Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000     

Fast repair of purine deoxynucleotide radical cations by rutin and quercetin

Repair effects of rutin and quercetin on purine deoxynucleotide radical cations were studied using pulse radiolysis technique. On electron pulse irradiation of N₂ saturated deoxynucleotide aqueous solution containing 20 mmol/L K₂S₂O₈, 200 mmol/Lt-BuOH and rutin or quercetin, the transient absorption spectra of the deoxynucleotide radical cations decayed quickly. At the same time, the spectra of flavonoid phenoxyl radicals formed within several dozen microseconds. The results indicated that deoxynucleotide radical cations can be repaired by flavonoids. The rate constants of the repair reactions were 3.8 ×10⁸-4.4 ×10⁸ mol⁻¹ · L · s⁻¹ and 1.3×10⁸-1.8×10⁸ mol⁻¹ · L · s⁻¹ for dAMP and dGMP radical cations, respectively.     

Kinetics of N-substituted phenothiazines and N-substituted phenoxazines oxidation catalyzed by fungal laccases

Laccase-catalyzed oxidation of N-substituted phenothiazines and N-substituted phenoxazines was investigated at pH 5.5 and 25°C. The recombinant laccase from Polyporus pinsitus (rPpL) and the laccase from Myceliophthora thermophila (rMtL) were used. The dependence of initial reaction rate on substrate concentration was analyzed by applying the laccase action scheme in which the laccase native intermediate (NI) reacts with a substrate forming reduced enzyme. The reduced laccase produces peroxide intermediate (PI) which in turn decays to the NI. The calculated constant (k_ox) values of the PI formation are (6.1±3.1)×10⁵ M⁻¹s⁻¹ for rPpL and (2.5±0.9)×10⁴ M⁻¹s⁻¹ for rMtL. The bimolecular constants of the reaction of the native intermediate with electron donor (kred) vary in the interval from 2.2×10⁵ to 2.1×10⁷ M⁻¹s⁻¹ for rPpL and from 1.3×10² to 1.8×10⁵ M^-1s⁻¹ for rMtL. The larger reactivity of rPpL in comparison to rMtL is associated with the higher redox potential of type I Cu of rPpL. The variation of k_red values for both laccases correlates with the change of the redox potential of substrates. Following outer sphere (Marcus) electron transfer mechanism the calculated activationless electron transfer rate and the apparent reorganization energy are 5.0×107 M⁻¹s⁻¹ and 0.29 eV, respectively.     

Fast repair of purine deoxynucleotide radical cations by rutin and quercetin

Repair effects of rutin and quercetin on purine deoxynucleotide radical cations were studied using pulse radiolysis technique. On electron pulse irradiation of N2 saturated deoxynucleotide aqueous solution containing 20 mmol/L K2S2O8, 200 mmol/L f-BuOH and rutin or quercetin, the transient absorption spectra of the deoxynucleotide radical cations decayed quickly. At the same time, the spectra of flavonoid phenoxyl radicals formed within several dozen microseconds. The results indicated that deoxynucleotide radical cations can be repaired by flavonoids. The rate constants of the repair reactions were 3.8 × 108-4.4×108 mol-1 · L · s-1 and 1.3×108-1.8×108 mol-1 · L · s-1 for dAMP and dGMP radical cations, respectively.     

Oxyleghemoglobin scavenges nitrogen monoxide and peroxynitrite: a possible role in functioning nodules?

It has been demonstrated that the NO^• produced by nitric oxide synthase or by the reduction of nitrite by nitrate reductase plays an important role in plants’ defense against microbial pathogens. The detection of nitrosyl Lb in nodules strongly suggests that NO^• is also formed in functional nodules. Moreover, NO^• may react with superoxide (which has been shown to be produced in nodules by various processes), leading to the formation of peroxynitrite. We have determined the second-order rate constants of the reactions of soybean oxyleghemoglobin with nitrogen monoxide and peroxynitrite. At pH 7.3 and 20 °C, the values are on the order of 10⁸ and 10⁴ M⁻¹ s⁻¹, respectively. In the presence of physiological amounts of CO₂ (1.2 mM), the second-order rate constant of the reaction of oxyleghemoglobin peroxynitrite is even larger (10⁵ M⁻¹ s⁻¹). The results presented here clearly show that oxyleghemoglobin is able to scavenge any NO^• and peroxynitrite formed in functional nodules. This may help to stop NO^• triggering a plant defense reaction.     

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.     

Kinetic study of peroxidase-catalyzed oxidation of 1-hydroxypyrene. Development of a nanomolar hydrogen peroxide detection system

Kinetics of 1-hydroxypyrene (1-HP) oxidation catalyzed with recombinant Coprinus cinereus (rCiP) and horseradish (HRP) peroxidases was investigated with a special emphasis for developing a nanomolar hydrogen peroxide (H₂O₂) detection system. At pH 8.0 the bimolecular constants of 1-HP oxidation with the ferryl compounds of rCiP and HRP were equal to (1.0 ± 0.3) × 10⁸ M⁻¹ s⁻¹ and (0.6 ± 0.2) × 10⁸ M⁻¹ s⁻¹, respectively. High bimolecular constants and fluorescence quantum yield of 1-HP (0.66) permitted detection as low as 21 nM of H₂O₂. To optimize the detection system 1-HP oxidation was modeled at steady-state conditions in the range pH 5.0 to pH 8.0. The 1-HP based detection system was compared with the Amplex Red system. The peroxidase-catalyzed 1-HP oxidation system was used for determination of ozone in the air.     

Stereoselective and driving-force-dependent photoinduced electron-transfer reactions of zinc myoglobin with optically active N,N′-dimethylcinchoninium and N,N′-dimethylcinchonidinium ions

In order to understand the detailed mechanism of the stereoselective photoinduced electron-transfer (ET) reactions of zinc-substituted myoglobin (ZnMb) with optically active molecules by flash photolysis, we designed and prepared new optically active agents, such as N,N′-dimethylcinchoninium diiodide ([MCN]I₂) and N,N′-dimethylcinchonidinium diiodide ([MCD]I₂). The photoexcited triplet state of ZnMb, ³(ZnMb)*, was successfully quenched by [MCN]²⁺ and [MCD]²⁺ ions to form the radical pair of ZnMb cation (ZnMb^·+) and reduced [MCN]^·+ and [MCD]^·+, followed by a thermal back ET reaction to the ground state. The rate constants (k _q) for the ET quenching at 25 °C were obtained as k _q(MCN)=(1.9±0.1)×10⁶ M⁻¹ s⁻¹ and k _q(MCD)=(3.0±0.2)×10⁶ M⁻¹ s⁻¹, respectively. The ratio of k _q(MCD)/k _q(MCN)=1.6 indicates that the [MCD]²⁺ preferentially quenches ³(ZnMb)*. The second-order rate constants (k _b) for the thermal back ET reaction from [MCN]^·+ and [MCD]^·+ to ZnMb^·+ at 25 °C were k _b(MCN)=(0.79±0.04)×10⁸ M⁻¹ s⁻¹ and k _b(MCD)=(1.0±0.1)×10⁸ M⁻¹ s⁻¹, respectively, and the selectivity was k _q(MCD)/k _q(MCN)=1.3. Both quenching and thermal back ET reactions are controlled by the ET step. In the quenching reaction, the energy differences of ΔΔH ^≠(MCD–MCN) and ΔΔS ^≠(MCD–MCN) at 25 °C were obtained as −1.1 and 0 kJ mol⁻¹, respectively. On the other hand, ΔΔH ^≠(MCD–MCN)=11±2 kJ mol⁻¹ and TΔΔS ^≠(MCD–MCN)=−10±2 kJ mol⁻¹ were given in the thermal back ET reaction. The highest stereoselectivity of 1.7 for [MCD]^·+ found at low temperature (10 °C) was due to the ΔΔS ^≠ value obtained in the thermal back ET reaction. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Comparative analysis of four <Emphasis Type="Italic">β</Emphasis>-galactosidases from <Emphasis Type="Italic">Bifidobacterium bifidum</Emphasis> NCIMB41171: purification and biochemical characterisation

Four different β-galactosidases (previously named BbgI, BbgII, BbgIII and BbgIV) from Bifidobacterium bifidum NCIMB41171 were overexpressed in Escherichia coli, purified to homogeneity and their biochemical properties and substrate preferences comparatively analysed. BbgI was forming a hexameric protein complex of 875 kDa, whereas BbgII, BbgIII and BbgIV were dimers with native molecular masses of 178, 351 and 248 kDa, respectively. BbgII was the only enzyme that preferred acidic conditions for optimal activity (pH 5.4–5.8), whereas the other three exhibited optima in more neutral pH ranges (pH 6.4–6.8). Na⁺ and/or K⁺ ions were prerequisite for BbgI and BbgIV activity in Bis–Tris-buffered solutions, whereas Mg⁺⁺ was strongly activating them in phosphate-buffered solutions. BbgII and BbgIII were slightly influenced from the presence or absence of cations, with Mg⁺⁺, Mn⁺⁺ and Ca⁺⁺ ions exerting the most positive effect. Determination of the specificity constants (k _cat/K _m) clearly indicated that BbgI (6.11 × 10⁴ s⁻¹ M⁻¹), BbgIII (2.36 × 10⁴ s⁻¹ M⁻¹) and especially BbgIV (4.01 × 10⁵ s⁻¹ M⁻¹) are highly specialised in the hydrolysis of lactose, whereas BbgII is more specific for β-d-(1→6) galactobiose (5.59 × 10⁴ s⁻¹ M⁻¹) than lactose (1.48 × 10³ s⁻¹ M⁻¹). Activity measurements towards other substrates (e.g. β-d-(1→6) galactobiose, β-d-(1→4) galactobiose, β-d-(1→4) galactosyllactose, N-acetyllactosamine, etc.) indicated that the β-galactosidases were complementary to each other by hydrolysing different substrates and thus contributing in a different way to the bacterial physiology.     

The influence of differentiation-inducing agents on plasma membrane surface characteristics of THP-1 cells

Undifferentiated THP-1 cells from Cell Culture Collection of the Institute of Cytology, RAS (St. Petersburg), are characterized by weak expression of Toll-like receptor-4 (TLR4) on the cell surface (up to 2%) and by almost undetectable expression of CD14 and CD11b receptors. Differentiation agent phorbol-12-myristate-13-acetate independently of its concentration (2 × 10⁻⁷ M or 10⁻⁸ M) and incubation time (24 or 48 h) did not initiate CD11b surface expression and did not change the parameter S_app (0.605 ± 0.005 at 37°C) reflecting the cell membrane viscosity. Differentiation of THP-1 cells induced by another differentiation agent, 1α,25-dihydroxyvitamin D₃, caused expression of CD14 (up to 70–80%) and CD11b (up to 15–20%) receptors, again without changes in plasma membrane viscosity. The rate constants of the reduction of 5- and 16-doxyl-stearic acids by THP-1 cells were in the range of 6–8 × 10⁻³ s⁻¹ at 37°C. During cell differentiation significant changes in cell electrophoretic mobility (EM, μm s⁻¹ V⁻¹ cm) were observed. Mean value of EM for undifferentiated THP-1 cells was −1.332 ± 0.011, whereas for phorbol-12-myristate-13-acetate- and 1α,25-dihydroxyvitamin D₃-treated cells it was −1.432 ± 0.030 and −1.212 ± 0.016, respectively.     

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

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

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

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

Gallium uptake by transferrin and interaction with receptor 1

The kinetics and thermodynamics of Ga(III) exchange between gallium mononitrilotriacetate and human serum transferrin as well as those of the interaction between gallium-loaded transferrin and the transferrin receptor 1 were investigated in neutral media. Gallium is exchanged between the chelate and the C-site of human serum apotransferrin in interaction with bicarbonate in about 50 s to yield an intermediate complex with an equilibrium constant K ₁ = (3.9 ± 1.2) × 10⁻², a direct second-order rate constant k ₁ = 425 ± 50 M⁻¹ s⁻¹ and a reverse second-order rate constant k ₋₁ = (1.1 ± 3) × 10⁴ M⁻¹ s⁻¹. The intermediate complex loses a single proton with proton dissociation constant K _1a = 80 ± 40 nM to yield a first kinetic product. This product then undergoes a modification in its conformation which lasts about 500 s to produce a second kinetic intermediate, which in turn undergoes a final extremely slow (several hours) modification in its conformation to yield the gallium-saturated transferrin in its final state. The mechanism of gallium uptake differs from that of iron and does not involve the same transitions in conformation reported during iron uptake. The interaction of gallium-loaded transferrin with the transferrin receptor occurs in a single very fast kinetic step with a dissociation constant K _d = 1.10 ± 0.12 μM and a second-order rate constant k _d = (1.15 ± 0.3) × 10¹⁰ M⁻¹ s⁻¹. This mechanism is different from that observed with the ferric holotransferrin and suggests that the interaction between the receptor and gallium-loaded transferrin probably takes place on the helical domain of the receptor which is specific for the C-site of transferrin and HFE. The relevance of gallium incorporation by the transferrin receptor-mediated iron-acquisition pathway is discussed.     

Purification and characterisation of lignin peroxidase from <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> MTCC-137

Extracellular secretion of lignin peroxidase from Pycnoporus sanguineus MTCC-137 in the liquid culture growth medium amended with lignin containing natural sources has been shown. The maximum secretion of lignin peroxidase has been found in the presence of saw dust. The enzyme has been purified to homogeneity from the culture filtrate of the fungus using ultrafiltration and anion exchange chromatography on DEAE-cellulose. The purified lignin peroxidase gave a single protein band in sodium dodecylsulphate polyacrylamide gel electrophoresis corresponding to the molecular mass 40 kDa. The K _m, k _cat and k _cat/K _m values of the enzyme using veratryl alcohol and H₂O₂ as the substrate were 61 M, 2.13 s⁻¹, 3.5 × 10⁴ M⁻¹s⁻¹ and 71 M, 2.13 s⁻¹, 3.0 × 10⁴ M⁻¹ s⁻¹ respectively at the optimum pH of 2.5. The temperature optimum of the enzyme was 25°C.     

Production,purification and partial characterisation of a novel laccase from the white-rot fungus <Emphasis Type="Italic">Panus tigrinus</Emphasis> CBS 577.79

Extracellular laccase from Panus tigrinus CBS 577.79 was produced in a bubble-column reactor using glucose-containing medium supplemented with 2,5-xylidine under conditions of nitrogen sufficiency. The main laccase isoenzyme was purified to apparent homogeneity by ultra-filtration, anion-exchange chromatography and gel filtration that led to a purified enzyme with a specific activity of 317 IU (mg protein)⁻¹ and a final yield of 66%. Laccase was found to be a monomeric protein with a molecular mass of 69.1 kDa, pI of 3.15 and 6.9% N-glycosylation of the high mannose type. Temperature and pH optima were 55°C and 3.75 (2,6-dimethoxyphenol as substrate). At 50 and 60°C, the enzyme half-lives were 281 and 25 min, respectively. The P. tigrinus laccase oxidized a wide range of both naturally occurring and synthetic aromatic compounds: the highest catalytic efficiencies were for 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic) acid and 2,6-dimethoxyphenol (5.99 × 10⁶ and 3.07 × 10⁶ M⁻¹ s⁻¹, respectively). Catalytic rate constants for typical N–OH redox mediators, such as 1-hydroxybenzotriazole (2.6 s⁻¹), violuric acid (8.4 s⁻¹) and 2,2,6,6-tetramethylpiperidin-N-oxide radical (7.8 s⁻¹), were found to be higher than those reported for other high redox potential fungal laccases.     

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

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

Involvement of carboxyl groups in chloride transport and reversible DIDS binding to band 3 protein in human erythrocytes

Noncovalent DIDS binding to Band 3 (AE1) protein in human erythrocyte membranes, modified by non-penetrating, water soluble 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide iodide (EAC), was studied at 0°C in the presence of 165 mM KCl. Under experimental conditions applied up to (48 ± 5) % of irreversible chloride self-exchange inhibition was observed. The apparent dissociation constant, KD, for “DIDS-Band 3” complex, determined from the chloride transport experiments, was (34 ± 3) nM and (80 ± 12) nM for control and EAC-treated resealed ghosts, respectively. The inhibition constant, Ki, for DIDS was (35 ± 6) nM and (60 ± 8) nM in control and EAC-treated ghosts, respectively. The reduced affinity for DIDS reversible binding was not a result of negative cooperativity of DIDS binding sites of Band 3 oligomer since Hill’s coefficients were indistinguishable from 1 (within the limit error) both for control and EAC-treated ghosts. By using tritium-labeled DIDS, 4,4’-diisothiocyanato-2,2’-stilbenedisulfonate ([³H]DIDS), the association rate constant, k₊₁ (M⁻¹s⁻¹), was measured. The mean values of (4.3 ± 0.7) × 10⁵ M⁻¹s⁻¹ for control and (2.7 ± 0.7) × 10⁵ M⁻¹s⁻¹ for EAC-treated ghosts were obtained. The mean values for K_D, evaluated from [³H]DIDS binding measurements, were (37 ± 9) nM and (90 ± 21) nM for control and EAC-modified ghosts, respectively. The results demonstrate that EAC modification of AE1 reduces about 2-fold the affinity of AE1 for DIDS. It is suggested that half of the subunits are modified near the transport site by EAC.     

A hydrogen-bonding network formed by the B10–E7–E11 residues of a truncated hemoglobin from <Emphasis Type="Italic">Tetrahymena pyriformis</Emphasis> is critical for stability of bound oxygen and nitric oxide detoxification

Abstract  

Truncated hemoglobins (trHbs) are distributed from bacteria to unicellular eukaryotes and have roles in oxygen transport and nitric oxide detoxification. It is known that trHbs exist in ciliates of the Tetrahymena group, but trHb structure and function remain poorly understood. To investigate trHb function with respect to stability of bound oxygen and protein structure, we measured the oxygen binding kinetics of Tetrahymena pyriformis trHb, and determined the crystal structure of the protein. The O₂ association and dissociation rate constants of T. pyriformis trHb were 5.5 μM⁻¹ s⁻¹ and 0.18 s⁻¹, respectively. The autooxidation rate constant was 3.8 × 10⁻³ h⁻¹. These values are similar to those of HbN from Mycobacterium tuberculosis. The three-dimensional structure of an Fe(II)–O₂ complex of T. pyriformis trHb was determined at 1.73-? resolution. Tyr25 (B10) and Gln46 (E7) were hydrogen-bonded to a heme-bound O₂ molecule. Tyr25 donated a hydrogen bond to the terminal oxygen atom, whereas Gln46 hydrogen-bonded to the proximal oxygen atom. Furthermore, Tyr25 was hydrogen-bonded to the Gln46 and Gln50 (E11) residues. Mutations at Tyr25, Gln46, and Gln50 increased the O₂ dissociation and autooxidation rate constants. An Fe(III)–H₂O complex of T. pyriformis trHb was formed following reaction of the Fe(II)–O₂ complex of T. pyriformis trHb, in a crystal state, with nitric oxide. This suggests that T. pyriformis trHb functions in nitric oxide detoxification.     

Characterization of a recombinant endo-type alginate lyase (Alg7D) from <Emphasis Type="Italic">Saccharophagus degradans</Emphasis>

A gene, alg7D, from Saccharophagus degradans, coding for a putative alginate lyase belonging to the family of polysaccharide lyase-7, was overexpressed in Escherichia coli. The properties of the recombinant Alg7D were characterized. The enzyme endolytically depolymerized alginate by β-elimination into oligo-alginates with degrees of polymerization of 2–5. Its activity was maximal at 50°C and pH 7 and was slightly increased in the presence of Na⁺. The K _M , V _max , k _cat , and k _cat /K _M values were: 3 mg ml⁻¹, 6.2 U mg⁻¹, 1.9 × 10⁻² s⁻¹, and 6.3 × 10⁻³ mg⁻¹ ml s⁻¹, respectively.     

A steady-state and pre-steady-state kinetics study of the tungstoenzyme formaldehyde ferredoxin oxidoreductase from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis>

Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a homotetrameric protein with one tungstodipterin and one [4Fe–4S] cubane per 69-kDa subunit. The enzyme kinetics have been studied under steady-state conditions at 80 °C and pre-steady state conditions at 50 °C, in the latter case via monitoring of the relatively weak (ε ≈ 2 mM⁻¹ cm⁻¹) optical spectrum of the tungsten cofactor. The steady-state data are consistent with a substrate substituted-enzyme mechanism for three substrates (formaldehyde plus two ferredoxin molecules). The K _M value for free formaldehyde (21 μM) with ferredoxin as an electron acceptor is approximately 3 times lower than the value measured when benzyl viologen is used as an acceptor. The K _M of ferredoxin (14 μM) is an order of magnitude less than previously reported values. An explanation for this discrepancy may be the fact that high concentrations of substrate are inhibitory and denaturing to the enzyme. Pre-steady-state difference spectra reveal peak shifts and a lack of isosbestic points, an indication that several processes happen in the first seconds of the reaction. Two fast processes (k _obs1 = 4.7 s⁻¹, k _obs2 = 1.9 s⁻¹) are interpreted as oxidation of the substrate followed by rearrangement of the active site. Alternatively, these processes could be the entry/binding of the substrate followed by its oxidation. The release of the product and the electron shuffling over the tungsten and iron–sulfur center in the absence of an external electron acceptor are slower (k _obs3 = 6.10 × 10⁻² s⁻¹, k _obs4 = 2.18 × 10⁻² s⁻¹). On the basis of these results in combination with results from previous electron paramagnetic resonance studies an activation route plus catalytic redox cycle is proposed.