Other Radical Enzymes

The mechanism proposed for the pyruvate ferredoxin oxidoreductase is drastically different from that of the pyruvate dehydrogenase enzyme complexes, as radical intermediates are proposed, suggesting stepwise one-electron transfers. Furthermore, the preliminary work by Uyeda and Rabinowitz (211) on the mechanism of C. acidiurici pyruvate ferredoxin oxidoreductase indicates that there may be subtle differences in the mechanism of the oxidoreductase from different organisms. The key points and contrasts in the mechanism of these enzymes may be summarized as follows. Addition of pyruvate to a stoichiometric amount of enzyme leads to the formation of an equimolar amount of CO2. In H. halobium (213) a enzyme-mediated one-electron transfer to an exogenous electron acceptor occurs at this stage, with formation of a stable enzyme-bound radical intermediate, whereas the enzyme from C. acidiurici remains in the oxidized state. Addition of CoA leads to the subsequent formation of acetyl-CoA from both enzymes, which reduction (two electrons ) of an unknown chromophore in the 

acidiurici enzyme, and transfer of a second electron in the H. halobium reaction. The mechanistic cycle is then completed with the reoxidation of both enzymes by suitable electron acceptors. 

Recently, Wahl and Orme-Johnson reported their studies on the characterization and mechanism of the pymvate flavodoxin (ferredoxin) oxidoreductase from Klebsiella pneumoniae and Clostridium thermoacetium (214). These oxi-doreductases appear to be closely related to that from C. acidiurici (215) in that the iron is present in two Fe4S4 clusters, which act as election acceptors in the catalytic mechanism. However the K. pneumoniae and C. thermoaceticum enzymes may be mechanistically distinct from the H. halobium oxidoreductase (213) in that free radical intermediates are not detected for the former enzymic reaction. EPR signals in the Klebsiella or C. thermoaceticum oxidoreductases are only observed in the fully reduced enzyme when the reductants dithionite or pyruvate and CoASH are present (214). The suggested mechanism for the pyruvate oxidoreductase from K. pneumoniae and C. thermoaceticum is initially similar to the mechanism for all TPP enzymes in that decarboxylation of pyra-vate leads to the formation of hydroxyethyl-TPP. Two one-electron transfers to each of the two Fe-S clusters occur on the binding of CoASH. However, the mechanism for the formation of acetyl-CoA from the hydroxyethyl-TPP intermediate and of the CoASH-induced electron transfers is not yet clear. 

Although the chemistry for this mechanism has not been worked out in detail, the idea of a carbon-centered radical at the C-3 position of pyruvate is consistent with EPR studies on the pyruvate ferredoxin oxidoreductase from H. halobium discussed above, where the hyperfine splitting was dependent on the substrate structure and the number of hydrogen nuclei near C-3 (209). 

The lysine 2,3-aminomutase reaction is reminiscent of the 1,2 rearrangement reactions catalyzed by B -dependent enzymes. The B12 dependence of several enzymic lysine 2,3-aminomutase reactions has been characterized in a variety of animals and microorganisms (222). However, the enzyme from C. subterminale SB4 is apparently not B12 requiring (220), suggesting that the enzymes catalyzing a- to j8-amino transfers are not all evolutionarily related. Given the unique cofactor requirements of the C. subterminale SB4 enzyme, i.e., SAM and Fe(ll), several studies aimed at elucidating the reaction mechanism have recently been reported. 

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EPR and other spectroscopic work on the tyrosine radicals and their function in PS II has recently been reviewed 345-385-387 in a special issue of Biochim. Biophys. Acta. The authors give their specific views on the research efforts which led to our present knowledge of these highly interesting radicals that are also found in many other radical enzymes .385,388 389 In the following some of the important results described in recent EPR papers are summarized. For further details and references to the earlier literature the reader is referred to the cited review articles. 

In addition, several other radical enzymes have been investigated theoretically by us and others, such as DNA photolyase, Cu amine oxidase and prostaglandine H synthase, but we have found it beyond the scope of the present chapter to include aU of these. 

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]. 

The splitting of H2 by hydrogenases is heterolytic (into H and H" ), rather than homolytic (into two H. radicals). The hydride is considered to deliver two electrons at a time to the enzyme. A [4Fe-4S] cluster in proteins can, however, accept only one electron. Other redox enzymes, e.g. flavoproteins, dealing with two-electron donors (like NADH) solve this problem by first accommodating both electrons onto the flavin, whereafter these electrons are transferred to an Fe-S cluster one at a time. 

Xanthine oxidase and other radical-producing enzymes activity ... 

Any chemical process in which one reactant removes an atom (neutral or charged) from the other reacting entity. An example is the generation of a free radical by the action of an initiator on another molecule. If abstraction takes place at a chiral carbon, racemization is almost always observed in nonenzymic processes. On the other hand, enzymes frequently abstract and reattach atoms or groups of atoms in a fashion that maintains stereochemistry. 

This survey has been concerned with the enumeration of all possible mechanisms for a complex chemical reaction system based on the assumption of given elementary reaction steps and species. The procedure presented for such identification has been directly applied to a number of examples in the field of heterogeneous catalysis. Application to other areas is clearly indicated. These would include complex homogeneous reaction systems, many of which are characterized by the presence of intermediates acting as catalysts or free radicals. Enzyme catalysis should also be amenable to this approach. 

They were found not to react with BESOD, the rate constant was estimated to be < 10 M s , if there was a reaction at all The reaction of BESOD was also investigated with several other radicals generated by pulse radiolysis. With the semiquinone of riboflavin 5 -phosphate no reaction was detected. The semiquinone of 9,10-anthraquinone-2-sulfonate and the radical anion of 4-nitroacetophenone converted the enzyme into an unreactive form... 

Tyrosinase is both an oxidase and a hydroxylase. Some other copper enzymes have only a hydroxylase function. One of the best understood of these is the peptidylglycine a-hydroxylating monoxygenase, which catalyzes the first step of the reaction of Eq. 10-11. The enzyme is a colorless two-copper protein but the copper atoms are 1.1 nm apart and do not form a binuclear center.570 Ascorbate is an essential cosubstrate, with two molecules being oxidized to the semidehydro-ascorbate radical as both coppers are reduced to Cu(I). A ternary complex of reduced enzyme, peptide, and 02 is formed and reacts to give the hydroxylated product.570 A related two-copper enzyme is dopamine (J-monooxygenase, which utilizes 02 and ascorbate to hydroxylate dopamine to noradrenaline (Chapter 25).571/572 These and other types of hydroxylases are compared in Chapter 18. 

Cells have substantial chemical defenses against the UV photoproducts produced in seawater and intracellular fluids. Many organisms have antioxidants (e.g., carotenoids, ascorbate, tocopherols, anthocyanins, and tridentatols) that quench photo-oxidative reactions.64-67 Cells also have enzymes (e.g., catalase and superoxide dismutase) that can counteract the oxidative nature of peroxides and other radicals.26 Some compounds, such as the UV-absorbing pigment melanin, can act as both optical filter and antioxidant.68 The MAA mycosporine-glycine (Figure 15.3) functions in a similar dual capacity.69 The role of UV-mediated reactions in seawater relative to biological effects is an important current area of study. 

The number of enzymes discovered to harbour and employ a metastable radical site for the catalytic activity has steadily increased over the past decades [1]. Besides pure radical enzymes, i.e., systems that use a stable radical for the catalytic action at the active site, theoretical studies indicate that radical intermediates are also employed in several other systems - see e.g. the chapter by Siegbahn and Blomberg. In addition, many key reactions in biology make use of radical forms of cofactors such as quinones in photosynthesis (see chapter by Wheeler), the vitamin E controlled quenching of lipid peroxidation or the various catalytic mechanisms involving radical forms of coenzyme B12 (see chapter by Radom et al). The form in which the radical nature is stored and employed hence differs significantly from system to system, and the aim of the present chapter is to give a flavour of some of these aspects with key focus on radical enzymes. 

In addition to the peroxidase and laccases discussed above, a range of other, radical-forming enzymes have been reported in the context of vinyl polymerizations (Table 6.1). 

Class II RTPR has in common with other coenzyme Bi2-dependent enzymes the use of the 5 -deoxyade-nosyl radical from adenosylcobalamin as a radical initiator. The elimination of water from the 3 -radical intermediate might in part be analogous to the elimination of water in the reaction of DDH. The variant function of RTPR can be viewed as foreshadowing the wider functions of the 5 -deoxyadenosyl radical in other radical reactions, in particular the radical SAM enzymes. ... 

Another indication of complexity is provided when intermediates can be detected by chemical or other means during the course of reaction. When this can be done a kinetic scheme must be developed which will account for the existence of these intermediates. Sometimes these intermediates are relatively stable substances, while in other cases they are labile substances such as atoms and free radicals. Enzyme-substrate complexes are of the latter class, in that they usually cannot be isolated and preserved and can be detected only by special methods such as spectroscopic ones. Free radicals can sometimes be observed by spectroscopic methods, and evidence for their existence may be obtained by causing them to undergo certain specific reactions which less active substances cannot bring about. 

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