Synthetic Model Complex of the Key Intermediate in Cytochrome P450 Nitric Oxide Reductase

Ashley B. McQuarters, Elizabeth J. Blaesi, Jeff W. Kampf, E. Ercan Alp, Jiyong Zhao, Michael Hu, Carsten Krebs, Nicolai Lehnert

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)1398-1413
Number of pages16
JournalInorganic Chemistry
Volume58
Issue number2
DOIs
StatePublished - Jan 22 2019

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cytochromes
nitric oxide
Cytochrome P-450 Enzyme System
Electronic structure
Oxidoreductases
electronic structure
Vibrational spectroscopy
Denitrification
Heme
spectroscopy
Catalysis
catalysis
Paramagnetic resonance
soils
electron paramagnetic resonance
Nitrogen
Iron
reactivity
Nuclear magnetic resonance
Spectroscopy

All Science Journal Classification (ASJC) codes

  • Physical and Theoretical Chemistry
  • Inorganic Chemistry

Cite this

McQuarters, A. B., Blaesi, E. J., Kampf, J. W., Alp, E. E., Zhao, J., Hu, M., ... Lehnert, N. (2019). Synthetic Model Complex of the Key Intermediate in Cytochrome P450 Nitric Oxide Reductase. Inorganic Chemistry, 58(2), 1398-1413. https://doi.org/10.1021/acs.inorgchem.8b02947
McQuarters, Ashley B. ; Blaesi, Elizabeth J. ; Kampf, Jeff W. ; Alp, E. Ercan ; Zhao, Jiyong ; Hu, Michael ; Krebs, Carsten ; Lehnert, Nicolai. / Synthetic Model Complex of the Key Intermediate in Cytochrome P450 Nitric Oxide Reductase. In: Inorganic Chemistry. 2019 ; Vol. 58, No. 2. pp. 1398-1413.
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abstract = "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 {\AA} 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 M{\"o}ssbauer 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.",
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McQuarters, AB, Blaesi, EJ, Kampf, JW, Alp, EE, Zhao, J, Hu, M, Krebs, C & Lehnert, N 2019, 'Synthetic Model Complex of the Key Intermediate in Cytochrome P450 Nitric Oxide Reductase', Inorganic Chemistry, vol. 58, no. 2, pp. 1398-1413. https://doi.org/10.1021/acs.inorgchem.8b02947

Synthetic Model Complex of the Key Intermediate in Cytochrome P450 Nitric Oxide Reductase. / McQuarters, Ashley B.; Blaesi, Elizabeth J.; Kampf, Jeff W.; Alp, E. Ercan; Zhao, Jiyong; Hu, Michael; Krebs, Carsten; Lehnert, Nicolai.

In: Inorganic Chemistry, Vol. 58, No. 2, 22.01.2019, p. 1398-1413.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Synthetic Model Complex of the Key Intermediate in Cytochrome P450 Nitric Oxide Reductase

AU - McQuarters, Ashley B.

AU - Blaesi, Elizabeth J.

AU - Kampf, Jeff W.

AU - Alp, E. Ercan

AU - Zhao, Jiyong

AU - Hu, Michael

AU - Krebs, Carsten

AU - Lehnert, Nicolai

PY - 2019/1/22

Y1 - 2019/1/22

N2 - 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.

AB - 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.

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