Ferric enzyme

MnP is the most commonly widespread of the class II peroxidases [72, 73], It catalyzes a PLC -dependent oxidation of Mn2+ to Mn3+. The catalytic cycle is initiated by binding of H2O2 or an organic peroxide to the native ferric enzyme and formation of an iron-peroxide complex the Mn3+ ions finally produced after subsequent electron transfers are stabilized via chelation with organic acids like oxalate, malonate, malate, tartrate or lactate [74], The chelates of Mn3+ with carboxylic acids cause one-electron oxidation of various substrates thus, chelates and carboxylic acids can react with each other to form alkyl radicals, which after several reactions result in the production of other radicals. These final radicals are the source of autocataly tic ally produced peroxides and are used by MnP in the absence of H2O2. The versatile oxidative capacity of MnP is apparently due to the chelated Mn3+ ions, which act as diffusible redox-mediator and attacking, non-specifically, phenolic compounds such as biopolymers, milled wood, humic substances and several xenobiotics [72, 75, 76]. 

Several compounds can be oxidized by peroxidases by a free radical mechanism. Among various substrates of peroxidases, L-tyrosine attracts a great interest as an important phenolic compound containing at 100 200 pmol 1 1 in plasma and cells, which can be involved in lipid and protein oxidation. In 1980, Ralston and Dunford [187] have shown that HRP Compound II oxidizes L-tyrosine and 3,5-diiodo-L-tyrosine with pH-dependent reaction rates. Ohtaki et al. [188] measured the rate constants for the reactions of hog thyroid peroxidase Compounds I and II with L-tyrosine (Table 22.1) and showed that Compound I was reduced directly to ferric enzyme. Thus, in this case the reaction of Compound I with L-tyrosine proceeds by two-electron mechanism. In subsequent work these authors have shown [189] that at physiological pH TPO catalyzed the two-electron oxidation not only L-tyrosine but also D-tyrosine, A -acetyltyrosinamide, and monoiodotyrosine, whereas diiodotyrosine was oxidized by a one-electron mechanism. 

LOXs are proteins containing a single atom of nonheme iron in catalytic center, with the ferric enzyme in an active form. The free radical-mediated mechanism of LOX-catalyzed process may be presented as follows (see also Figure 26.1) ... 

In accord with this mechanism, free peroxyl radical of the reaction product hydroperoxide activates the inactive ferrous form of enzyme (Reaction (1)). Then, active ferric enzyme oxidizes substrate to form a bound substrate radical, which reacts with dioxygen (Reaction (4)). The bound peroxyl radical may again oxidize ferrous enzyme, completing redox cycling, or dissociate and abstract a hydrogen atom from substrate (Reaction (6)). 

It has already been mentioned earlier that similar to LOXs, prostaglandin H synthases can be activated or inhibited by reactive nitrogen species. Nitric oxide may exhibit the inhibitory [58,65,86,101 104] or stimulatory effects [105 110] on PGHSs. Inhibitory effects depend on the ability of nitric oxide to reduce the ferric enzyme to the inactive ferrous form, competition... 

Fig. 4. Visible spectra of catalase, compound I, and compound II 5 [xM (heme) beef liver catalase (Boehringer-Mannheim) in 0.1 M potassium phosphate buffer pH 7.4, 30°C. Compound I was formed by addition of a slight excess of peroxoacetic acid. Compound II was formed from peroxoacetic acid compound I by addition of a small excess of potassium ferrocyanide. Absorbance values are converted to extinction coefficients using 120 mM for the coefficient at 405 nm for the ferric enzyme (confirmed by alkaline pyridine hemochromogen formation). Spectra are corrected to 100% from occupancies of f 90% compound I, 10% ferric enzyme (steady state compound I) and 88% compound II, 12% compound I (steady state compound II). The extinction coefficients for the 500 to 720 nm range have been multiplied by 10. Unpublished experiments (P.N., 1999).

The donor types D3, D4, and D6 of Keilin and Nicholls (37) all reduce compound I of Type A enzymes directly to the ferric state in a two-electron process without detectable intermediates. Each of these donors is probably also able to bind in the heme pocket of the free enzyme. Alcohols (type D3) form complexes with free ferric Type A enzymes whose apparent affinities parallel the effectiveness of the same alcohols as compound I donors (39). Formate (type D3) reacts with mammalian ferric enzyme at a rate identical to the rate with which it reduces compound I to free enz5mie (22). Its oxidation by compound I may thus share an initial step analogous to its complex formation with ferric enzyme. Formate also catalyzes the reduction of compound II to ferric enzyme by endogenous donors in the enz5mie (40, 41). Both compound I and compound II may thus share with the free enzyme the ability to ligate formate in the heme pocket. Nitrite, which is oxidized to nitrate by a two-electron reaction with compoimd I (type D4), also forms a characteristic complex with free enzyme (42). In both cases the reaction involves the donor in its protonated (HNO2) form. 

Effect of pH on Lignin Peroxidase Catalysis. The oxidation of organic substrates by lignin peroxidase (Vmax) has a pH optimum equal to or possibly below 2. Detailed studies have been performed on the pH dependency of many of the individual reactions involved in catalysis. The effect of pH on the reaction rates between the isolated ferric enzyme, compounds I or II and their respective substrates has been studied. Rapid kinetic data indicate that compound I formation from ferric enzyme and H2O2 is not pH dependent from pH 2.5-7.5 (75,16). Similar results are obtained with Mn-dependent peroxidase (14). This is in contrast to other peroxidases where the pKa values for the reaction of ferric enzyme with H2O2 are usudly in the range of 3 to 6 (72). 

It is interesting to compare the oxygenase activities of binuclear and mononuclear iron enzymes. The iron in mononuclear oxygenases may serve either as a Lewis acid to activate the substrate (ferric enzymes) or as a Lewis base to activate oxygen (ferrous enzymes). It appears that in the binuclear enzymes the iron center performs both functions. The difer-rous center first activates oxygen to the hydroperoxide and is converted... 

The P. chlororaphis B23 NHase is the ferric enzyme [50], which has been characterized in detail [51,52]. (i) The NHase is the first known nonheme iron enzyme containing a typical low-spin Fe(III) site, (ii) the axial position of the Fe(III) site in the enzyme may be occupied by aquo and sulfhydryl groups, and (iii) aliphatic nitrile substrates directly bind to the Fe(III)-active center through H20-substrate replacement. The NHase also seems to contain pyrroloquinoline quinone (PQQ) or a PQQ-like compound [53]. 

Redox titrations involve determination of equilibrium between the enzyme and a redox agent of known redox potential. The method requires a redox agent with redox potential close to the protein of interest, to ensure reversibility. The protein is exposed to different concentrations of the redox agent, and once equilibrium is attained, the half cell potential is measured with electrodes and the oxidation-reduction state of the proteins is measured by some physical technique, usually UV-Vis spectrophotometry. The concentration of the oxidized and reduced forms is determined at isosbestic points, and thus spectral characterization of redox species (ferric enzyme,... 

Enzyme Compound 1/ Compound II Compound 11/ ferric enzyme... 

The EPR spectra of native LiP (34) and MnP (35) are also typical of high-spin ferric heme, with g values 5.83 and 1.99 (LiP) and 5.79 and 1.99 (MnP). These values are essentially identical to those of aquometmyoglobin but differ somewhat from those of HRP for which a large rhombic component is observed. Resonance Raman (RR) studies (34-37) also indicate that native LiP and MnP are high-spin ferric enzymes which are predominantly pentacoordinate at room temperature. RR studies confirm that the native enzymes form low-spin complexes with CN and N3 and that the reduced enzymes have high-spin... 

The oxidation of phenols and other organic substrates by MnP Is dependent on Mnll (23,30). Apparently the enzyme first oxidizes Mnll to Mnlll, and Mnlll subsequently oxidizes the organic substrates (30,31,47). As shown In Figure 3, addition of one equivalent of Mnll rapidly reduces MnP compound I to compound II (41). A second equivalent of Mnll reduces MnP compound II to the native ferric enzyme. Similarly, MnP compound I Is reducible by phenolic substrates, albeit at a slower rate. However, phenolic substrates are not able to reduce MnP compound II efficiently (41). Thus the enzyme Is unable to complete Its catalytic cycle efficiently In the absence of Mnll. This would seem to explain the absolute Mnll requirement for catalytic activity. In the conversion of MnP compound I to compound II, the porphyrin tt-cation Is reduced back to a normal porphyrin. This suggests that the porphyrin radical Is exposed In a peripheral site as recently suggested for HRP (48) and that this site may be available to organic substrates and to Mnll. In contrast, the FeIV 0 center of MnP compound II may be partially burled and only available to Mnll Ions. 

Whilst of mechanistic interest these reactions of compound III are not used by most peroxidases (although myeloperoxidase and some plant peroxidases may be exceptions). The formation of compound III is usually inhibitory, as is the case for lignin peroxidase [17]. Indeed, in some enzymes that use two-electron oxidation by compound I the single-electron reduction to compound II is also deleterious and leads to an inactive enzyme (e.g. myeloperoxidase). The activity of such enzymes is enhanced in the presence of excess reductants such as ascorbate which convert compound II back to the ferric enzyme and allow another reaction cycle to take place [21],... 

Compound I crystals of cytochrome c peroxidase can be generated by the direct addition of H2O2 to crystals of the ferric complex [7]. These crystals are stable for four hours at — 15°C, which with the improvements in data collection afforded by area detectors [103] allows a difference crystal structure to be determined relative to that of the ferric enzyme. Unfortunately the resolution... 

The first information on the mechanism of catalysis in wild type pAPX was published in 1996 (Marquez et al., 1996). The kinetic data are consistent with a scheme in which the ferric enzyme is oxidized by two electrons to a so-called Compound I intermediate with eoncomitant release of one mole of water, followed by two successive single electron reductions of the intermediate by ascorbate (S) to regenerate ferric enzyme, Eqs. (3)n(5). 

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