This proposal seeks to advance ongoing studies as a collaborative consortium between the Gray and Goodin laboratories to develop and use synthetic molecular wires to study the structure and mechanism of several heme enzymes including cytochrome P450, nitric oxide synthase, and cytochrome c peroxidase. The overall goal is to use these novel structures to introduce redox-active substrate analogs into the active site of an enzyme where it is used to initiate rapid photo-induced electron transfer reactions or to directly couple the active site to electrode surfaces. In the last period, our aims were focused on targeting families of molecular wires to a number of distinct heme enzyme active sites, and several strategies for the design and construction of these molecules were developed. Crystallographic studies were undertaken to determine how the proteins respond to small variations in the chemical composition of the wires. In the current period, we will use these molecular wires in photochemical and electrochemical experiments to initiate electron transfer into or out of an enzyme under conditions that will allow us to answer fundamental questions about chemical mechanism. The studies are organized around three specific aims: 1) Wires for the active sites of P450cam and P450BM3 will be used in photochemical and electrochemical studies that will provide insight into high-valent heme intermediates that have been proposed in models for the enzyme mechanism. 2) Molecular wires for the active site of nitric oxide synthase will be used to induce electron transfer to the heme. These complexes may enable the trapping of proposed reactive intermediates in the catalytic cycle and provide tests of models for O-O bond cleavage. 3) Molecular wires that replace the native electron transfer pathway in cytochrome c peroxidase will be used to examine the efficiency and coupling of electron transfer in these systems. Overall, this proposal will contribute to the development of a new approach to control and understand the behavior of redox active metalloenzymes.
The structure, function and mechanism of heme enzymes play a central role in biology and human health. For example, cytochrome P450s in the liver are responsible for metabolizing most of the drugs in clinical use. Nitric oxide synthase catalyzes the production of the universally important messenger, nitric oxide, which plays a critical role in memory, blood pressure, and the immune response. This proposal is focused on the development of molecular tools that will allow a new approach to understanding how these enzymes function at a fundamental level.
| Whited, Charlotte A; Belliston-Bittner, Wendy; Dunn, Alexander R et al. (2009) Nanosecond photoreduction of inducible nitric oxide synthase by a Ru-diimine electron tunneling wire bound distant from the active site. J Inorg Biochem 103:906-11 |
| Hays Putnam, Anna-Maria A; Lee, Young-Tae; Goodin, David B (2009) Replacement of an electron transfer pathway in cytochrome c peroxidase with a surrogate peptide. Biochemistry 48:1-3 |
