Kinetic properties of Cu,Zn-superoxide dismutase as a function of metal content--order restored

In a recent publication (Michel et al. Arch. Biochem. Biophys. 439:234-240; 2005) the authors argued that the catalytic rate constant, k(cat), for wild-type Cu,Zn-superoxide dismutase (Cu,Zn-SOD), determined previously by pulse radiolysis, was overestimated due to contamination with excess copper. They reported that addition of 0.1 mM EDTA to a sample that already contained excess copper did not remove spurious activity, which is incompatible with well-known stability constants of copper complexes and contradicts previous observations. In the present study we verified that the addition of EDTA eliminates completely the effect of excess copper on the decomposition rate of O2*- in the presence of Cu,Zn-SOD. We determined that k(cat) = (2.82 +/- 0.02) x 10(9) M(-1) s(-1) at low ionic strength (2 < I < 15 mM) and (1.30 +/- 0.02) x 10(9) M(-1) s(-1) in the presence of 50 mM phosphate at pH 7.8 (I = approximately 150 mM), which are about twice higher than those reported by Michel et al. We also determined k(cat) by the cytochrome c assay and demonstrated the correlation between these direct and indirect assays. The phenotypic deficits imposed by deletion of SODs, and the oxygen dependence of these deficits, have repeatedly demonstrated that the several SODs do in fact, as well as is theory, provide an important protection against that facet of oxidative stress imposed by O2*-.     

Catalysis of superoxide dismutation by manganese aminopolycarboxylate complexes

Complexes of manganese, copper, cobalt, and iron with a variety of aminopolycarboxylates at concentrations from 2 X 10(-7) to 3 X 10(-6) M were tested for superoxide dismutase activity with horse ferricytochrome c as the competing reagent for superoxide. In the presence of excess ligand only manganous nitrilotriacetate and manganous ethylenediaminediacetate showed activity with catalytic rate constants of 2.2 X 10(7) and 1.8 X 10(7) M-1 S-1, respectively, at pH 6, 22 +/- 1 degree C, and 10 mM ionic strength. These rate constants decrease considerably at higher pH. Manganous N-hydroxyethylethylenediaminetriacetate is oxidized by superoxide, but does not appear to have catalytic activity. From the experimental conditions under which the two complexes mentioned above exhibit catalysis, and the inactivity of other metal chelates, it is concluded that an open coordination site is essential but not sufficient to catalyze the dismutation reaction.     

Stopped-flow kinetic analysis for monitoring superoxide decay in aqueous systems.

We have utilized a commercially available, computer-driven stopped-flow spectrophotometer to rapidly measure the self-dismutation or catalyzed decay of superoxide in aqueous buffers. In the self-dismutation assay, a dimethyl sulfoxide solution of superoxide is mixed in less than 2 ms with an aqueous buffer. The decay of superoxide is monitored directly by its absorbance at 245 nm and the data is processed by computer. By careful purification of the water and the use of metal-free buffers, a decay of superoxide that fits second-order kinetics is obtained without using metal ion chelators in the buffer. The second-order rate constant for superoxide decreased with increasing pH and decreased by a factor of 3.3 by using D2O in place of H2O in the buffer. The rapid mixing time makes it possible to determine rate constants for active superoxide dismutase catalysts at a pH as low as 7. A first-order decay of superoxide is obtained when the aqueous buffer contains bovine Cu/Zn superoxide dismutase or aquo copper(II), which are known catalysts of superoxide dismutation. The rate of superoxide decay was established to be first-order in catalyst. The catalytic rate constant for bovine Cu/Zn superoxide dismutase was determined to be 2.3 x 10(9) M-1 s-1 in H2O and D2O-based buffers and was independent of pH over the range 7-9. Aquo copper(II) gave a catalytic rate constant of 1.2 x 10(8) M-1 s-1, but was ineffective in the presence of EDTA. The catalytic rate constants obtained by stopped-flow kinetics are in excellent agreement with studies carried out by the direct method of pulse radiolysis.     

The iron superoxide dismutase from the filamentous cyanobacterium Nostoc PCC 7120. Localization, overexpression, and biochemical characterization

The nitrogen-fixing filamentous cyanobacterium Nostoc PCC 7120 (formerly named Anabaena PCC 7120) possesses two genes for superoxide dismutase, a unique membrane-associated manganese superoxide dismutase (MnSOD) and a soluble iron superoxide dismutase (FeSOD). A phylogenetic analysis of FeSODs shows that cyanobacterial enzymes form a well separated cluster with filamentous species found in one subcluster and unicellular species in the other. Activity staining, inhibition patterns, and immunogold labeling show that FeSOD is localized in the cytosol of vegetative cells and heterocysts (nitrogenase containing specialized cells formed during nitrogen-limiting conditions). The recombinant Nostoc FeSOD is a homodimeric, acidic enzyme exhibiting the characteristic iron peak at 350 nm in its ferric state, an almost 100% occupancy of iron per subunit, a specific activity using the ferricytochrome assay of (2040 +/- 90) units mg(-1) at pH 7.8, and a dissociation constant Kd of the azide-FeSOD complex of 2.1 mM. Using stopped flow spectroscopy it was shown that the decay of superoxide in the presence of various FeSOD concentrations is first-order in enzyme concentration allowing the calculation of the catalytic rate constants, which increase with decreasing pH: 5.3 x 10(9) M(-1) s(-1) (pH 7) to 4.8 x 10(6) M(-1) s(-1) (pH 10). FeSOD and MnSOD complement each other to keep the superoxide level low in Nostoc PCC 7120, which is discussed with respect to the fact that Nostoc PCC 7120 exhibits oxygenic photosynthesis and oxygen-dependent respiration within a single prokaryotic cell and also has the ability to form differentiated cells under nitrogen-limiting conditions.     

Superoxide levels and function of cerebral blood vessels after inhibition of CuZn-SOD

The goal of this study was to examine the role of endogenous copper/zinc (CuZn)-superoxide dismutase (SOD) on superoxide levels and on responses of cerebral blood vessels to stimuli that are mediated by nitric oxide (acetylcholine) and cyclooxygenase-dependent mechanisms (bradykinin and arachidonic acid). Levels of superoxide in the rabbit basilar artery were measured using lucigenin-enhanced chemiluminescence (5 microM lucigenin). Diethyldithiocarbamate (DDC; 10 mM), an inhibitor of CuZn-SOD, increased superoxide levels by approximately 2.4-fold (P < 0.05) from a baseline value of 1.0 +/- 0.2 relative light units x min(-1) x mm(-2) (means +/- SE). The diameter of cerebral arterioles (baseline diameter, 99 +/- 3 microm) was also measured using a closed cranial window in anesthetized rabbits. Topical application of DDC attenuated responses to acetylcholine, bradykinin, and arachidonate, but not nitroprusside. For example, 10 microM arachidonic acid dilated cerebral arterioles by 40 +/- 5 and 2 +/- 2 microm under control conditions and after DDC, respectively (P < 0.05). These inhibitory effects of DDC were reversed by the superoxide scavenger 4,5-dihydroxy-1,3-benzenedisulfonic acid (10 mM). Arachidonate increased superoxide levels in the basilar artery moderately under normal conditions and this increase was greatly augmented in the presence of DDC. These findings suggest that endogenous CuZn-SOD limits superoxide levels under basal conditions and has a marked influence on increases in superoxide in vessels exposed to arachidonic acid. The results also suggest that nitric oxide- and cyclooxygenase-mediated responses in the cerebral microcirculation are dependent on normal activity of CuZn-SOD.     

Oxidation of bis(terpyridine)cobalt(II) chloride by cytochrome c peroxidase compounds I and II

The bis(terpyridine)cobalt(II), Co(terpy)2(2+), reduction of cytochrome c peroxidase compound I, CcP-I, has been investigated using stopped-flow techniques as a function of ionic strength in pH 7.5 buffers at 25 degrees C. Co(terpy)2(2+) initially reduces the Trp191 radical site in CcP-I with an apparent second-order rate constant, k2, equal to 6.0+/-0.4x10(6) M(-1)s(-1) at 0.01 M ionic strength. A pseudo-first-order rate constant of 480 s(-1) was observed for the reduction of CcP-I by 79 microM Co(terpy)2(2+) at 0.01 M ionic strength. The one-electron reduction of CcP-I produces a second enzyme intermediate, CcP compound II (CcP-II), which contains an oxyferryl, Fe(IV), heme. Reduction of the Fe(IV) heme in CcP-II by Co(terpy)2(2+) shows saturation kinetics with a maximum observed rate constant, k3max, of 24+/-2 s(-1) at 0.01 M ionic strength. At low reductant concentrations, the apparent second-order rate constant for Co(terpy)2(2+) reduction of CcP-II, k3, is 1.2+/-0.5x10(6) M(-1) s-1. All three rate constants decrease with increasing ionic strength. At 0.10 M ionic strength, values of k2, k3, and k3max decrease to 6.0+/-0.8x10(5) M(-1) s(-1), 1.2+/-0.5x10(5) M(-1) s(-1), and 11+/-3 s(-1), respectively. Both the product, Co(terpy)2(3+), and ferricytochrome c inhibit the rate of Co(terpy)2(2+) reduction of CcP-I and CcP-II. Gel-filtration studies show that a minimum of two Co(terpy)2(3+) molecules bind to the native enzyme in low ionic strength buffers.     

Antioxidant activity of hemocyanin; a pulse radiolysis study

In order to determine the reactivity on hemocyanin from Androctonus australis, the reaction of superoxide anion has been investigated using pulse radiolysis. The kinetics of O2- decays have been studied in aqueous buffered media at various basic pH (8, 8.5 and 9), first in the absence and then in the presence of hemocyanin (in oxygenated solutions containing formate anion 0.16 mol.l-1). We have shown that, in the presence of hemocyanin, O2- decay is a first-order process whose apparent rate constant is proportional to protein concentration (10(-7) to 10(-6) mol.l-1) and pH independent between 8 to 9. A second-order rate constant of 3.5 +/- 0.1.10(7) mol-1.l.s-1, has been deduced for the catalytic rate constant of hemocyanin with O2-. Meanwhile, this activity is smaller than that described for free copper, eukaryotic Cu-Zn-SOD or some copper chelates. We have verified that apohemocyanin--the copper deprived protein--does not exhibit such an activity vs. SOD (superoxide dismutase).     

The kinetics of the reduction of cytochrome c by the superoxide anion radical.

1. At neutral pH ferricytochrome c is reduced by the superoxide anion radical (O2-), without loss of enzymatic activity, by a second order process in which no intermediates are observed. The yield of ferrocytochrome c (82-104%), as related to the amount of O2- produced, is slightly dependent on the concentration of sodium formate in the matrix solution. 2. The reaction (k1 equals (1.1+/-0.1) - 10(6) M-1 - s-1 at pH 7.2, I equals 4 mM and 21 degrees C) can be inhibited by superoxide dismutase and trace amounts of copper ions. The inhibition by copper ions is removed by EDTA without interference in the O2- reduction reaction. 3. The second-order rate constant for the reaction of O2- with ferricytochrome c depends on the pH of the matrix solution, decreasing rapidly at pH greater than 8. The dependence of the rate constant on the pH can be explained by assuming that only the neutral form of ferricytochrome c reacts with O2- and that the alkaline form of the hemoprotein is unreactive. From studies at pH 8.9, the rate for the transition from the alkaline to the neutral form of ferricytochrome c can be estimated to be 0.3 s-1 (at 21 degrees C and I equals 4 mM). 4. The second-order rate constant for the reaction of O2- with ferricytochrome c is also dependent on the ionic strength of the medium. From a plot of log k1 versus I1/2-(I + alphaI1/2)-1 we determined the effective charge on the ferricytochrome c molecule as +6.3 and the rate constant at I equals 0 as (3.1+/-0.1) - 10(6) M-1 - s-1 (pH 7.1, 21 degrees C). 5. The possibility that singlet oxygen is formed as a product of the reaction of O2- with ferricytochrome c can be ruled out on thermodynamic grounds.     

Electrostatic dependence of the thrombin-thrombomodulin interaction

The rate constants for the binding interaction between thrombin and a fully active fragment of its anticoagulant cofactor, thrombomodulin, have been determined by surface plasmon resonance. At physiological ionic strength, the k(a) was 6.7x10(6) M(-1) s(-1 )and the dissociation rate constant was 0.033 s(-1). These extremely fast association and dissociation rates resulted in an overall binding equilibrium constant of 4.9 nM, which is similar to previously reported values. Changing the ionic strength from 100 mM to 250 mM NaCl caused a tenfold decrease in the association rate while the dissociation rate did not change significantly. A similar effect was observed with tetramethylammonium chloride. A Debye-Hückel plot of the data had a slope of -6 and an intercept at 0 ionic strength of 10(9) M(-1) s(-1). The same slope and intercept were obtained for data that was collected in the presence of glycerol to slow the association rates. These results show that the thrombin-TM456 interaction is extremely rapid and nearly completely electrostatically steered. An association model is presented in which TM456 approaches thrombin along the direction of the thrombin molecular dipole.     

A flash-photometric method for determination of reactivity of superoxide: application to superoxide dismutase assay

Pulse-generation of O2- by a flash was used to determine the reactivity of O2-, O2- was produced within 10 ms by a flash of light through the excitation of FMN in the presence of N,N,N',N'-tetramethylethylenediamine and oxygen. Kinetic analysis of cytochrome c reduction by O2- generated by flash yielded the reaction rate constant between cytochrome c and O2- and the spontaneous disproportionation rate constant of O2-. We applied it for superoxide dismutase assay using a linear relation between superoxide dismutase concentration and the apparent rate constant of cytochrome c reduction by O2-. The catalytic rate constant and activation energy at pH 7.3 of bovine liver Cu,Zn-superoxide dismutase were found to be 1.75 x 10(9) M-1 . s-1 at 25 degrees C and 26.9 kJ . M-1, respectively. The kinetics of O2- decay can be also monitored at 240 nm in this flash-photometric system and gave the spontaneous disproportionation rate constant of O2- and the catalytic rate constant of superoxide dismutase.     

Binding of ellipticine base and ellipticinium cation to calf-thymus DNA. A thermodynamic and kinetic study

The acid-basic properties of ellipticine have been re-estimated. The apparent pK of protonation at 3 microM drug concentration is 7.4 +/- 0.1. The ellipticine free base (at pH 9, I = 25 mM) intercalates into calf-thymus DNA with an affinity constant of 3.3 +/- 0.2 X 10(5) M-1, and a number of binding sites per phosphate of 0.23. The ellipticinium cation (pH 5, I = 25 mM) binds also to DNA with a constant of 8.3 +/- 0.2 x 10(5) M-1 and at a number of binding sites (n = 0.19). It is postulated that the binding of the drug to DNA at pH 9 is driven by hydrophobic and/or dipolar effects. Even at pH 5, where ellipticine exists as a cation, it is thought that the hydrophobic interaction is the main contribution to binding. The neutral and cationic forms share common binding within DNA sites but yield to structurally different complexes. The free base has 0.04 additional specific binding sites per phosphate. As determined from temperature-jump experiments, the second-order rate constant of the binding of the free base (pH 9) is 3.4 x 10(7) M-1 s-1 and the residence time of the base within the DNA is 8 ms. The rate constant for the binding of the ellipticinium cation is 9.8 x 10(7) M-1 s-1 when it is assumed that drug attachment occurs via a pathway in which the formation of an intermediate ionic complex is not involved (competitive pathway).     

Kinetics and mechanism of the reduction of ferricytochrome c by the superoxide anion

The temperature and pH dependence of the reaction of the superoxide radical anion with ferricytochrome c have been measured using the pulse-radiolysis technique. The temperature dependence of the reaction at low ionic strength yields an activation energy of 31 +/- 5 kJ/mol as compared to 14 +/- 3 kJ/mol for the reaction of CO2.(-) under the same conditions. The pH dependence fits the single pK'a of ferricytochrome c of 9.1. The bimolecular rate constant for the reaction of the superoxide anion with ferricytochrome c at pH 7.8, 21 +/- 2 degrees C, in the presence of 50 mM phosphate and 0.1 mM EDTA is (2.6 +/- 0.1) X 10(5) M-1 s-1. Using this value, 1 unit of superoxide dismutase activity (McCord, J. M., and Fridovich, I. (1969) J. Biol. Chem. 244, 6049-6055) is calculated to be 3.6 +/- 0.3 pmol of enzyme if the assay is performed in a total volume of 3.0 ml. Copper ions reduce the yield of the reaction of ferricytochrome c with CO2.(-). The reactivities of native and singly modified 4-carboxy-2,4-dinitrophenyllysine cytochromes c towards the superoxide anion radical are in the order native greater than 4-carboxy-2,4-dinitrophenyllysine 60 greater than lysine 13 greater than lysine 87 greater than lysine 27 greater than lysine 86 greater than lysine 72, indicating that electron transfer takes place at or close to the solvent accessible heme edge. The mechanism of the reaction is discussed in terms of the approach of superoxide anion radicals to the heme edge and the available molecular orbitals of both heme and free radicals.     

Osmium tetroxide, used in the treatment of arthritic joints, is a fast mimic of superoxide dismutase

Aqueous solutions of osmium tetroxide (OsO4) have been injected into arthritic knees for the past 45 years to chemically destroy diseased tissue, in a procedure termed "chemical synovectomy." Arthritis is an inflammatory disease. The primary inflammatory chemical species are the superoxide anion radical (O2.-) and nitric oxide (.NO), which combine to form the peroxynitrite anion (ONOO-). Here we show that OsO4 does not react with ONOO- but very efficiently catalyzes the dismutation of O2.- to O2 and H2O2. Using the pulse-radiolysis technique, the catalytic rate constant has been determined to be (1.43+/-0.04) x 10(9) M-1 s-1, independent of the pH in the 5.1-8.7 range. This value is about half that for the natural Cu,Zn-superoxide dismutase (Cu,Zn-SOD). Per unit mass, OsO4 is about 60 times more active than Cu,Zn-SOD. The catalytically active couple is OsVIII/OsVII, OsVIII oxidizing O2.- to O2 with a bimolecular rate constant of k=(2.6+/-0.1)x10(9) M-1 s-1 and OsVII reducing it to H2O2 with a bimolecular rate constant of (1.0+/-0.1)x10(9) M-1 s-1. Although lower valent osmium species are intrinsically poor catalysts, they are activated through oxidation by O2.- to the catalytic OsVIII/OsVII redox couple. The OsVIII/OsVII catalyst is stable to biochemicals other than proteins and peptides comprising histidine, cysteine, and dithiols.     

Role of the electrostatic loop charged residues in Cu,Zn superoxide dismutase.

We have expressed and characterized a mutant of Xenopus laevis Cu,Zn superoxide dismutase in which four highly conserved charged residues belonging to the electrostatic loop have been replaced by neutral side chains: Lys120 --> Leu, Asp130 --> Gln, Glu131 --> Gln, and Lys134 --> Thr. At low ionic strength, the mutant enzyme is one of the fastest superoxide dismutases ever assayed (k = 6.7 x 10(9) M(-1) s(-1), at pH 7 and mu = 0.02 M). Brownian dynamics simulations give rise to identical enzyme-substrate association rates for both wild-type and mutant enzymes, ruling out the possibility that enhancement of the activity is due to pure electrostatic factors. Comparative analysis of the experimental catalytic rate of the quadruple and single mutants reveals the nonadditivity of the mutation effects, indicating that the hyperefficiency of the mutant is due to a decrease of the energy barrier and/or to an alternative pathway for the diffusion of superoxide within the active site channel. At physiological ionic strength the catalytic rate of the mutant at neutral pH is similar to that of the wild-type enzyme as it is to the catalytic rate pH dependence. Moreover, mutation effects are additive. These results show that, at physiological salt conditions, electrostatic loop charged residues do not influence the diffusion pathway of the substrate and, if concomitantly neutralized, are not essential for high catalytic efficiency of the enzyme, pointing out the role of the metal cluster and of the invariant Arg141 in determining the local electrostatic forces facilitating the diffusion of the substrate towards the active site.     

Oxidation of cytochrome c peroxidase to compound I by peroxyacids: evidence for rate-limiting diffusion through the protein matrix

The rate of the reaction between p-nitroperoxybenzoic acid and cytochrome c peroxidase (CcP) has been investigated as a function of pH and ionic strength. The pH dependence of the reaction between CcP and peracetic acid has also been determined. The rate of the reactions are influenced by two heme-linked ionizations in the protein. The enzyme is active when His-52 (pK(a) 3.8 +/- 0.1) is unprotonated and an unknown group with a pK(a) of 9.8 +/- 0.1 is protonated. The bimolecular rate constant for the reaction between peracetic acid and CcP and between p-nitroperoxybenzoic acid and CcP are (1.8 +/- 0.1) x 10(7) and (1.6 +/- 0.2) x 10(7) M(-)(1) s(-)(1), respectively. These rates are about 60% slower than the reaction between hydrogen peroxide and CcP. A critical comparison of the pH dependence of the reactions of hydrogen peroxide, peracetic acid, and p-nitroperoxybenzoic acid with CcP provides evidence that both the neutral and anionic forms of the two peroxyacids react directly with the enzyme. The peracetate and p-nitroperoxybenzoate anions react with CcP with rates of (1.5 +/- 0.1) x 10(6) and (1.6 +/- 0.2) x 10(6) M(-)(1) s(-)(1), respectively, about 10 times slower than the neutral peroxyacids. These data indicate that CcP discriminates between the neutral peroxyacids and their negatively charged ions. However, the apparent bimolecular rate constant for reaction between p-nitroperoxybenzoate and CcP is independent of ionic strength in the range of 0.01-1.0 M, suggesting that electrostatic repulsion between the anion and CcP is not the cause of the lower reactivity for the peroxybenzoate anion. The data are consistent with the hypothesis that the rate-limiting step for the oxidation of CcP to compound I by both neutral peroxyacid and the negatively charged peroxide ion is diffusion of the reactants through the protein matrix, from the surface of the protein to the distal heme pocket.     

Benefits of membrane electrodes in the electrochemistry of metalloproteins: mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by horse cytochrome c: a case study

A comparative study of direct and mediated electrochemistry of metalloproteins in bulk and membrane-entrapped solutions is presented. This work reports the first electrochemical study of the electron transfer between a bacterial cytochrome c peroxidase and horse heart cytochrome c. The mediated catalysis of the peroxidase was analysed both using the membrane electrode configuration and with all proteins in solution. An apparent Michaelis constant of 66 +/- 4 and 42 +/- 5 microM was determined at pH 7.0 and 0 M NaCl for membrane and bulk solutions, respectively. The data revealed that maximum activity occurs at 50 mM NaCl, pH 7.0, with intermolecular rate constants of (4.4 +/- 0.5) x 10(6) and (1.0 +/- 0.5) x 10(6) M(-1) s(-1) for membrane-entrapped and bulk solutions, respectively. The influence of parameters such as pH or ionic strength on the mediated catalytic activity was analysed using this approach, drawing attention to the fact that careful analysis of the results is needed to ensure that no artefacts are introduced by the use of the membrane configuration and/or promoters, and therefore the dependence truly reflects the influence of these parameters on the (mediated) catalysis. From the pH dependence, a pK of 7.5 was estimated for the mediated enzymatic catalysis.     

Reaction of peroxynitrite with Mn-superoxide dismutase. Role of the metal center in decomposition kinetics and nitration

Manganese superoxide dismutase (Mn-SOD), a critical mitochondrial antioxidant enzyme, becomes inactivated and nitrated in vitro and potentially in vivo by peroxynitrite. Since peroxynitrite readily reacts with transition metal centers, we assessed the role of the manganese ion in the reaction between peroxynitrite and Mn-SOD. Peroxynitrite reacts with human recombinant and Escherichia coli Mn-SOD with a second order rate constant of 1.0 +/- 0.2 x 10(5) and 1.4 +/- 0.2 x 10(5) m(-)1 s(-)1 at pH 7.47 and 37 degrees C, respectively. The E. coli apoenzyme, obtained by removing the manganese ion from the active site, presents a rate constant <10(4) m(-)1 s(-)1 for the reaction with peroxynitrite, whereas that of the manganese-reconstituted apoenzyme (apo/Mn) was comparable to that of the holoenzyme. Peroxynitrite-dependent nitration of 4-hydroxyphenylacetic acid was increased 21% by Mn-SOD. The apo/Mn also promoted nitration, but the apo and the zinc-substituted apoenzyme (apo/Zn) enzymes did not. The extent of tyrosine nitration in the enzyme was also affected by the presence and nature (i.e. manganese or zinc) of the metal center in the active site. For comparative purposes, we also studied the reaction of peroxynitrite with low molecular weight complexes of manganese and zinc with tetrakis-(4-benzoic acid) porphyrin (tbap). Mn(tbap) reacts with peroxynitrite with a rate constant of 6.8 +/- 0.1 x 10(4) m(-)1 s(-)1 and maximally increases nitration yields by 350%. Zn(tbap), on the other hand, affords protection against nitration. Our results indicate that the manganese ion in Mn-SOD plays an important role in the decomposition kinetics of peroxynitrite and in peroxynitrite-dependent nitration of self and remote tyrosine residues.     

Properties of purified cytosolic isoenzyme I of Cu,Zn-superoxide dismutase from Nicotiana plumbaginifolia leaves.

The isoenzyme I of cytosolic Cu,Zn-superoxide dismutase (SOD) from Nicotiana plumbaginifolia (tobacco) leaves has been purified to apparent homogeneity. The relative molecular mass of the native isoenzyme, determined by gel filtration chromatography, is about 33.2 kDa. SDS-polyacrylamide gel electrophoresis shows that the enzyme is composed of two equal subunits of 16.6 kDa The isolectric point, assayed by isoelectric focusing, in the pH range of 3.5-6.5, is 4.3. The enzyme stability was tested at different temperatures, pH, and concentration of inhibitors (KCN and H(2)O(2)). The catalytic constant (k(cat)) was 1.17 +/- 0.14 x 10(9) M(-1) s(-1) at pH 9.9 and 0.1 M ionic strength. The activation energy of the thermal denaturation process is 263 kJ mol(-1). The electrostatic surface potential of the modeled tobacco Cu,Zn-SOD I was calculated showing that the functional spatial network of charges on the protein surface has been maintained, independently of the amino acid substitution around the active sites.     

Autoxidation of soluble trypsin-cleaved microsomal ferrocytochrome b5 and formation of superoxide radicals.

The rate and mechanism of autoxidation of soluble ferrocytochrome b5, prepared from liver microsomal suspensions, appear to reflect an intrinsic property of membrane-bound cytochrome b5. The first-order rate constant for autoxidation of trypsin-cleaved ferrocytochrome b5, prepared by reduction with dithionite, was 2.00 X 10(-3) +/- 0.19 X 10(-3) S-1 (mean +/- S.E.M., n =8) when measured at 30 degrees C in 10 mM-phosphate buffer, pH 7.4. At 37 degrees C in aerated 10 mM-phosphate buffer (pH 7.4)/0.15 M-KCl, the rate constant was 5.6 X 10(-3) S-1. The autoxidation reaction was faster at lower pH values and at high ionic strengths. Unlike ferromyoglobin, the autoxidation reaction of which is maximal at low O2 concentrations, autoxidation of ferrocytochrome b5 showed a simple O2-dependence with an apparent Km for O2 of 2.28 X 10(-4) M (approx. 20kPa or 150mmHg)9 During autoxidation, 0.25 mol of O2 was consumed per mol of cytochrome oxidized. Cyanide, nucleophilic anions, EDTA and catalase each had little or no effect on autoxidation rates. Adrenaline significantly enhanced autoxidation rates, causing a tenfold increase at 0.6 mM. Ferrocytochrome b5 reduced an excess of cytochrome c in a biphasic manner. An initial rapid phase, independent of O2 concentration, was unaffected by superoxide dismutase. A subsequent slower phase, which continued for up to 60 min, was retarded at low O2 concentrations and inhibited by 65% by superoxide dismutase at a concentration of 3 mug/ml. It is concluded that autoxidation is responsible for a significant proportion of electron flow between cytochrome b5 and O2 in liver endoplasmic membranes, this reaction being capable of generating superoxide anions. A biological role for the reaction is discussed.     

Microtubule assembly kinetics. Changes with solution conditions.

The assembly kinetics of microtubule protein are altered by ionic strength, temperature and Mg2+, but not by pH. High ionic strength (I0.2), low temperature (T less than 30 degrees C) and elevated Mg2+ (greater than or equal to 1.2 mM) induce a transition from biphasic to monophasic kinetics. Comparison of the activation energy obtained for the fast biphasic step at low ionic strength (I0.069) shows excellent agreement with the values obtained at high ionic strength, low temperature and elevated Mg2+. From this observation it can be implied that the tubulin-containing reactant of the fast biphasic event is also the species that elongates microtubules during monophasic assembly. Second-order rate constants for biphasic assembly are 3.82(+/- 0.72) x 10(7) M-1.s-1 and 5.19(+/- 1.25) x 10(6) M-1.s-1, and for monophasic assembly the rate constant is 2.12(+/- 0.56) x 10(7) M-1.s-1. The microtubule number concentration is constant during elongation of microtubules for biphasic and monophasic assembly.