GM65440 Renewal Resubmission Project Summary The element iron plays an enormous role in biology, including electron transport reactions (ferredoxins), oxygen transport and storage (hemoglobin and myoglobin), catalysis (nitrogenase, P-450, hydrogenase, and thousands of other enzymes), small molecule sensing (especially O2, CO, and NO), and DNA processing and repair. The health-relatedness of a better understanding of the structure and function of biological Fe is clear. The general theme of research in the Cramer lab involves the use spectroscopy as a tool for characterization of metals in biological systems. Much of this work involves the application of synchrotron radiation x-ray sources. Thanks to enormous improvements of these sources, experiments with exquisite sensitivity can now be conducted on dilute Fe samples. These include the technique Nuclear Resonance Vibrational Spectroscopy (NRVS), which is sensitive to the motion of 57Fe in a sample. Another new technique, nuclear-resonant time-domain interferometry (NR-TDI) can probe motion of all atoms in a sample. The x-ray experiments at international facilities are complemented by IR and Raman measurements at the home lab. The goals for the next 4 years can be divided into 3 main themes: · better understanding of the catalytic intermediates of the enzymes that process hydrogen – [NiFe] and [FeFe] hydrogenases, · information about structural changes that occur when Fe-S cluster proteins sense their environment, including mitoNEET and NAF-1, two health-related proteins which sense and communicate pH and redox status, and · characterization of protein dynamics in extremophiles (organisms that live at extremes of temperature, pressure, pH and other stressors) using the protein rubredoxin as a model system and using NRVS and NR-TDI to respectively measure the motion of Fe or the entire protein. The overall vision of this research program is to use spectroscopic methods to better understand how iron is used in important proteins, in ways that complement the information that can be gathered from diffraction and microscopy. In the course of research on specific topics, spectroscopic techniques such as NRVS will be further developed for the bioinorganic community. The novel NR-TDI technique will be assessed as a complementary probe of dynamical information about proteins.