Project Summary The function of cytochromes P450 can be classified into two major divisions, those that operate in xenobiotic degradation and those that play critical roles in steroid hormone synthesis. Due to the critical role of cytochrome P450 in human health, these enzyme systems have occupied NIH supported investigators for many decades. The general long term goals of the research program are to gain a molecular level insight into the impressive range of functional diversity displayed by these enzymes. The essential focus of the work proposed deals with two important human enzymes involved in steroid biosynthesis, CYP17 and CYP51, both current drug targets for various diseases, including atherosclerosis and prostate cancer, progress in this endeavor ultimately depends on gaining a deeper understanding of their structure and precise reaction pathways. In order to fully understand the pertinent relationship of structure to function, it is important to acquire structural information for not only the stable terminal states in a reaction, but also for the reaction intermediates, investigation of the latter having been historically impeded, owing to their fleeting existence. Also, the fact that these are membrane‐bound proteins has complicated their investigation owing to problems associated with aggregation. In order to address these obstacles, this research program employs an approach using innovative nanodisc technology which not only provides an environment mimicking the natural cell membrane for the isolated CYP enzyme, but also enables the synthesis of unique functional dyads of the CYP with its natural or alternative reductases. Also of immense importance, a powerful combination of resonance Raman spectroscopy with the cryoradiolysis technique permits trapping and detailed structural characterization of otherwise elusive, but crucial reaction intermediates, thereby providing a heretofore unattainable level of insight into the structure and function of these enzymes.