# Coordinate control of hemeprotein maturation and function by cell chaperones, heme, and nitric oxide

> **NIH NIH R01** · CLEVELAND CLINIC LERNER COM-CWRU · 2020 · $465,616

## Abstract

ABSTRACT
Heme proteins are fundamental in biology yet we do not understand the mechanisms that regulate their
maturation and function in cells, or how they become dysfunctional in disease. Our lab is discovering new roles
for cell chaperones and nitric oxide (NO) in regulating these processes. Soluble guanylate cyclase (sGC),
myoglobin (Mb), and NO synthase (NOS) will serve as the model heme proteins in our proposal. sGC
becomes dysfunctional in inflammatory diseases with higher NO production. Our work reveals that cells
chaperones (hsp90) govern the maturation of all three hemeproteins, but the mechanisms driving their heme
insertion, and in the case of sGC, that drive its inactivation, are unclear. We hypothesize: (i) In health, hsp90
supports maturation of sGC, Mb, and NOS through making a direct interaction with each client protein that
enables their heme insertion, likely operating in concert with co-chaperone proteins. (ii) In disease, higher NO
inactivates sGC by causing oxidation of its heme and S-nitrosation of its protein Cys groups (SNO). The SNO
modifications in turn causes sGC heterodimer breakup, possible heme loss from sGC, and sGC re-association
with hsp90. (iii) Cell denitrosylase (thioredoxin 1, Trx1) and sGC heme reductase (cytochrome b5r) enzymes
may protect and/or repair sGC. To understand the molecular basis for these events, our Aims coordinate
biochemical, biophysical, & cell-based approaches.
AIM 1. How do chaperones drive heme insertion? Determine regions in apo-Mb and apo-NOS that enable
complex formation with hsp90, build structural model of the complexes, test if interaction as predicted by models
enable complex formation and heme insertion to occur in mammalian cells. Identify the co-chaperones and
proteins that may assist hsp90 during heme insertions into the sGC, Mb, and NOS clients. Create defined
protein systems to study their heme insertion. Characterize structure of hsp90-apo-hemeprotein client complexes
by HxD MS and EM.
AIM 2. How is sGC inactivated & how might its recovery take place? In cells and in a purified system,
determine the importance of: (i) sGCβ heme occupancy & heme oxidation state in catalyzing specific SNO in
modifications in sGC/, (ii) SNO modifications vs heme oxidation or loss in driving sGC heterodimer breakup
& the rebinding of sGCβ to hsp90; (iii) protective/recovery mechanisms, including SNO removal by Trx1, hsp90-
mediated heme reinsertion, sGCβ heme reduction by cytochrome b5r, & binding of a drug that occupies the
sGCβ heme site (BAY 60).
By defining the molecular & cellular mechanisms of chaperone-driven heme insertion during sGC, Mb, and NOS
maturation, and the mechanisms causing sGC inactivation and recovery, our study will make fundamental
contributions to our understanding of hemeprotein maturation and function, and will reveal new ways to optimize
hemeprotein functions in health and disease.

## Key facts

- **NIH application ID:** 9997979
- **Project number:** 5R01GM130624-02
- **Recipient organization:** CLEVELAND CLINIC LERNER COM-CWRU
- **Principal Investigator:** DENNIS J STUEHR
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $465,616
- **Award type:** 5
- **Project period:** 2019-09-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9997979

## Citation

> US National Institutes of Health, RePORTER application 9997979, Coordinate control of hemeprotein maturation and function by cell chaperones, heme, and nitric oxide (5R01GM130624-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9997979. Licensed CC0.

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