Carbon monoxide (CO) poisoning remains a major cause of death and disability, affecting 50,000 persons each year in the U.S. alone. Patients removed from fires or following exposure to car and home generator exhaust are placed on 100% oxygen and transferred to a facility with a hyperbaric oxygen delivery system. Despite the availability of hyberbaric therapy centers, inherent delays in access to and initiation of therapy greatly limit efficacy. Even with hyberbaric oxygen therapy, 1-2% of patients die and >25% of surviving patients exhibit neurocognitive impairments. There is currently no point-of-care antidote for CO poisoning available clinically. In our initial work we discovered a surprising and near-irreversible CO-binding affinity of mutationally engineered human neuroglobin (Ngb). Ngb is a six-coordinate hemoprotein, with the heme iron coordinated by two histidine residues. We mutated the distal histidine to glutamine (H64Q) and three surface-thiols to form a five-coordinate heme protein (Ngb-H64Q-CCC) that has very high solubility (>10mM), allowing for high concentration and intravenous infusion. This molecule binds CO ≈ 500 times more strongly than Hb. Infusions of Ngb-H64Q-CCC in CO-poisoned mice enhanced CO removal from red blood cells in vivo from 25 minutes to 25 seconds, restored heart rate and blood pressure, increased survival from less than 10% to over 85%, and were followed by rapid renal elimination of CO-bound Ngb-H64Q-CCC. These findings provided proof of concept that heme-based scavenger molecules with very high CO binding affinity can be developed as potential antidotes for CO poisoning. In the previous funding period, we continued the development of our Ngb-H64Q-CCC molecule, evaluating efficacy on the restoration of cellular aerobic respiration, safety, and acute and long-term effects on cardiovascular and cognitive function and survival in pre-clinical models, and scaling production of recombinant protein for clinical development. We showed how infusion of Ngb-H64Q-CCC can restore mitochondrial respiration in tissues and reverse CO-induced effects. We also set out to discover novel CO scavenger molecules which may have improved properties over our lead molecule. Our studies uncovered that RcoM, a bacterial CO sensor, has a high affinity towards CO and presents promising safety profiles in mouse models. In the present proposal we plan to further develop our Ngb-H64Q-CCC molecule, adding modifications that improve its CO affinity and stability for a safer toxicity profile. We also will engineer RcoM to obtain the smallest unit that can scavenge CO with high affinity and present optimal stability and safety properties. Finally, we will leverage all the knowledge on CO and oxygen binding achieved during our research program to develop novel oxygen carrier molecules that can serve as blood substitutes. Overall, these proposed studies are in keeping with the mission of the NHLBI and NIH to advance highly impactful, significant...