# P450 and NO Synthase Regulation by Multiprotein Complexes

> **NIH NIH R01** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2022 · $391,812

## Abstract

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.

## Key facts

- **NIH application ID:** 10335136
- **Project number:** 5R01GM077430-16
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** YOICHI OSAWA
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $391,812
- **Award type:** 5
- **Project period:** 2006-05-01 → 2023-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10335136

## Citation

> US National Institutes of Health, RePORTER application 10335136, P450 and NO Synthase Regulation by Multiprotein Complexes (5R01GM077430-16). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10335136. Licensed CC0.

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