Kinetic studies of electron transfer in the cyt b 5 : metHb system

 The kinetics of methemoglobin reduction by cytochrome b ₅ has been studied by stopped-flow and saturation transfer NMR. A forward rate constant k _f = 2.44×10⁴ M^–1 s^–1 and a reverse rate constant k _b = 540 M^–1s^–1 have been observed at 10 mm, pH 6.20, 25  °C. The ratio k _f/k _b = k _eq = 43.6 is in good agreement with the equilibrium constant calculated from the electrochemical potential between cyt b ₅ and methemoglobin. A bimolecular collisional mechanism is proposed for the electron transfer from cyt b ₅ to methemoglobin based on the kinetic data analysis. The dependence of the rate constants on ionic strengths supports such collisional mechanism. It is also found that the reaction rate strongly depends on the conformations of methemoglobin. Received: 20 February 1996 / Accepted: 4 June 1996     

Spectroscopic characterization and intramolecular electron transfer processes of native and type 2 Cu-depleted nitrite reductases

 Native nitrite reductases (NIRs) containing both type 1 and 2 Cu ions and type 2 Cu-depleted (T2D) NIRs from three denitrifying bacteria (Achromobacter cycloclastes IAM 1013, Alcaligenes xylosoxidans NCIB 11015, and Alcaligenes xylosoxidans GIFU 1051) have been characterized by electronic absorption, circular dichroism, and electron paramagnetic resonance spectra. The characteristic visible absorption spectra of these NIRs are due to the type 1 Cu centers, while the type 2 Cu centers hardly contribute in the same region. The intramolecular electron transfer (ET) process from the type 1 Cu to the type 2 Cu in native NIRs has been observed as the reoxidation of the type 1 Cu(I) center by pulse radiolysis, whereas no type 1 Cu in T2D NIRs exhibits the same reoxidation. The ET process obeys first-order kinetics, and observed rate constants are 1400–1900 s^–1 (t_1/2 = ca. 0.5 ms) at pH 7.0. In the presence of nitrite, the ET process also obeys first-order kinetics, with rate constants decreased by factors of 1/12–1/2 at the same pH. The redox potential of the type 2 Cu site is estimated to be +0.24 - +0.28 V, close to that of the type 1 Cu site. Nitrate and azide ions bound to the type 2 Cu site change the redox potential. Nitrite also would shift the redox potential of the type 2 Cu by coordination, and hence the intramolecular ET rate constant is decreased. Pulse radiolysis experiments on T2D NIRs in the presence of nitrite demonstrate that the type 1 Cu(I) site is slowly oxidized with a first-order rate constant of 0.03 s^–1 at pH 7.0, suggesting that nitrite bound to the protein accepts an electron from the type 1 Cu. This result is in accord with the finding that T2D NIRs show enzymatic activities, although they are lower than those of the native enzymes. Received: 9 July 1996 / Accepted: 30 January 1997     

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     

Spectroscopic and electrochemical properties of two azurins (Az-iso1 and Az-iso2) from the obligate methylotroph Methylomonas sp. strain J and the structure of novel Az-iso2

Methylomonas sp. strain J gives rise to two azurins (Az-iso1 and Az-iso2) with methylamine dehydrogenase (MADH-Mj). The intense blue bands characteristic of Az-iso1 and Az-iso2 are observed at 621 and 616 nm in the visible absorption spectra respectively, being revealed at 620−630 nm in those of usual azurins. The EPR signal of Az-iso1, similar to usual azurins, shows axial symmetry, while the axial EPR signal of Az-iso2 involves a slightly rhombic character. The half-wave potentials (E _1/2) of the two azurins and the intermolecular electron-transfer rate constants (k _ET) from MADH-Mj to each azurin were determined by cyclic voltammetry. The E _1/2 values of Az-iso1 and Az-iso2 are +321 and +278 mV vs NHE at pH 7.0, respectively. The k _ET value of Az-iso2 is larger than that of Az-iso1 by a factor of 5. However, the electron-transfer rate of Az-iso2 is interestingly slower than those of the azurins from a denitrifying bacterium, Alcaligenes xylosoxidans NCIB 11015, and the amicyanin from a different methylotroph, Methylobacterium extorquens AM1. The structure of Az-iso2 has been determined and refined against 1.6 Å X-ray diffraction data. The whole structure of Az-iso2 is quite similar to those of azurins reported already. The Cu(II) site of Az-iso2 is a distorted trigonal bipyramidal geometry like those of other azurins, but some of the Cu-ligand distances and ligand-Cu-ligand bond angle parameters are slightly different. These findings suggest that Az-iso2 is a novel azurin and perhaps functions as an electron acceptor for MADH. Received: 23 February 1999 / Accepted: 9 September 1999     

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     

Pyranose 2-oxidase from Phanerochaete chrysosporium– further biochemical characterisation

Pyranose 2-oxidase (P2O) was purified 43-fold to apparent homogeneity from the basidiomycete Phanerochaete chrysosporium using liquid chromatography on phenyl Sepharose, Mono Q (twice) and phenyl Superose. The native enzyme has a molecular mass of about 250 kDa (based on native PAGE) and is composed of four identical subunits of 65 kDa. It contains three isoforms of isoelectric point (pI) 5.0, 5.05 and 5.15 and does not appear to be a glycoprotein. P2O is optimally stable at pH 8.0 and up to 60 °C. It is active over a broad pH range (5.0–9.0) with maximum activity at pH 8.0–8.5 and at 55 °C, and a broad substrate specificity. d-Glucose is the preferred substrate, but 1-β-aurothioglucose, 6-deoxy-d-glucose, l-sorbose, d-xylose, 5-thioglucose, d-glucono-1,5-lactone, maltose and 2-deoxy-d-glucose are also oxidised at relatively high rates. A Ping Pong Bi Bi mechanism was demonstrated for the P2O reaction at pH 8.0, with a catalytic constant (k _cat) of 111.0 s⁻¹ and an affinity constant (K _m) of 1.43 mM for d-glucose and 83.2 μM for oxygen. Whereas the steady-state kinetics for glucose oxidation were unaffected by the medium at pH ≥ 7.0, at low pH both pH and buffer composition affected the P2O kinetics with the k _cat/K _m value decreasing with decreasing pH. The greatest effect was observed in acetate buffer (0.1 M, pH 4.5), where the k _cat decreased to 60.9 s⁻¹ and the K _m increased to 240 mM. The activity of P2O was completely inhibited by 10 mM HgCl₂, AgNO₃ and ZnCl₂, and 50% by lead acetate, CuCl₂ and MnCl₂. Received: 28 August 1996 / Received revision: 25 November 1996 / Accepted: 29 November 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.     

Type I blue copper proteins as enantioselective quenchers of the photoluminescence of Δ,Λ-Eu(pyridine-2,6-dicarboxylate)3 3–: azurin from Pseudomonas aeruginosa and its Met44→Lys mutant, amicyanin from Paracoccus versutus and parsley plastocyanin

 The dynamic quenching of the luminescence of racemic Eu(III)(pyridine-2,6-dicarboxylate=dpa)₃ ^3– by the title proteins is investigated and the enantioselectivity of the proteins in the quenching of the Δ and Λ enantiomers of Eu(dpa)₃ ^3– is determined. The two diastereomeric quenching rate constants pertaining to azurin (k _q ^Δ=3.3×10⁶, k _q ^Λ=2.7×10⁶ M^–1s^–1, pH 7.2, ionic strength I=22 mM) are lower than for its Met→44Lys mutant (k _q ^Δ=1.9×10⁷, k _q ^Λ=1.4×10⁷ M^–1 s^–1, same pH and I), indicating that energy transfer occurs from Eu(dpa)₃ ^3– to the Cu(II) centre when the luminophore is bound to the hydrophobic patch of the protein near residue 44. The enantioselectivity remains unaltered by the mutation: k _q ^Δ/k _q ^Λ=1.27±0.04, so Lys44 is probably not in direct contact with the Eu chelate. The I and pH dependence of k _q indicate that the lysine residue interacts electrostatically with Eu(dpa)₃ ^3–. For plastocyanin the quenching rates are of the order of 10⁶ M^–1 s^–1; for amicyanin they are two orders of magnitude larger (k _q ^Δ=12×10⁷, k _q ^Λ=11×10⁷ M^–1 s^–1, pH 7.2, I=22 mM). The variation of k _q is attributed to differences in the charge distribution on the proteins, which influences the binding of the luminophore to the protein surface. For amicyanin the anion binding site near Lys59 and Lys60 may be involved in the energy transfer. Received: 16 June 1998 / Accepted: 18 September 1998     

Azide adducts of stearoyl-ACP desaturase: a model for μ-1,2 bridging by dioxygen in the binuclear iron active site

 The stearoyl-acyl carrier protein Δ⁹ desaturase (Δ9D) uses an oxo-bridged diiron center to catalyze the NAD(P)H– and O₂–dependent desaturation of stearoyl-ACP. Δ9D, ribonucleotide reductase, and methane monooxygenase have substantial similarities in their amino acid primary sequences and the physical properties of their diiron centers. These three enzymes also appear to share common features of their reaction cycles, including the binding of O₂ to the diferrous state and the subsequent generation of transient diferric-peroxo and diferryl species. In order to investigate the coordination environment of the proposed diferric-peroxo intermediate, we have studied the binding of azide to the diiron center of Δ9D using optical, resonance Raman (RR), and transient kinetic spectroscopic methods. The addition of azide results in the appearance of new absorption bands at 325 nm and 440 nm (k _app≈3.5 s^–1 in 0.7 M NaN₃, pH 7.8). RR experiments demonstrate the existence of two different adducts: an η¹–terminal structure at pH 7.8 (¹⁴N₃ ^– asymmetric stretch at 2073 cm^–1, resolved into two bands with ¹⁵N¹⁴N₂ ^–) and a μ-1,3 bridging structure at pH<7 (¹⁴N₃ ^– asymmetric stretch at 2100 cm^–1, shifted as a single band with ¹⁵N¹⁴N₂ ^–). Both adducts also exhibit an Fe–N₃ stretching mode at ≈380 cm^–1, but no accompanying Fe–O–Fe stretching mode, presumably due to either protonation or loss of the oxo bridge. The ability to form a μ-1,3 bridging azide supports the likelihood of a μ-1,2 bridging peroxide as a catalytic intermediate in the Δ9D reaction cycle and underscores the adaptability of binuclear sites to different bridging geometries. Received: 23 August 1996 / Accepted: 4 October 1996     

Intramolecular electron transfer rate between active-site copper and TPQ in Arthrobacter globiformis amine oxidase

Copper amine oxidases catalyze the oxidative deamination of primary amines operating through a ping-pong bi bi mechanism, divided into reductive and oxidative half-reactions. Considerable debate still exists regarding the role of copper in the oxidative half-reaction, where O₂ is reduced to H₂O₂. Substrate-reduced amine oxidases display an equilibrium between a Cu(II) aminoquinol and a Cu(I) semiquinone, with the magnitude of the equilibrium constant being dependent upon the enzyme source. The initial electron transfer to dioxygen has been proposed to occur from either the reduced Cu(I) center or the reduced aminoquinol cofactor. In order for Cu(I) to be involved, it must be shown that the rate of electron transfer (k _ET) between the aminoquinol and Cu(II) is sufficiently rapid to place the Cu(I) semiquinone moiety on the mechanistic pathway. To further explore this issue, we measured the intramolecular electron transfer rate for the Cu(II) aminoquinol ⇆ Cu(I) semiquinone equilibrium in Arthrobacter globiformis amine oxidase (AGAO) by temperature-jump relaxation techniques. The results presented herein establish that k _ET is greater than the rate of catalysis (k _cat) for the preferred amine substrate β-phenylethylamine at three pH values, thereby permitting the Cu(I) semiquinone to be a viable catalytic intermediate during enzymatic reoxidation in this enzyme. The data show that k _ET is approximately equivalent at pH 6.2 and 7.2, being 2.5 times k _cat for these pH values. At pH 8.2, however, k _ET decreases, becoming comparable to k _cat. Potential reasons for the decreased k _ET at basic pH are presented. The implications of these results in light of a previously published study measuring reoxidation rates of substrate-reduced AGAO are also addressed.     

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

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

Interprotein metal ion exchange between cadmium-carbonic anhydrase and apo- or zinc-metallothionein

 − 1  s^− 1 at 25 °C and pH 7.4 in Tris.HCl buffer and 0.1 M KCl. At 25 °C, Zn₇-metallothionein also exchanged metal ions with Cd-carbonic anhydrase with a rate constant of 0.33 ± 0.02 M^− 1 s^− 1 to reconstitute enzymatically active protein. Cd-carbonic anhydrase reacted within the time of mixing with the peptide sequence 49–61 of rabbit metallothionein 2 which contains four cysteinyl residues, leading to the exchange of most of the Cd²⁺ into the peptide. At pH 7.4 and 25 °C, Cd²⁺ has higher affinity for apometallothionein than for the apo-peptide. Received: 25 February 1999 / Accepted: 17 September 1999     

Production, purification and characterization of glucose oxidase from a newly isolated strain of Penicillium pinophilum

A number of nutritional factors influencing growth and glucose oxidase (EC 1.1.3.4) production by a newly isolated strain of Penicillium pinophilum were investigated. The most important factors for glucose oxidase production were the use of sucrose as the carbon source, and growth of the fungus at non-optimal pH 6.5. The enzyme was purified to apparent homogeneity with a yield of 74%, including an efficient extraction step of the mycelium mass at pH 3.0, cation-exchange chromatography and gel filtration. The relative molecular mass (M _r) of native glucose oxidase was determined to be 154 700 ± 4970, and 77 700 for the denatured subunit. Electron-microscopic examinations revealed a sandwich-shaped dimeric molecule with subunit dimensions of 5.0 × 8.0 nm. Glucose oxidase is a glycoprotein that contains tightly bound FAD with an estimated stoichiometry of 1.76 mol/mol enzyme. The enzyme is specific for d-glucose, for which a K _m value of 6.2 mM was determined. The pH optimum was determined in the range pH 4.0–6.0. Glucose oxidase showed high stability on storage in sodium citrate (pH 5.0) and in potassium phosphate (pH 6.0), each 100 mM. The half-life of the activity was considerably more than 305 days at 4 °C and 30 °C, and 213 days at 40 °C. The enzyme was unstable at temperatures above 40 °C in the range pH 2.0–4.0 and at a pH above 7.0. Received: 18 November 1996 / Received revision: 3 March 1997 / Accepted: 7 March 1997     

Simultaneous utilization of glucose and D-xylose by Candida shehatae in a chemostat

The kinetics of biomass formation, D-xylose utilization, and mixed substrate utilization were determined in a chemostat using the yeast Candida shehatae. The maximum growth rate of C. shehatae grown aerobically on D-xylose was 0.42 h⁻¹ and the Monod constant, K _s, was 0.06 g L⁻¹. The biomass yield, Y _{X/S}, ranged from 0.40 to 0.50 g g⁻¹ over a dilution rate range of 0.2–0.3 h⁻¹, when C. shehatae was grown on pure D-xylose. Mixtures of D-xylose and glucose (∼1 : 1) were simultaneously utilized over a dilution rate from 0.15 to 0.35 h⁻¹ at pH 3.5 and 4.5, but pH 3.5 reduced μ_max and reduced the dilution rate range over which D-xylose was utilized in the presence of glucose. At pH 4.5, μ_max was not reduced with the mixed sugar feed and the overall or lumped K _s value was not significantly increased (0.058 g L⁻¹ vs 0.06 g L⁻¹), when compared to a pure D-xylose feed. Kinetic data indicate that C. shehatae is an excellent candidate for chemostat production of value added products from renewable carbon sources, since simultaneous mixed substrate utilization was observed over a wide range of growth rates on a 1 : 1 mixture of glucose and D-xylose. Received 21 August 1997/ Accepted in revised form 28 May 1998     

Kinetic studies on the reactions of Fusarium galactose oxidase with five different substrates in the presence of dioxygen

 Reactions (25  °C) of galactose oxidase, GOase_ox from Fusarium NRRL 2903 with five different primary-alcohol-containing substrates RCH₂OH:- D-galactose (I) and 2-deoxy-d-galactose (II) (monosaccharides); methyl-β-d-galactopyranoside (III) (glycoside);d-raffinose (IV) (trisaccharide); and dihydroxyacetone (V) have been studied in the presence of O₂. The GOase_ox state has a tyrosyl radical coordinated at a square-pyramidal Cu^II active site, and is a two-equivalent oxidant. Reactant concentrations were [GOase_ox] (0.8–10 μM), RCH₂OH (1.0–6.0 mM), and O₂ (0.14–0.29 mM), with I=0.100 M (NaCl). The reactions, monitored at 450 nm by stopped-flow spectrophotometry, terminated with depletion of the O₂. Each trace was fitted to the competing reactions GOase_ox+RCH₂ OH → GOase_redH₂+RCHO (k ₁), and GOase_redH₂+O₂→ GOase_ox+H₂O₂ (k ₂), with GOase_redH₂ written as the doubly protonated two-electron-reduced Cu^I product. It was necessary to avoid auto-redox interconversion of GOase_ox and GOase_semi . Information obtained at pH 7.5 indicates a 5 : 95 (ox : semi) "native" mix equilibration complete in ∼3 h. At pH >7.5, rate constants 10^–4 k ₁ / M^–1 s^–1 for the reactions of GOase_ox with (I) (1.19), (II) (1.07), (III) (1.29), (IV) (1.81), (V) (2.94) were determined. On decreasing the pH to 5.5, k ₁ values decreased by factors of up to a half, and acid dissociation pK _as in the range 6.6–6.9 were obtained. UV-Vis spectrophotometric studies on GOase_ox gave an independently determined pK _a of 6.7. No corresponding reactions of the Tyr495Phe variant were observed, and there are no similar UV-Vis absorbance changes for this variant. The pK _a is therefore assigned to protonation of Tyr-495 which is a ligand to the Cu. The rate constant k ₂ (1.01×10⁷ M^–1 s^–1) is independent of pH in the range 5.5–9.0 investigated, suggesting that H⁺ (or H-atoms) for the O₂ → H₂O₂ change are provided by the active site of GOase_red . The Cu^I of GOase_red is less extensively complexed, and a coordination number of three is likely. Received: 4 February 1997 / Accepted: 16 May 1997     

The reaction mechanism of nitrosothiols with copper(I)

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

Catalase Activity of Cytochrome c Oxidase Assayed with Hydrogen Peroxide-Sensitive Electrode Microsensor

An iron-hexacyanide-covered microelectrode sensor has been used to continuously monitor the kinetics of hydrogen peroxide decomposition catalyzed by oxidized cytochrome oxidase. At cytochrome oxidase concentration ≈1 μM, the catalase activity behaves as a first order process with respect to peroxide at concentrations up to ≈300–400 μM and is fully blocked by heat inactivation of the enzyme. The catalase (or, rather, pseudocatalase) activity of bovine cytochrome oxi- dase is characterized by a second order rate constant of ≈2•10₂ M^-1•sec^-1 at pH 7.0 and room temperature, which, when divided by the number of H₂O₂ molecules disappearing in one catalytic turnover (between 2 and 3), agrees reasonably well with the second order rate constant for H₂O₂-dependent conversion of the oxidase intermediate F_I-607 to F_II-580. Accordingly, the catalase activity of bovine oxidase may be explained by H₂O₂ procession in the oxygen-reducing center of the enzyme yielding superoxide radicals. Much higher specific rates of H₂O₂ decomposition are observed with preparations of the bacterial cytochrome c oxidase from Rhodobacter sphaeroides. The observed second order rate constants (up to ≈3000 M^-1•sec^-1) exceed the rate constant of peroxide binding with the oxygen-reducing center of the oxidized enzyme (≈500 M^-1•sec^-1) several-fold and therefore cannot be explained by catalytic reaction in the a ₃/Cu_B site of the enzyme. It is proposed that in the bacterial oxidase, H2O2 can be decomposed by reacting with the adventitious transition metal ions bound by the polyhistidine-tag present in the enzyme, or by virtue of reaction with the tightly-bound Mn²⁺, which in the bacterial enzyme substitutes for Mg²⁺ present in the mitochondrial oxidase.     

Characterization of the cadmium complex of peptide 49–61: a putative nucleation center for cadmium-induced folding in rabbit liver metallothionein IIA

 The synthetic peptide fragment containing residues 49–61 of rabbit liver metallothionein II (MT-II) (Ac-Ile-Cys-Lys-Gly-Ala-Ser-Asp-Lys-Cys-Ser-Cys-Cys-Ala-COOH), which includes the only sequential four cysteines bound to the same metal ion in Cd₇MT, forms a stable, monomeric Cd-peptide complex with 1 : 1 stoichiometry (Cd:peptide) via Cd-thiolate interactions. This represents the first synthesis of a single metal-binding site of MT independent of the domains. The ¹¹¹Cd NMR chemical shift at 716 ppm indicates that the ¹¹¹Cd²⁺ in the metal site is terminally coordinated to four side-chain thiolates of the cysteine residues. The pH of half dissociation for this Cd-peptide derivative, ∼3.3, demonstrates an affinity similar to that for Cd₇MT. Molecular mechanics calculations show that the thermodynamically most stable folding for this isolated Cd²⁺ center has the same counterclockwise chirality (Λ or S) observed in the native holo-protein. These properties are consistent with its proposed role as a nucleation center for cadmium-induced protein folding. However, the kinetic reactivity of the CdS₄ structure toward 5,5′-dithiobis(5-nitrobenzoate) (DTNB) and EDTA is greatly increased compared to the complete cluster (α-domain or holo-protein). The rate law for the reaction with DTNB is rate=(k _uf +k _1,f +k _2,f [DTNB])[peptide], where k _uf=0.15 s^–1, k _1,f=2.59×10^–3s^–1, and k _2,f=0.88 M^–1s^–1. The ultrafast step (uf), observable only by stopped-flow measurement, is unprecedented for mammalian (M₇MT) and crustacean (M₆MT) holo-proteins or the isolated domains. The accommodation of other metal ions by the peptide indicates a rich coordination chemistry, including stoichiometries of M-peptide for Hg²⁺, Cd²⁺, and Zn²⁺, M₂-peptide for Hg²⁺ and Au⁺, and (Et₃PAu)₂-peptide. Received: 9 December 1998 / Accepted: 20 May 1999     

Steady-state kinetics, micellar effects, and the mechanism of peroxidase-catalyzed oxidation of n -alkylferrocenes by hydrogen peroxide

 Kinetics of the steady-state oxidation of n–alkylferrocenes (alkyl = H, Me, Et, Bu and C₅H₁₁) by H₂O₂ to form the corresponding ferricenium cations catalyzed by horseradish peroxidase has been studied in micellar systems of Triton X-100, CTAB, and SDS, mostly at pH 6.0 and 25  °C. The rate of oxidation of ferrocenes with longer alkyl radicals is too slow to be measured. The reaction obeying the [RFc]:[H₂O₂] = 2 : 1 stoichiometry is strictly first-order in both HRP and RFc in a wide concentration range. The corresponding observed second-order rate constants k, which refer to the interaction of the peroxidase compound II (HRP-II) with RFc, decrease with the elongation of the alkyl substituent R, and this in turn is accompanied by an increase in the formal redox potentials E°′ in the same medium. Increasing the surfactant concentration lowers the rate constants k, the effect being due to the nonproductive binding of RFc to micelles rather than to enzyme inactivation. The micellar effects are accounted for in terms of the Berezin pseudo-phase model of micellar catalysis applied to the interaction of enzyme with organometallic substrates. The oxidation was found to occur primarily in the aqueous pseudo-phase and the calculated intrinsic second-order rate constants k _w are (1.9 ± 0.5)×10⁵, (2.7 ± 0.1)×10⁴, and (5.9 ± 0.6)×10³ M^–1 s^–1 for HFc, EtFc, and n–BuFc, respectively. The data obtained were used for estimating the self-exchange rate constants for the HRP-II/HRP couple in terms of the Marcus formalism. Received: 15 July 1996 / Accepted: 15 November 1996     

A novel cold-adapted lipase from <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. XMZ-26: gene cloning and characterisation

Acinetobacter sp. XMZ-26 (ACCC 05422) was isolated from soil samples obtained from glaciers in Xinjiang Province, China. The partial nucleotide sequence of a lipase gene was obtained by touchdown PCR using degenerate primers designed based on the conserved domains of cold-adapted lipases. Subsequently, a complete gene sequence encoding a 317 amino acid polypeptide was identified. Our novel lipase gene, lipA, was overexpressed in Escherichia coli. The recombinant protein (LipA) was purified by Ni-affinity chromatography, and then deeply characterised. The LipA resulted to hydrolyse pNP esters of fatty acids with acyl chain length from C2 to C16, and the preferred substrate was pNP octanoate showing a k _cat = 560.52 ± 28.32 s⁻¹, K _m = 0.075 ± 0.008 mM, and a k _cat/K _m = 7,377.29 ± 118.88 s⁻¹ mM⁻¹. Maximal LipA activity was observed at a temperature of 15°C and pH 10.0 using pNP decanoate as substrate. That LipA peaked at such a low temperature and remained most activity between 5°C and 35°C indicated that it was a cold-adapted enzyme. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents, including Ninol, Triton X-100, methanol, PEG-600, and DMSO. This cold-adapted lipase may find applications in the detergent industry and organic synthesis.