Denitrifying NO reductases (denNORs) are integral membrane proteins that catalyze the two-electron reduction of NO to N2O with high turnover numbers. These enzymes protect symbiotic and pathogenic microorganisms against micromolar concentrations of NO and lead to resistance to the mammalian immune response. DenNORs are analogous to the heme/copper terminal oxidases of aerobic respiration but their active site is composed of a high-spin heme/nonheme (heme/FeB) binuclear center. Despite years of spectroscopic and kinetic analyses, the outlines of their catalytic cycle remain largely undefined. Results from stopped-flow absorption, FTIR and rapid-freeze-quench resonance Raman studies on engineered myoglobin system (FeBMbs) designed by Yi Lu’s group at the University of Illinois will be presented. These engineered proteins mimic the diiron center of denNORs and have allowed the characterization of transient iron-nitrosyl species that lead to either dead-end complexes or successful production of N2O. Specifically, two successive steps lead to the formation of a [6-coordinate low-spin heme FeII(NO)/high-spin FeBIINO] ternary complex which decays either productively to form N2O, or unproductively by reacting with a third NO to form a [5-coordinate low-spin heme FeII(NO)/high-spin FeBII(NO)] dead-end complex. These published data strongly support a “trans mechanism” where each iron binds one NO before formation of an N-N bond between two nitroxyl-like groups. However, recent experiments with other FeBMb constructs suggest that alternative mechanisms could also lead to N2O production. These unpublished results will be discussed along with recent findings relevant to single-turnover reaction and substrate inhibition in Paracoccus denitrificans cNOR and efforts to adapt rapid-freeze-quench samples to FTIR analysis.

Vibrational studies of the single turnover reaction cycle and substrate inhibition reaction in denitrifying NO reductases

253rd National Meeting (2017)

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