9405281 Poulos The overall goal of this project is to further our understanding on the mechanism of heme containing enzymes using protein crystallography as our main tool. These enzymes carry out two processes fundamental to all living systems. First, the heme group activates either O2 or H2O2 and stores the oxidizing equivalents of the oxygen or peroxide in the active site. Second, the oxidizing equivalents now stored at the heme active center are used to oxidize other molecules. These other molecules can be another redox protein or an organic molecule. Our goal is to compare several crystal structures of different heme enzymes in order to understand how these enzymes catalyze cleavage of the peroxide or O2 O-O bond. Because the heme group and its activated oxointermediates exhibit distinct spectral properties, it is possible to correlate the crystal structure information with the spectral data to obtain a deeper insight into the precise mechanism of oxygen activation and the structure of the resulting activated intermediate. The second part of the reaction involves oxidation of substrate by the enzyme. If the substrate is another protein, the problem becomes one of protein-protein recognition and long-range electron transfer through proteins. Small molecule oxidation also involves electron transfer from the substrate to the enzyme but in this case, we expect the enzyme to possess a more conventional small molecule binding pocket which specifically recognizes the substrate. Comparative crystal structure information is essential to understand the diversity of reactions and substrates used by various peroxidases and oxygenases. %%% Peroxidases and oxidases also are attracting increasing attention by the biotechnology community. These enzymes are, in some cases, able to oxidize toxic pollutants such as chlorinated biphenyls. One of the more important advances in this area was our recent structure determination of lignin peroxidases supported by NSF. Lignin perox idase is secreted by certain fungi that are able to degrade lignin, a complex aromatic polymer that provides the outer coating around cellulose in woody plants. Lignin is the second most abundant polymer on earth and presents a major problem in the paper pulp industry since lignin degradation requires rather harsh chemical treatments. There is considerable interest in developing less harsh biological processes for lignin degradation. Moreover, because of the unusual reactivity of lignin peroxidase, there is optimism that this peroxidase can be utilized for the degradation of toxic pollutants. Our structural work has provided the basis for understanding how such reactions occur. Of particular importance are recent developments in our ability to clone and overexpress heme enzymes. This opens the way for using protein engineering to alter the active sites of peroxidases and other heme enzymes in order to optimize their use for degrading molecules of interest such as toxic pollutants. The crystal structure data is essential for such an undertaking. It also should be emphasized that over the years, NSF has been the primary means of support for heme enzyme crystallography. The first two heme enzyme crystal structures solved were cytochrome c peroxidase and cytochrome P450, both of which were solved in our lab with support provided by NSF. These two structures alone have provided the basis for research in dozens of other labs. and countless spin-off projects. NSF also supported solution of the lignin peroxidase structure. Hence, it is not an understatement to claim that NSF is directly responsible for a majority of what we know today about heme enzyme structure and function and it is very likely that there will be significant and important practical applications of this work in the near future. ***
