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Spectroscopic Characterization of Mononitrosyl Complexes in Heme–Nonheme Diiron Centers within the Myoglobin Scaffold (FeBMbs): Relevance to Denitrifying NO Reductase

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journal contribution
posted on 05.07.2011, 00:00 by Takahiro Hayashi, Kyle D. Miner, Natasha Yeung, Ying-Wu Lin, Yi Lu, Pierre Moënne-Loccoz
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 FeB 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 CuI- or ZnII-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 FeII-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 CuI-bound proteins exhibit ν(FeNO) and ν(NO) that are only marginally distinct from those reported for native myoglobin. However, binding of FeII at the FeB 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 FeII. Detection of a similarly low ν(NO) in the ZnII-loaded protein supports this interpretation.