PHYSICAL-CHEMICAL ANALYSIS OF PEROXIDASES

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Ligninase, the lignin degrading enzyme which catalyzes the oxidative cleavage of the propyl backbone of lignin, also catalyzes the H2O2 dependent oxidation of a wide range of substrates, including the often most difficult and rate limiting initial oxidative step in the degradation of many environmentally persistent xenobiotics. It is not only able to initiate degradation of many substrates but to catalyze all these steps to CO2. These enzymes offer great potential in the management of environmental waste. Although these lignin degrading enzymes have many characteristics and properties similar to other peroxidases, other peroxidases do not exhibit ligninase activity and these enzymes are unique in their ability to oxidize substrates of extremely high reduction potential. This activity has a low pH optimum and is controlled by an ionization with a pK in the range of carboxyl ionization. The initial step of H2O2 activation is not controlled by a single ionizable group having a pK in the slightly acidic region, unlike all other peroxidase, suggesting a different mechanism which may account for the difference in reactivity. The proposed studies attempt to clarify on a molecular level, the structure -- function relationship in the vicinity of the active site of the ligninase enzymes and to compare and contrast these with other peroxidases to provide the basic design principles for synthetic analogs and genetic engineering of this ubiquitous function. The local structure of the active site in the intermediate states formed in the H2O2 dependent primary reactions of ligninase and other peroxidases will be investigated using x-ray absorption spectroscopy while the distal pocket ligand environment will be probed with Fourier transformed infrared spectroscopy. Comparison of the isoenzymes and structure- - function changes produced by site directed mutagenesis offer a unique approach to identify the structural basis for reactivity and substrate specificity and establish a structure based mechanism for these potentially useful waste degrading enzymes.