Fungal denitrification plays a crucial role in the nitrogen cycle and contributes to the total N 2 O emission from agricultural soils. Here, cytochrome P450 NO reductase (P450nor) reduces two NO to N 2 O using a single heme site. Despite much research, the exact nature of the critical "Intermediate I" responsible for the key N-N coupling step in P450nor is unknown. This species likely corresponds to a Fe-NHOH-type intermediate with an unknown electronic structure. Here we report a new strategy to generate a model system for this intermediate, starting from the iron(III) methylhydroxylamide complex [Fe(3,5-Me-BAFP)(NHOMe)] (1), which was fully characterized by 1 H NMR, UV-vis, electron paramagnetic resonance, and vibrational spectroscopy (rRaman and NRVS). Our data show that 1 is a high-spin ferric complex with an N-bound hydroxylamide ligand that is strongly coordinated (Fe-N distance, 1.918 Å Fe-NHOMe stretch, 558 cm -1 ). Simple one-electron oxidation of 1 at -80 °C then cleanly generates the first model system for Intermediate I, [Fe(3,5-Me-BAFP)(NHOMe)] + (1 + ). UV-vis, resonance Raman, and Mössbauer spectroscopies, in comparison to the chloro analogue [Fe(3,5-Me-BAFP)(Cl)] + , demonstrate that 1 + is best described as an Fe III -(NHOMe) • complex with a bound NHOMe radical. Further reactivity studies show that 1 + is highly reactive toward NO, a reaction that likely proceeds via N-N bond formation, following a radical-radical-type coupling mechanism. Our results therefore provide experimental evidence, for the first time, that an Fe III -(NHOMe) • electronic structure is indeed a reasonable electronic description for Intermediate I and that this electronic structure is advantageous for P450nor catalysis because it can greatly facilitate N-N bond formation and, ultimately, N 2 O generation.
All Science Journal Classification (ASJC) codes
- Physical and Theoretical Chemistry
- Inorganic Chemistry