Dismutation rate

Attempts to optimize the stability and SOD activity of C-substi-tuted (R = methyl and fused cycloalkyl) [Mn(II)[15]aneN5)Cl2] complexes 95 have shown that increasing the number of hydrocarbon substituents greatly increases the kinetic stability of the complex toward dissociation via protonation (Fig. 19). (443). There is also some enhancement of thermodynamic stability. The trans-fused endohexano Mn(II) complex 96 has a faster dismutation rate constant (9.09 X 107M-1 s1, pH 7.4) and a 10 times higher thermodynamic stability than the unsubstituted complex. 

A series of 4- and 5-coordinated Mn11 complexes of salen Schiff bases (Figure 5) [20] have either N202 or N202C1 chromophores and dismutation rates similar to those of the previously reported desferrioxamine [20,32a,45,46] complexes. 

The dismutation rate constant includes in it the fraction of H-H additions.) Here Xn depends on monomer concentration which is contrary to fact. There are about 1-2% H-H additions along the PVAc backbone (31), so it does not dismute as soon as one monomer adds backward. 

The dismutation rate of 05 catalyzed by CuZn-SOD, 2X109 M-1 s l, is 10% of the rate of diffusion-limited reaction expected for a small molecule (05) and a macromolecule (SOD), whereas the Cu site comprises only 0.1% of the total surface area of CuZn-SOD. This indicates that the interaction of 05 with the Cu site is facilitated 100-fold, for which an electrostatic interaction is responsible. 

The overall advantage of enzymatic dismutation of superoxide is illustrated in Fig. 2, where the rate constants for dismutation for three of the enzyme classes are plotted as a function of pH on a graph that also has the self-dismutation rate constants for superoxide/perhy-droxyl radical plotted as a function of pH. It should be noted, though, that the enzymatic rate constants are catalytic and therefore only depend upon the enzyme concentiation whereas the self-dismu-tation is bimolecular in superoxide. This leads to very important ramifications with regards to the enzyme-substrate concentrations. Figure 3 illustrates three different scenarios and shows the relative rate constants at different extremes of [S0D] [02 ]. The in vivo implications of this are profound. 

One of the earliest demonstrations by ESR of the peroxidase oxidation of drugs involved the tranquilizer, chlorpromazine [171]. The stoichiometry of the reaction forming the chlorpromazine radical cation (13) was established, and kinetics of the transient radical were shown to be identical to those of an optically detected intermediate absorbing at 530 nm. Measurements were made of the pH-dependent radical dismutation rate and the rate of reaction of compound II with chlorpromazine. At high enzyme concentrations and low dismutation rates, the radical cation undergoes further oxidation by the enzyme, to form the thionium ion. 

The dismutation rate constants (2kd) for TNT and other radicals of explosives are not reported however, for the radicals of o-, m-, p-dinitrobcnzcncs, 2kd are equal to 2.4 x 106 M 1s 1, 8.0 x 106 M 1s 1, and 3.3 x 108 M ls, respectively, at pH 7.0 [40], It is important to note that the fraction of stable reduction products of nitroaromatics may be formed under aerobic conditions due to the competition between the nitroradical oxidation by oxygen and its dismutation. The rate of dismutation of free radicals (Vdism) may be expressed as... 

SODs are differentiated mainly by the redox-active metal in the active site copper, manganese, or iron. The iron and manganese SODs are structurally similar (5-11) and are structurally distinct from the Cu,Zn SOD (12). The dramatic features of these enzymes are that they catalytically dismutate superoxide at rates that are not only diffusion controlled but have been shown to be electrostatically facilitated (13). In these systems, modifications of amino acid residues near the active site have been shown to alter the enzymatic activity, indicating that superoxide is electrostatically drawn into the active site channel (14). In addition, in contrast to the spontaneous dismutation rate of 02 and the dismutation rates of 02 by many metal complexes, all of which are pH dependent, the enzymatic dismutation rate is largely pH independent over the pH range (5-10). 

The methylene blue semiquinone protonation states assigned by Keene et al. (12) differ from the results of Matsumoto (15), who proposed the following four states MB (pH > 9), MBH+ (pH 3-8), MBH22+ (in 0.1 N H2S04) and MBH >3+ (in cone. H2S04). Recent measurements by J. Faure, R. Bonneau, and J. Joussot-Dubien [I. Chim. Phys. 65, 369 (1968)] on the effect of the ionic strength on the dismutation rate constant support the conclusion of Matsumoto that the neutral semiquinone MB- is stable only above pH 9. Keene, Land, and Swallow... 

These direct assays provide immediate answers about the rate of dismutation of superoxide by SOD. Activity assays performed on the same sample with different direct methods (i.e., pulse radiolysis and polarography) in most cases give similar dismutation rate constants... 

Fig. 16. Effect of ionic strength on dismutation rate of O2 by superoxide dismutase as measured experimentally by pulse radiolysis at pH 8. Ionic strengths were adjusted using NaCl. O, Bovine SOD , human SOD expressed in yeast , mutant Cys-6Ala, Cys-lllSer of human SOD expressed in yeast , Thr-137Ile SOD A, Arg-143Lys.

The Cu2ZnjSuperoxide dismutase has dismutation rates for the superoxide ion which are near the diffusion control (2.0 0.5 x 10 M values. The reaction catalyzed can be summarized as follows ... 

Curve a) shows the reduction to O2" in the presence of triphenylphosphinoxide, whereas curve d) corresponds to the reduction to H2O2. The addition of superoxide dismutase converts part of the OJ to dioxygen which increases the limiting current. By that means, the dropping mercury electrode is at the same time both a a>urce of O2 and a detector of its dismutation rate according to ... 

Figure lb shows the transient absorption spectra of RF (i.e. the difference between the ground singlet and excited triplet states) obtained by laser-flash photolysis using a Nd Yag pulsed laser operating at 355 nm (10 ns pulse width) as excitation source. At short times after the laser pulse, the transient spectrum shows the characteristic absorption of the lowest vibrational triplet state transitions (0 <— 0) and (1 <— 0) at approximately 715 and 660 nm, respectively. In the absence of GA, the initial triplet state decays with a lifetime around 27 ps in deoxygenated solutions by dismutation reaction to form semi oxidized and semi reduced forms with characteristic absorption bands at 360 nm and 500-600 nm and (Melo et al., 1999). However, in the presence of GA, the SRF is efficiently quenched by the gum with a bimolecular rate constant = 1.6x10 M-is-i calculated... 

Under conditions where the dismutation reaction is slow the exchange between Au(III) and Au(I) has been shown to proceed at a measurable rate at 0 °C in 0.09 M HCl, an exchange half-time of about 2 min was observed. The isotopic method ( Au) and a separation method based on the precipitation of dipyridine -chloroaurate(III) was used to obtain data. An acceleration in the exchange rate was observed as the HCl concentration was increased. ... 

Scheme B) is considered unlikely on the grounds that, at the low concentrations of U(V) involved, the latter would disappear by oxidation with Fe(ni) rather by dismutation. The hydrogen-ion dependence suggests that the rate-controlling step between Fe(irf) and U([V) can be visualised in terms of a series of competitive reactions of hydrolysed species of both reactants viz.

Another area of active research is the development of stable low molecular weight metal complexes, which could serve as SOD mimics. Fridovich has described a complex of mangsmese (III) with desferral, which can catalyse the dismutation of superoxide anion in vitro and can protect green algae against paraquat toxicity (Beyer and Fridovich, 1989). This manganese-desferral complex was evaluated in models of circulatory shock and also found to improve survival rate (de Garavilla etal., 1992). 

The rate constant of electron transfer (ks) and anodic and cathodic electron transfer coefficients (aa and ac) of the SODs at various pH values were estimated with Laviron s equation and summarized in Table 6.5. Interestingly, the fastest electron transfer of the SODs was essentially achieved in a neutral solution, probably in agreement with the biological conditions for the inherent catalytic mechanisms of the SODs for 02" dismutation, although the electrode processes of the SODs follow a different mechanism. 

Similar reactions are catalyzed by Mn and Fe centers of MnSOD and FeSOD. It is obvious that before participation in Reaction (2), superoxide must be protonized to form hydroper-oxyl radical HOO by an outer-sphere or an intra-sphere mechanisms. All stages of dismuting mechanism, including the measurement of elementary rate constants, have been thoroughly studied earlier (see, for example, Ref. [2]). 

High values of reaction rates for the two dismutation steps confirm the ability of both nitroxides TPO and 3-CP to be SOD mimics. However, as follows from the above mechanism, hydroperoxyl radical and not superoxide must participate in the first dismutation step (Reaction (5)). (As expected, a rate constant for the reaction of nitroxides with superoxide is very low <103 1 mol 1 s-1 [27].) Therefore, superoxide had to be protonated before participating in Reaction (5), which will diminish the total catalytic process at physiological pH and increase it at lower pH values. 

Many transition metal complexes have been considered as synzymes for superoxide anion dismutation and activity as SOD mimics. The stability and toxicity of any metal complex intended for pharmaceutical application is of paramount concern, and the complex must also be determined to be truly catalytic for superoxide ion dismutation. Because the catalytic activity of SOD1, for instance, is essentially diffusion-controlled with rates of 2 x 1 () M 1 s 1, fast analytic techniques must be used to directly measure the decay of superoxide anion in testing complexes as SOD mimics. One needs to distinguish between the uncatalyzed stoichiometric decay of the superoxide anion (second-order kinetic behavior) and true catalytic SOD dismutation (first-order behavior with [O ] [synzyme] and many turnovers of SOD mimic catalytic behavior). Indirect detection methods such as those in which a steady-state concentration of superoxide anion is generated from a xanthine/xanthine oxidase system will not measure catalytic synzyme behavior but instead will evaluate the potential SOD mimic as a stoichiometric superoxide scavenger. Two methodologies, stopped-flow kinetic analysis and pulse radiolysis, are fast methods that will measure SOD mimic catalytic behavior. These methods are briefly described in reference 11 and in Section 3.7.2 of Chapter 3. 

Since it has been reported that in the inner-sphere SOD catal5rtic pathway (Scheme 5) the water-exchange process is the rate-limiting one, the inner-sphere catalytic rate constants is were correlated with the water-exchange rate constants on [Mn(H20)6l (22,31). However, it seems that it is not possible to draw a direct correlation between these rate constants. Firstly, is (which is pH independent) according to the observed rate law for dismutation of superoxide (V — —d[02 ]/ d = [Mn][02 ] H[H+]+ ind>, ind 2kis, ku = 2kos/KJ has the unit... 

In the literature there are no quantitative studies on the kinetics and thermodynamics of stoichiometric superoxide reactions with metal centers in general, and metalloporphyrins in particular. More precisely, superoxide concentration and temperature dependent kinetic and thermodynamic measurements were never reported and consequently the rate constants, activation parameters or binding constants for this t5rpe of reactions (Scheme 15) are not known. (The catalytic rate constants for the superoxide disproportionation, i.e., dismutation, by metal complexes are known (see earlier), however in those measurements the concentration of a catalytic amount... 

The most efficient catalyst for the dismutation of O, is the [CufHjO) ] complex with a second-order rate constant of 8x 10 s more than twice... 

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