Spectroscopic characterization of mononitrosyl complexes in heme-nonheme diiron centers within the myoglobin scaffold (Fe_BMbs): Relevance to denitrifying No reductase

Denitrifying NO reductases are evolutionarily related to the superfamily of heme-copper terminal oxidases. These transmembrane protein complexes utilize a heme-nonheme diiron center to reduce two NO molecules to N₂O. To understand this reaction, the diiron site has been modeled using sperm whale myoglobin as a scaffold and mutating distal residues Leu-29 and Phe-43 to histidines and Val-68 to a glutamic acid to create a nonheme Fe_B site. The impact of incorporation of metal ions at this engineered site on the reaction of the ferrous heme with one NO was examined by UV-vis absorption, EPR, resonance Raman, and FTIR spectroscopies. UV-vis absorption and resonance Raman spectra demonstrate that the first NO molecule binds to the ferrous heme, but while the apoproteins and Cu^I- or Zn^II-loaded proteins show characteristic EPR signatures of S = ¹/₂ six-coordinate heme {FeNO}⁷ species that can be observed at liquid nitrogen temperature, the Fe^II-loaded proteins are EPR silent at ≥30 K. Vibrational modes from the heme [Fe-N-O] unit are identified in the RR and FTIR spectra using ¹⁵NO and ¹⁵N¹⁸O. The apo and Cu^I-bound proteins exhibit ν(FeNO) and ν(NO) that are only marginally distinct from those reported for native myoglobin. However, binding of Fe^II at the Fe_B site shifts the heme ν(FeNO) by 17 cm^-1 and the ν(NO) by -50 cm^-1 to 1549 cm ^-1. This low ν(NO) is without precedent for a six-coordinate heme {FeNO}⁷ species and suggests that the NO group adopts a strong nitroxyl character stabilized by electrostatic interaction with the nearby nonheme Fe^II. Detection of a similarly low ν(NO) in the Zn ^II-loaded protein supports this interpretation.