposure at subzero temperatures. Given a short-time exposure, mortality in wet soil is lower than in dry soil due to the buffering effect of soil water against temperature change. The pattern of egg mortality is reversed after long-time exposure at low temperature, suggesting that inoculative freezing may be an important mortality factor. It is suggested that interactions between soil moisture and low temperature can influence the cold hardiness of locust eggs, and partial dehydration is beneficial to over-wintering eggs of the migratory locust.     

A system for studying the effects of naturally-occurring subzero temperatures on winter barley

Thermostatically-controlled, electrical soil heating cables were used to examine the effects of subzero temperatures on winter barley growing in plots outdoors. Plants in plots without heating cables were exposed to naturally-occurring subzero temperatures (unheated), while those in corresponding plots with the cables (heated) were protected from such temperatures. Measurements of soil, plant and air temperatures in heated and unheated plots showed that the system can, at least with the temperatures encountered during the measurement periods, prevent plant temperature from falling below the temperature set on the thermostat. A field experiment involving different cultivars and sowing dates showed that subzero temperatures experienced did not significantly affect individual plant grain yield or the number of fertile ears produced per plant. However, small, but statistically significant, effects of naturally-occurring subzero temperatures were found in relation to the number of grains per ear, thousand grain weight and harvest index. The nature of these effects varied, and were dependent upon cultivar and/or sowing date. Subzero temperatures were also responsible for reducing the numbers of established plants in late-sown plots through soil heaving. Possible uses of the soil heating cable system for temperature-related studies in the Gramineae are discussed.     

Cold tolerance during larval development: effects on the thermal distribution limits of Leptinotarsa decemlineata

Insects' cold tolerance during their development is a surprisingly understudied subject in ecology, despite the fact that subzero temperatures during the growing season are common at high altitudes and latitudes. Subzero temperatures can have detrimental effects on organisms, restricting a species' range. This study addresses the question whether night frosts during the growing season have an instant or delayed negative impact on larval mortality of the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae). We also tested whether populations from the centre (Poland) and margins (Russia) of the distribution range of L. decemlineata differ in their responses to subzero exposure and a low rearing temperature. Larvae of three ages were subjected to a subzero temperature (−4 °C for 3 h simulating night frost) twice, after which they were reared on a fluctuating temperature regime of 10–15 °C. These rearing conditions imitated cool summer temperatures beyond the beetles' current range, such as in Finland. Individuals of both populations were highly cold tolerant, as only 3.1% of larvae died immediately following the subzero treatment. Nonetheless, the low rearing temperature was harmful to beetles of both populations. It caused high larval (ca. 90%) and overwintering (ca. 80%) mortality. As beetle performance was affected solely by rearing temperature, low temperatures during the growing season rather than night frosts apparently retard the beetle's northern expansion.     

The effect of low temperatures on the viability of human epidermal keratinocytes found at different stages of differentiation

The aim of this study was a comparative analysis to the degree of stability of human epidermal cells found at different stages of differentiation to low temperatures. The effect of different subzero temperatures of liquid nitrogen vapor on keratinocytes found both in human skin fragments and as isolated cells extracted from skin fragments has been studied. The degree of stability of epidermal cells low temperatures was evaluated by their ability to form a multilayer stratum in culture; hence this phenomenon explains the survival of a sufficient amount of proliferative cells after exposure to subzero temperatures. Quantitative analysis of the ratio of epidermal stem, transitory and differentiated cells in a population of viable cells before and after exposure to low temperatures were determined using antibodies corresponding to their different stages of differentiation. The results of this research show that the stability of human epidermal cells to low temperature differs depending on their stage of differentiation both in situ and in vitro. Epidermal stem cells and transitory cells are more stable than differentiated cells.     

Exposure to subfreezing temperature and a freeze-thaw cycle affect freezing tolerance of winter wheat in saturated soil

Winter wheat is sown in the autumn and harvested the following summer, necessitating the ability to survive subfreezing temperatures for several months. Autumn months in wheat-growing regions typically experience significant rainfall and several days or weeks of mild subfreezing temperatures at night, followed by above-freezing temperatures in the day. Hence, the wheat plants usually are first exposed to potentially damaging subfreezing temperatures when they have high moisture content, are growing in very wet soil, and have been exposed to freeze-thaw cycles for a period of time. These conditions are conducive to freezing stresses and plant responses that are different from those that occur under lower moisture conditions without freeze-thaw cycles. This study was conducted to investigate the impact of mild subfreezing temperature and a freeze-thaw cycle on the ability of 22 winter wheat cultivars to tolerate freezing in saturated soil. Seedlings that had been acclimated at +4°C for 5 weeks in saturated soil were frozen to potentially damaging temperatures under three treatment conditions: (1) without any subzero pre-freezing treatment; (2) with a 16-h period at ?3°C prior to freezing to potentially damaging temperatures; and (3) with a freeze-thaw cycle of ?3°C for 24 h followed by +4°C for 24 h, followed by a 16-h period at ?3°C prior to freezing to potentially damaging temperatures. In general, plants that had been exposed to the freeze-thaw cycle survived significantly more frequently than plants frozen under the other two treatments. Plants that had been exposed to 16 h at ?3° (without the freeze-thaw cycle) before freezing to potentially damaging temperatures survived significantly more frequently than plants that were frozen to potentially damaging temperatures without a subzero pre-freezing treatment. These results indicated that cold-acclimated wheat plants actively acclimate to freezing stress while exposed to mild subfreezing temperatures, and further acclimate when allowed to thaw at +4°C for 24 h. The cultivar Norstar had the lowest LT₅₀ (temperature predicted to be lethal to 50% of the plants) of the 22 cultivars when frozen with either of the subzero pre-freezing treatments, but several cultivars had lower LT₅₀ scores than Norstar when frozen without a subzero pre-freezing treatment. We conclude it may be possible to improve winterhardiness of wheat grown in saturated soil by combining the ability to effectively respond to mild subzero pre-freezing temperatures with a greater ability to withstand freezing to damaging temperatures without a subzero pre-freezing exposure.     

Proteomic analysis of <Emphasis Type="Italic">Psychrobacter</Emphasis><Emphasis Type="Italic"> cryohalolentis</Emphasis> K5 during growth at subzero temperatures

It is crucial to examine the physiological processes of psychrophiles at temperatures below 4°C, particularly to facilitate extrapolation of laboratory results to in situ activity. Using two dimensional electrophoresis, we examined patterns of protein abundance during growth at 16, 4, and −4°C of the eurypsychrophile Psychrobacter cryohalolentis K5 and report the first identification of cold inducible proteins (CIPs) present during growth at subzero temperatures. Growth temperature substantially reprogrammed the proteome; the relative abundance of 303 of the 618 protein spots detected (∼31% of the proteins at each growth temperature) varied significantly with temperature. Five CIPs were detected specifically at −4°C; their identities (AtpF, EF-Ts, TolC, Pcryo_1988, and FecA) suggested specific stress on energy production, protein synthesis, and transport during growth at subzero temperatures. The need for continual relief of low-temperature stress on these cellular processes was confirmed via identification of 22 additional CIPs whose abundance increased during growth at −4°C (relative to higher temperatures). Our data suggested that iron may be limiting during growth at subzero temperatures and that a cold-adapted allele was employed at −4°C for transport of iron. In summary, these data suggest that low-temperature stresses continue to intensify as growth temperatures decrease to −4°C.     

Networks of polysaccharides with hydrophilic and hydrophobic characteristics in the presence of co-solute

The present investigation deals with the changing network morphology of agarose and high methoxy pectin when mixed with polydextrose as co-solute at concentrations varying up to high level of solids. Thermomechanical analysis and micro-imaging were performed using small deformation dynamic oscillation in shear, modulated differential scanning calorimetry and environment scanning electron microscopy. Fourier transform infrared spectroscopy and wide angle X-ray diffraction were practised to examine the nature of interactions between polymer and co-solute, and the extent of amorphicity of preparations. We observed a decline in the mechanical strength of aqueous agarose preparations upon addition of high levels of polydextrose, which should be attributed to reduced enthalpic content of the coil-to-helix transition of the polysaccharide network. Glass transition phenomena were observed at subzero temperatures in condensed preparations, hence further arguing for the formation of a lightly cross-linked agarose network with changing solvent quality. High levels of co-solute induce formation of weak pectin gels at elevated temperatures (even at 95°C), which with lowering temperature exhibit increasing strength. This results in the formation of rubbery pectin gels at ambient temperature, which upon controlled cooling to subzero temperatures convert to a clear glass earlier than the agarose counterparts.     

Water permeability of yeast cells at sub-zero temperatures

Summary A combined cryomicroscopic-multiple nonlinear regression analysis technique has been used to determine the water permeability of the yeast cellSaccharomyces cerevisiae during freezing. The time rate of change in volume of supercooled yeast cells was photographically monitored using a cryomicroscope which is capable of controlling in a programmable manner both the temperature and the time rate of change in temperature of the cell suspension being studied. Multiple nonlinear regression analysis together with a thermodynamic model of cell water transport during freezing was then used to statistically deduce the subzero temperature dependence of the cell water permeability. The water permeability process forS. cerevisiae being cooled at subzero temperatures was found to be rate-limited by the passage of water through either the plasmalemma, the cell wall, or a combination of these two permeability barriers. The hydraulic water permeability coefficient for yeast at 20°C is approximately 1–2×10^–13 cm³/dyne sec, if extrapolation from subzero temperatures to room temperature is permissible, while the apparent activation energy governing the permeability process at subzero temperatures is approximately 45–68 kJ/mol (11–16 kcal/mol). Appendix I: Volumetric Changes in Yeast Cells during Freezing at Constant Cooling Rates     

Bacterial genome replication at subzero temperatures in permafrost

Microbial metabolic activity occurs at subzero temperatures in permafrost, an environment representing ∼25% of the global soil organic matter. Although much of the observed subzero microbial activity may be due to basal metabolism or macromolecular repair, there is also ample evidence for cellular growth. Unfortunately, most metabolic measurements or culture-based laboratory experiments cannot elucidate the specific microorganisms responsible for metabolic activities in native permafrost, nor, can bulk approaches determine whether different members of the microbial community modulate their responses as a function of changing subzero temperatures. Here, we report on the use of stable isotope probing with ¹³C-acetate to demonstrate bacterial genome replication in Alaskan permafrost at temperatures of 0 to −20 °C. We found that the majority (80%) of operational taxonomic units detected in permafrost microcosms were active and could synthesize ¹³C-labeled DNA when supplemented with ¹³C-acetate at temperatures of 0 to −20 °C during a 6-month incubation. The data indicated that some members of the bacterial community were active across all of the experimental temperatures, whereas many others only synthesized DNA within a narrow subzero temperature range. Phylogenetic analysis of ¹³C-labeled 16S rRNA genes revealed that the subzero active bacteria were members of the Acidobacteria, Actinobacteria, Chloroflexi, Gemmatimonadetes and Proteobacteria phyla and were distantly related to currently cultivated psychrophiles. These results imply that small subzero temperature changes may lead to changes in the active microbial community, which could have consequences for biogeochemical cycling in permanently frozen systems.     

Cryoenzymology of human plasmin catalysis: comparison of cryosolvents and reactions with nitrophenyl ester and anilide substrates

A comparative study of nucleophilic (methanol), aprotic (dimethyl sulfoxide), and protic but non nucleophilic (ethylene glycol, ethylene glycol/dimethylformamide) solvents on the catalytic and structural properties of human plasmin has been made. All four solvent systems are potentially suitable as cryosolvents for plasmin catalysis at subzero temperatures although the solubility of plasmin is limited in the methanol and dimethyl sulfoxide systems. Each cryosolvent system caused minor effects on the catalytic properties of the enzyme, which could be rationalized in terms of the known physical properties of the cosolvent. Solvent systems containing ethylene glycol induce a minor conformational change which increases the catalytic efficiency of plasmin. The cosolvent effects on Km and Ki indicate that electrostatic interactions dominate the binding of both substrates and inhibitors such as benzamidine. A change in slope of the Arrhenius plots for catalysis, reflecting a temperature-induced isomerization, is observed around 0 degree C; the energies of activation being 13 +/- 2 kcal mol-1 at higher temperatures and 19 +/- 2 kcal mol-1 at subzero temperatures, and essentially independent of solvent. Deacylation was shown to be the rate-limiting step in the hydrolysis of specific p-nitrophenyl ester substrates. Previous stopped-flow studies at room temperature provided observations suggesting that a tetrahedral intermediate could be detected in the plasmin-catalyzed hydrolysis of p-nitroanilide substrates. Experiments at subzero temperatures with such substrates failed to reveal any buildup of a tetrahedral intermediate under the experimental conditions.     

Membrane permeability parameters for freezing of stallion sperm as determined by Fourier transform infrared spectroscopy

Cellular membranes are one of the primary sites of injury during freezing and thawing for cryopreservation of cells. Fourier transform infrared spectroscopy (FTIR) was used to monitor membrane phase behavior and ice formation during freezing of stallion sperm. At high subzero ice nucleation temperatures which result in cellular dehydration, membranes undergo a profound transition to a highly ordered gel phase. By contrast, low subzero nucleation temperatures, that are likely to result in intracellular ice formation, leave membrane lipids in a relatively hydrated fluid state. The extent of freezing-induced membrane dehydration was found to be dependent on the ice nucleation temperature, and showed Arrhenius behavior. The presence of glycerol did not prevent the freezing-induced membrane phase transition, but membrane dehydration occurred more gradual and over a wider temperature range. We describe a method to determine membrane hydraulic permeability parameters (E_Lp, Lpg) at subzero temperatures from membrane phase behavior data. In order to do this, it was assumed that the measured freezing-induced shift in wavenumber position of the symmetric CH₂ stretching band arising from the lipid acyl chains is proportional to cellular dehydration. Membrane permeability parameters were also determined by analyzing the H₂O-bending and -libration combination band, which yielded higher values for both E_Lp and Lpg as compared to lipid band analysis. These differences likely reflect differences between transport of free and membrane-bound water. FTIR allows for direct assessment of membrane properties at subzero temperatures in intact cells. The derived biophysical membrane parameters are dependent on intrinsic cell properties as well as freezing extender composition.     

Freezing-induced cellular and membrane dehydration in the presence of cryoprotective agents

Abstract

FTIR and cryomicroscopy have been used to study mouse embryonic fibroblast cells (3T3) during freezing in the absence and presence of DMSO and glycerol. The results show that cell volume changes as observed by cryomicroscopy typically end at temperatures above ?15°C, whereas membrane phase changes may continue until temperatures as low as ?30°C. This implies that cellular dehydration precedes dehydration of the bound water surrounding the phospholipid head groups. Both DMSO and glycerol increase the membrane hydraulic permeability at subzero temperature and reduce the activation energy for water transport. Cryoprotective agents facilitate dehydration to continue at low subzero temperatures thereby decreasing the incidence of intracellular ice formation. The increased subzero membrane hydraulic permeability likely plays an important role in the cryoprotective action of DMSO and glycerol. In the presence of DMSO water permeability was found to be greater compared to that in the presence of glycerol. Two temperature regimes were identified in an Arrhenius plot of the membrane hydraulic permeability. The activation energy for water transport at temperature ranging from 0 to ?10°C was found to be greater than that below ?10°C. The non-linear Arrhenius behavior of Lp has been implemented in the water transport model to simulate cell volume changes during freezing. At a cooling rate of 1°C min^-1, ～5% of the initial osmotically active water volume is trapped inside the cells at ?30°C.     

Synergistic and antagonistic effects of combined subzero temperature and high pressure on inactivation of Escherichia coli

The combined effects of subzero temperature and high pressure on the inactivation of Escherichia coli K12TG1 were investigated. Cells of this bacterial strain were exposed to high pressure (50 to 450 MPa, 10-min holding time) at two temperatures (-20 degrees C without freezing and 25 degrees C) and three water activity levels (a(w)) (0.850, 0.992, and ca. 1.000) achieved with the addition of glycerol. There was a synergistic interaction between subzero temperature and high pressure in their effects on microbial inactivation. Indeed, to achieve the same inactivation rate, the pressures required at -20 degrees C (in the liquid state) were more than 100 MPa less than those required at 25 degrees C, at pressures in the range of 100 to 300 MPa with an a(w) of 0.992. However, at pressures greater than 300 MPa, this trend was reversed, and subzero temperature counteracted the inactivation effect of pressure. When the amount of water in the bacterial suspension was increased, the synergistic effect was enhanced. Conversely, when the a(w) was decreased by the addition of solute to the bacterial suspension, the baroprotective effect of subzero temperature increased sharply. These results support the argument that water compression is involved in the antimicrobial effect of high pressure. From a thermodynamic point of view, the mechanical energy transferred to the cell during the pressure treatment can be characterized by the change in volume of the system. The amount of mechanical energy transferred to the cell system is strongly related to cell compressibility, which depends on the water quantity in the cytoplasm.     

Effect of subzero incubation on fluoride binding by laccase

We have refined a useful incubation method for preparing adducts of tree laccase with inhibitor anions; however, the technique should have wider application. The procedure involves incubating a previously frozen aqueous solution at subzero temperature. Factors influencing the amount of adduct that forms include the nature of the buffer, pH changes that occur at subzero temperatures and the presence of glycerol. In the absence of a glassing agent like glycerol, the phase separation and solute pooling that occur with ice formation help drive adduct formation. The results reveal several important factors to consider in designing or interpreting low-temperature spectroscopic investigations.     

Changes in the coordination geometry of the active-site metal during catalysis of benzylpenicillin hydrolysis by Bacillus cereus beta-lactamase II

Rapid-scanning stopped-flow spectroscopy (425-700 nm) has been used to study spectral changes in cobalt(II)-substituted Bacillus cereus beta-lactamase II during the binding and hydrolysis of benzylpenicillin. The experiments were carried out in aqueous solution over a temperature range of 3-20 degrees C. Three metallointermediates have been characterized by their visible absorption spectra. Two of them have visible absorption spectra identical with the intermediates ES1 and ES2 previously observed at subzero temperatures in a mixed aqueous/organic solvent [Bicknell, R., & Waley, S.G. (1985) Biochemistry 24, 6876-6887]. In addition, the branched kinetic pathway observed with the zinc(II) and cobalt(II) beta-lactamase II at subzero temperatures has been shown to occur with the cobalt(II)-substituted enzyme in aqueous solution at above-zero temperatures; thus, at pH 6.0 and 3 degrees C, the rate and equilibrium constants are readily determined for the reaction scheme: (Formula: see text). A third transient intermediate (called ES*) was found to precede ES1 in the pre-steady-state time period. The identity of the intermediates formed in aqueous solution with those previously observed in the cryostudy confirms that the mechanism is not changed either by the presence of an organic cosolvent or by subzero temperatures. Further characterization of ES1 and the steady-state intermediate ES2 at subzero temperatures, where their lifetime may be extended for up to several hours, has involved circular and magnetic circular dichroic studies. The magnetic circular dichroic spectra identify changes in the coordination sphere of the active-site metal during catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)