Radical Enzymes

Morasch B, HH Richnow, A Vieth, B Schink, RU Meckenstock (2004) Stable isotope fractionation caused by glycyl radical enzymes during bacterial degradation of aromatic compounds. Appl Environ Microbiol 70 2935-2940. 

Jeschke, G. 2005. EPR techniques for studying radical enzymes. Biochimica et Biophysica Acta 1707 91-102. 

The function of the metal site in the oxygen-dependent radical enzymes galactose oxidase, amine oxidases, ribonucleotide reductase, and cytochrome c oxidase is inter alia to bind 02 in their reduced forms and undergo the appropriate redox chemistry to generate a metal-bound, activated oxygen species of variable nature. 

Tyrosine-based radical enzymes are among the best characterized. We will briefly describe some of these emphasizing the nature of the one-electron oxidized tyrosine residue in the catalytic cycle. 

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. 

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. 

Gelius-Dietrich G, Henze (2004) Pyruvate formate lyase (PFL) and PFL activating enzyme in the chytrid fungus Neocallimastix frontalis A free-radical enzyme system conserved across divergent eukaryotic lineages. J Eukaryot Microbiol 51 456-463... 

B33. Burkhardt, H., Hartmann, F., and Schwingel, M. L., Activation of latent collagenase from polymorphonuclear leukocytes by oxygen radicals. Enzyme 36, 221—231 (1986). 

Eklund, H., Eriksson, M., Uhlin, U., Nordlimd, P., and Logan, D., 1997, Ribonucleotide reduc-taseoStructural studies of a radical enzyme. Biol. Chem. 378 821n825. 

Eklund, H., and Fontecave, M., 1999, Glycyl radical enzymes a conservative structural basis for radicals. Structure 7 R257nR262. 

Stubbe, J. A., and van der Donk, W. A., 1995, Ribonucleotide reductases Radical enzymes with suicidal tendencies. Chem. Biol. 2 793n801. 

Table 1 Classification of Coenzyme B12-dependent Radical Enzymes ...

Bnckel W, Golding BT. Radical enzymes in anaerobes. Annn. Rev. Microbiol. 2006 60 27 9. 

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. 

Radical enzymes are, like radical reactions in general, usually characterised by high turnover for the catalytic processes. It is hence rather difficult to study these reactions experimentally in order to gain direct insight into the mechanisms. In addition, several of the enzymes are membrane bound or anaerobic, why the determination of the crystal structures of many of these has been a formidable task. Most of the mechanistic proposals have thus been based on mutagenesis experiments (site specific exchange of an amino acid), kinetic measurements and isotope effects, and studies of inhibitor by-products . 

We have in the present chapter shown results from theoretical model system studies of the catalytic reaction mechanisms of three radical enzymes Galatose oxidase. Pyruvate formate-lyase and Ribonucleotide reductase. It is concluded that small models of the key parts of the active sites in combination with the DPT hybrid functional B3LYP and large basis sets provides a good description of the catalytic machineries, with low barriers for the rate determining steps and moderate overall exothermicity. The models employed are furthermore able to reproduce all the observed features in terms of spin distributions and reactive intermediates. 

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. 

Lklund, 11., Uhlin, U., I arnegardh, M., Logan, D. T. and Nordlund, P. jtlOL Structure and function ol the radical enzyme ribonucleotide reductase. Prog. Biophys, Mo/, Biol. 77 177—208. 

Reichard, P., and Ehrenbetg, A., Ribonucleotide Reductase—A Radical Enzyme, Science, 221 514—519, 1983. 

Treatment of the blue form of the lyase with dithionite or irradiation at wavelengths greater than 520 nm in the presence of DTT produces fully reduced flavin cofactor (FADH2) and results in a dramatic increase in activity and quantum yield (146, 153, 154). This suggests that the catalytically active oxidation state of the cofactor may be the reduced flavin, and that it is this form of the cofactor that functions as an electron donor in catalysis (155). Subsequent studies by Sancar and co-workers indicate that E. coli DNA-PL does not contain the flavin semiquinone radical in vivo, and that the blue, radical enzyme does not catalyze dimer cleavage (156). 

CoA thioesters are also the products of the oxidative decarboxylation reactions of a-keto acids, especially pyruvate and a-ketoglutarate, from which acetyl-CoA and succinyl-CoA are formed, respectively (Equation (14)). Three distinct types of enzymes catalyze such reactions however, the mechanistic involvement of CoA is generally rather limited for two of these, and only a brief discussion of each will be provided here. For more detailed information on these enzymes, the reader is referred to the relevant chapters on thiamin and lipoic acid enzymology and on radical enzymes in this series (see Chapters 1.08 and 7.03). 

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