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Electron Transfer in Iron and Copper Oxygenases and Oxidases

Award Number: R01GM159231
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 05/01/1979
PERIOD OF PERFORMANCE END DATE: 07/31/2029

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Electron Transfer in Iron and Copper Oxygenases and Oxidases - Oxygenase and oxidase enzymes must coordinate the delivery of four protons and four electrons to O2 to prevent the formation of harmful reactive oxygen species. The risks posed by reactive intermediates are so great that aerobic organisms need mechanisms to protect oxygen-utilizing enzymes from inactivation when primary electron/proton transfer mechanisms are disrupted. Radical transfer pathways that deliver oxidizing equivalents (holes) away from frustrated reactive intermediates in enzyme active sites to the protein surface for reaction with intracellular antioxidants can provide such protection. These radical transfer pathways are constructed from chains of tryptophan (W), tyrosine (Y), cysteine (C), and possibly methionine (M) residues. This research will focus on cytochromes P450 (P450) and Streptomyces coelicolor small laccase (SLAC). The cytochromes P450 are members of a superfamily of heme oxygenases involved in xenobiotic metabolic and biosynthetic pathways. In mammals these functions include drug metabolism, conversion of lipophilic molecules to more polar products for enhanced elimination, steroid biosynthesis, and eicosanoid synthesis and degradation. Cytochromes P450 also are responsible for 66% of enzymatic activation of carcinogens. Elucidating the mechanisms by which P450s avoid inactivation in the presence of diverse substrates can contribute to defining therapeutic drug efficacies and mitigating the risks of adverse drug-drug interactions. Chains of W and Y residues in three bacterial P450s have been characterized for their potential as protective electron transport conduits. The protective roles of individual amino acids in W/Y chains of the three cytochromes P450 will be evaluated in measurements of the total number of turnovers before enzyme deactivation. The mechanism of protection will be elucidated using colorimetric redox indicators bound near the surface termini of W/Y chains. These indicators will report on the arrival of holes at the end of a W/Y chain in single-turnover stopped-flow kinetics measurements. Evidence is emerging from in vitro studies of P. megaterium P450BM3 that hole transport along W/Y chains can extend the functional life of the enzyme. Research in this program aims to establish whether the extended P450BM3 survival resulting from these chains provides any measurable advantage to the bacterium. Preliminary studies of a P450BM3 deletion mutant of P. megaterium have identified a phenotype attributable to the enzyme. Investigations of wild-type and mutant P. megaterium strains aim to determine whether disruption of W/Y chains negatively impacts growth of the organism. The multicopper oxidase SLAC and human ceruloplasmin are structurally homologous enzymes in which a Tyr radical forms during catalysis. SLAC will be used as a surrogate for ceruloplasmin, owing to its efficient heterologous expression in E. coli. Measurements of the total number of turnovers in wild-type and mutant enzymes aim to elucidate the roles of W and Y radicals in SLAC catalysis.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = $438,263 )
2025	2025	CALIFORNIA INSTITUTE OF TECHNOLOGY	1200 E CALIFORNIA BLVD	PASADENA	CA	91125	LOS ANGELES	USA	Biomedical Research and Research Training	000	45	6/23/2025	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$438,263
														Subtotal = $438,263

Grand Total All Awards = $438,263

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Electron Transfer in Iron and Copper Oxygenases and Oxidases

Award Number: R01GM159231

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 05/01/1979

PERIOD OF PERFORMANCE END DATE: 07/31/2029

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