The Production of Nitrous Oxide by the Heme/Nonheme Diiron Center of Engineered Myoglobins (Fe_BMbs) Proceeds through a trans-lron-Nitrosyl Dimer

摘要

Denitrifying NO reductases are transmembrane protein complexes that are evolutionarily related to heme/ copper terminal oxidases. They utilize a hemeonheme diiron center to reduce two NO molecules to N_2O. 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 N_2O 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 υ(FeNO)_(heme) and υ(NO)_(heme) at 522 and 1660 cm~(-1), and a second NO molecule is bound to the nonheme Fe_B site with a υ(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/Fe_B-nitrosyl) transient dinitrosyl complex with characteristic υ(FeNO)_(heme) at 570 ± 2 cm~(-1) and υ(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_BMb2 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 N_2O at a rate of 0.7 s~(-1) at 4 ℃. Taken together, our results support the proposed trans mechanism of NO reduction in NORs.