The long range goal of this project is to study structure-function relationships in cytochromes P450 and related proteins using x-ray crystallography, molecular biology, and biochemistry. This competitive renewal focuses on the following areas: substrate binding, electron transfer, enzyme intermediates, and selected new structures. A comparison of the P450BM3 heme domain with and without substrate bound reveals large conformational differences, whose relevance will be probed by a combination of site directed mutagenesis and new crystal structures. During this past funding period, Dr. Poulos solved the first P450 electron transfer complex that formed between P450BM-3 and its FMN domain. The functional relevance of the model will be tested by a combination of mutagenesis, chemical modification, and laser flash photolysis. A new addition to the project is heme oxygenase, the enzyme responsible for the first step in heme degradation. Various intermediates can be trapped in the crystalline state and the structures of these intermediates will be determined. Lastly, Dr. Poulos will solve the structure of P450s that exhibit interesting and unusual properties. These studies will help to broaden our understanding on the diversity of P450 functional properties.
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