Nitric oxide reductases (NORs) from denitrifying bacteria catalyze the two-electron reduction of nitric oxide (NO) to nitrous oxide (N2O) as part of the global denitrification cycle that convers nitrite and nitrate to nitrogen. The active site of NOR consists of a high-spin heme b3 and nonheme FeB coordinated by three histidine and one glutamate residue. Despite the biochemical, biomedical, and environmental significance of NORs, structural features responsible for its activity and a clear mechanistic understanding of its reaction are not well understood. We have prepared structural and functional model of NOR by engineering a FeB center into the distal site of heme in sperm whale myoglobin. Spectroscopic studies of these biosynthetic models using Fourier transform infrared (FTIR) and resonance Raman (rR) have shown that heme-bound NO adopts a strong nitroxyl character through interactions with the nonheme iron, and time-resolved rapid-mixing experiments provided evidence for both heme and nonheme nitrosyl complexes, supporting the trans mechanism. Furthermore, replacing the native heme (iron protoporphyrin IX, FePPIX) in the model with ZnPPIX has allowed for a thorough spectroscopic and computational investigation into the FeB nitrosyl complex selectively, without interference from the heme nitrosyl. By using UV–vis absorbance, electron paramagnetic resonance, and Mössbauer spectroscopies, as well as X-ray crystallography and density functional theory (DFT) calculations, the nonheme nitrosyl was characterized as an S = 3/2 {FeNO}7 complex, using the Enemark–Feltham notation, best described as a HS ferrous iron antiferromagnetically coupled to an NO radical. Finally, by systematically decreasing E°' of heme in the biosynthetic models of NOR in myoglobin from +148 to -130 mV, we modulated their electronic and bonding character, resulting in 180-fold and 633-fold enhancements in NO association and heme-nitrosyl decay rates, respectively. In turn, the optimized NOR mimic displays multiple turnovers, unachievable in previous NOR models. Therefore, out results show that heme E°' is a critical factor in guiding NO reduction reactivity and can explain why the heme E°' in NORs is evolutionarily set in a narrow range with low values to facilitate N-O bond activation, NO binding and heme-nitrosyl decay.

Using biosynthetic models of nitric oxide reductase (NOR) in myoglobin to elucidate structural features responsible for NOR activity and its reaction mechanism

255th National Meeting (2018)

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