ABSTRACT We established that a multiprotein chaperone complex containing Hsp90/Hsp70 chaperones plays an essential role in maintaining protein quality control of neuronal NO synthase (nNOS) and other P450 cytochromes. In the last grant cycle, we turned our focus to determine the structure of nNOS by single particle EM and cryo-EM methods to help elucidate what triggers chaperone recognition of nNOS. In the course of these studies, we serendipitously discovered that full-length CYP2B4:cytochrome P450 reductase (CPR) complexes could form in amphipols, which are surfactants that self-assemble and stabilize membrane proteins in the absence of detergent. Remarkably, these complexes were fully functional, stable, and visible as tetramers by single particle EM methods, and found to contain equimolar amounts of P450 and CPR. Thus, in the current proposal, we aim to determine the first structure of a microsomal P450 in complex with CPR. Our prior success in elucidating the first full-length dimeric structure of nNOS and P450 BM3 by these single particle methods provides confidence in this undertaking. Currently we have resolved the structure of the oxygenase domain of full-length nNOS to 5 Å resolution by cryo-EM methods. It is noteworthy that a tetrameric complex of P450:CPR complex is analogous to the dimeric architecture of nNOS and BM3, both having a P450 and CPR domain in each monomer. We have a collaborative team of EM experts whose knowledge will synergize with our expertise in P450 and multi-protein complexes to tackle this exciting but challenging project. Also in prior grant cycles, we showed that Hsp90 is needed for cellular heme insertion into heme-deficient apo-nNOS and more recently the Stuehr lab has shown that Hsp90 is needed to insert heme into guanylate cyclase, hemoglobin, and inducible NO synthase. Thus, premise exists for a chaperone complex that is a ‘heme insertase’. Recently, we discovered that the cellular proteins that immunoprecipitate with apo-nNOS are stably bound and catalyze heme insertion into apo-nNOS. Hsp90 and Hsp70 are bound to apo-nNOS and play a key role in heme insertion but Hsp90 and Hsp70 chaperones alone are not sufficient. In the current proposal, we seek to identify the other proteins that make up the heme insertase activity, in part by identification of the co-immunoprecipitated proteins by LC-MS/MS. Moreover, we will define how the chaperone-based heme insertase complex is assembled. The successful completion of the aims will provide a groundbreaking new platform for the study of microsomal P450s, elucidate the first structure of a P450:CPR complex, and characterize the protein machinery that inserts heme into hemeproteins.