Probing Metalloenzymes with Sensitizer-Tethered Substrates

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This award in the Inorganic, Bioinorganic, and Organometallic Chemistry Program supports metalloenzyme research by Drs. Harry B. Gray and John H. Richards of the Beckman Institute, California Institute of Technology. The goal of the work is to develop a new method for probing the properties and reactivities of redox metalloenzymes. The prototypical heme oxygenase, cytochrome P450, will be the main system studied. This enzyme stereospecifically hydroxlates hydrocarbons using dioxygen as the oxidant. In many metalloenzymes, rate-limiting electron transfer (ET) steps preclude observation of other critical activation steps. To attack this problem, natural redox partners will be replaced with new reagents that are able to exchange electrons at much faster rates. Laser-initiated electron transfer reactions will be used to drive enzymatic reactions. In order to deliver the laser-generated redox equivalents, a direct covalent or hydrogen-bonded pathway has been shown to be essential. Here a direct link between a redox sensitizer and the enzyme active site will be formed by taking advantage of the affinity of the enzyme for its active site. Ruthenium-diimine sensitizers will be covalently tethered to cytochrome P450 substrates to provide a conduit for delivering oxiding and reducing equivalents to the heme cofactor. The initial goal is to prepare and characterize the oxo-iron (ferryl) intermediates that have been suggested as the active hydroxylating agents. Enzyme for this work will be expressed in E. coli, and site-directed mutagenesis will be employed to study the importance of specific residues in ferryl-formation reactions. The reactive intermediates will be studied using a variety of photochemical methods. Metalloenzymes catalyze many reactions involving small-molecule activation. For example, enzymes promote reduction of oxygen gas to water, reduction of nitrogen gas to ammonia, and use oxygen gas to add oxygen atoms to carbon chains. These reactions are important not only biologically but also commercially. This research aims at understanding the enzyme that promotes addition of oxygen to hydrocarbons by oxygen gas. The reactive enzyme intermediate has never been observed, so a stategy for replacing the reagents from the natural system with ones that can react more readily will be used to enable photochemical observation of reactions at the active site.