The production of nitrous oxide by the heme/nonheme diiron center of engineered myoglobins (Fe(B)Mbs) proceeds through a trans-iron-nitrosyl dimer.

Denitrifying NO reductases are transmembrane protein complexes that are evolutionarily related to heme/copper terminal oxidases. They utilize a heme/nonheme diiron center to reduce two NO molecules to N2O. Engineering a nonheme Fe(B) site within the heme distal pocket of sperm whale myoglobin has offered well-defined diiron clusters for the investigation of the mechanism of NO reduction in these unique active sites. In this study, we use FTIR spectroscopy to monitor the production of N2O in solution and to show that the presence of a distal Fe(B)(II) is not sufficient to produce the expected product. However, the addition of a glutamate side chain peripheral to the diiron site allows for 50% of a productive single-turnover reaction. Unproductive reactions are characterized by resonance Raman spectroscopy as dinitrosyl complexes, where one NO molecule is bound to the heme iron to form a five-coordinate low-spin {FeNO}(7) species with ν(FeNO)(heme) and ν(NO)(heme) at 522 and 1660 cm(-1), and a second NO molecule is bound to the nonheme Fe(B) site with a ν(NO)(FeB) at 1755 cm(-1). Stopped-flow UV-vis absorption coupled with rapid-freeze-quench resonance Raman spectroscopy provide a detailed map of the reaction coordinates leading to the unproductive iron-nitrosyl dimer. Unexpectedly, NO binding to Fe(B) is kinetically favored and occurs prior to the binding of a second NO to the heme iron, leading to a (six-coordinate low-spin heme-nitrosyl/FeB-nitrosyl) transient dinitrosyl complex with characteristic ν(FeNO)(heme) at 570 ± 2 cm(-1) and ν(NO)(FeB) at 1755 cm(-1). Without the addition of a peripheral glutamate, the dinitrosyl complex is converted to a dead-end product after the dissociation of the proximal histidine of the heme iron, but the added peripheral glutamate side chain in Fe(B)Mb2 lowers the rate of dissociation of the promixal histidine which in turn allows the (six-coordinate low-spin heme-nitrosyl/Fe(B)-nitrosyl) transient dinitrosyl complex to decay with production of N2O at a rate of 0.7 s(-1) at 4 °C. Taken together, our results support the proposed trans mechanism of NO reduction in NORs.