X-ray spectroscopy of Highly Reactive Catalytic Intermediates in Oxygen Activating Metallo-Enzymes

University Of Calif-Lawrenc Berkeley Lab, Berkeley CA

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Abstract

? DESCRIPTION (provided by applicant): Dioxygen activating enzymes are an import class of redox-active proteins that have critical roles in cellular function, aiding in key steps of the synthesis of biomolecules, signaling agents, and handling oxidative stress. Of these enzymes, Iron-containing Heme proteins have long been recognized as vital, notably in cellular respiration enzymes like cytochrome c oxidase. Nitric Oxide Synthase (NOS) is an Iron Heme enzyme, which catalyzes the synthesis of Nitric Oxide (NO), a small signaling molecule whose role in inflammation and vasodilation make it a popular drug target. In order to realize the benefit of our knowledge of NOS's function in the human body and to harness it as a tool for controlling disease it is necessary to have a deep understanding of the catalytic mechanism that underlies its function. Much about this enzyme is known. NOS activates O2 twice in order to release NO from the amino acid L-Arginine, and many of the intermediates to this reaction have been characterized by various methods. However, some key steps of the reaction remain hypothetical, either lacking direct experimental evidence or suffering from ambiguous interpretations. The sensitivity of the redox-active Fe to radiation damage by photoreduction has made difficult the collection of X-ray spectroscopic and crystallographic studies - key methods that could interrogate these unknown catalytic intermediates. Recently available ultra-short femtosecond pulse X-ray free electron laser (XFEL) user facilities are lifting the obstacles researchers have faced when trying to collect X-ray spectra and diffraction patterns at conventional long-pulse synchrotron sources. The femtosecond XFEL pulses avoid radiation damage, as the radiation damage happens on the picosecond time scale, resulting in a measurement paradigm known as signal before destruction. As a consequence, it is now possible to collect meaningful data from metalloenzymes with high photon flux at physiological conditions, rather than cryogenic temperatures, allowing for time resolved X-ray studies of weak, information rich spectral signals and even kinetic crystallography. The key objective of this proposal is to take advantage of this new paradigm and extend it to NOS so that direct observations of the catalytic metal's electronic state and surrounding environment can be made. To accomplish this, I propose the development of methods to introduce rapidly mixed solutions of NOS with L-arginine into the X-ray beam. Further, data analytic methods are proposed which take advantage of the unique XFEL pulse structure, permitting rapid collection of X-ray absorption and chemically specific resonant emission, an incisive probe of both the metal's electronic state and ligand environment.