Stepwise nitrosylation of the nonheme iron site in an engineered azurin and a molecular basis for nitric oxide signaling mediated by nonheme iron proteins

Abstract

Mononitrosyl and dinitrosyl iron species, such as {FeNO}⁷, {FeNO}⁸ and {Fe(NO)₂}⁹, have been proposed to play pivotal roles in the nitrosylation processes of nonheme iron centers in biological systems. Despite their importance, it has been difficult to capture and characterize them in the same scaffold of either native enzymes or their synthetic analogs due to the distinct structural requirements of the three species, using redox reagents compatible with biomolecules under physiological conditions. Here, we report the realization of stepwise nitrosylation of a mononuclear nonheme iron site in an engineered azurin under such conditions. Through tuning the number of nitric oxide equivalents and reaction time, controlled formation of {FeNO}⁷ and {Fe(NO)₂}⁹ species was achieved, and the elusive {FeNO}⁸ species was inferred by EPR spectroscopy and observed by Mössbauer spectroscopy, with complemental evidence for the conversion of {FeNO}⁷ to {Fe(NO)₂}⁹ species by UV-Vis, resonance Raman and FT-IR spectroscopies. The entire pathway of the nitrosylation process, Fe(ii) → {FeNO}⁷ → {FeNO}⁸ → {Fe(NO)₂}⁹, has been elucidated within the same protein scaffold based on spectroscopic characterization and DFT calculations. These results not only enhance the understanding of the dinitrosyl iron complex formation process, but also shed light on the physiological roles of nitric oxide signaling mediated by nonheme iron proteins.

Fig. 1. Metal-binding site in M121H/H46EAz. (a) Overlay of the structure and the 2F_o − F_c electron density map. (b) Hydrogen bond interactions around the active site. The backbone of M13 forms a hydrogen bond to the side chain of H121. The backbones of F114 and N47 form hydrogen bonds directly with Cys112. The hydrogen bonds between T113 and N47 are responsible for the rigidity of the metal-binding site (PDB: 4WKX, chain A).

Fig. 2. UV-Vis monitoring of nitrosyl iron complex formation at an engineered non-heme iron site in Az. (a) Kinetic UV-Vis profile of Fe(ii)-M121H/H46EAz reacting with 0.5 eq. of Proli NONOate in 50 mM BisTris buffer at pH 7. Black: Fe(ii)-M121H/H46EAz, blue: {FeNO}⁷ species 1. Inset: the time traces of absorbance at 337 nm (black), 425 nm (red) and 650 nm (red) upon Proli NONOate addition. (b) Kinetic UV-Vis profile of isolated {FeNO}⁷1 being reduced with excess NO. Blue: {FeNO}⁷, red: {Fe(NO)₂}⁹. Inset: the time traces of absorbance at 650 nm (black) and 720 nm (red) upon {FeNO}⁷ reduction with an excess amount of NO.

Fig. 3. 4.2 K variable field Mössbauer spectra of the Fe(ii)-M121H/H46EAz complex treated with NO (black) and the spectral simulations (red). The experimental data shown in this figure were obtained by subtracting 20% Fe(ii)-M121H/H46EAz spectra from the raw experimental data (see Fig. S5†). The simulations of the two S = 3/2 {FeNO}⁷ species are shown in purple solid lines (for the E/D = 0.033 species) and blue dashed lines (for the E/D = 0.007 species). The former species accounts for ∼40% of the total iron in the sample, and the latter one accounts for ∼30% of the total Fe. The relative ratio of the two {FeNO}⁷ species (40/30) determined by Mössbauer spectra is consistent with the 56/44 ratio observed in EPR. The green solid lines are the simulation of the {FeNO}⁸ species, which accounts for ∼10% of the iron in the sample. The magnetic broadening of this minor species observed in the experimental data with field strength > 0.5 T suggests the integer spin nature of this species. See the main text and Table S1 for detailed simulation parameters.

Fig. 4. CW-EPR investigation of the {Fe(NO)₂}⁹ species formed in an engineered non-heme iron Az. (a) X-band EPR spectrum of the {Fe(NO)₂}⁹ species (black) and the spectral simulation (red). (b) X-band EPR spectrum of {Fe(NO)₂}⁹ at room temperature. (c) X-band EPR spectra of {Fe(¹⁴NO)₂}⁹ (red) and {Fe(¹⁵NO)₂}⁹ species (black).

Fig. 5. Pulsed EPR investigation of dinitrosyl iron species formed in an engineered non-heme iron Az. (a) Q-band ¹H-ENDOR spectra of {Fe(NO)₂}⁹ species formed in an engineered nonheme iron Az collected at g_‖. (b) Q-band N-ENDOR spectra of {Fe(NO)₂}⁹ species formed in an engineered nonheme iron Az collected at g_‖, {Fe(¹⁴NO)₂}⁹ (black) and {Fe(¹⁵NO)₂}⁹ (red).

Fig. 6. RR spectra of the {FeNO}⁷ and {Fe(NO)₂}⁹ species. (a) Room-temperature RR spectra of Fe(ii)-azurin (top grey trace) and the {FeNO}⁷ complexes formed with ¹⁴NO (top black trace) and ¹⁵NO (top red trace). Lower traces correspond to the nitrosyl complexes minus Fe(ii)-azurin (¹⁴NO, black trace; ¹⁵NO red trace) and the double difference spectrum (green trace). These RR features overlap with non-resonant Raman vibrations from the protein matrix but protein bands are readily subtracted using the spectrum of Fe(ii)-azurin and the sharp 1002 cm⁻¹ ring vibration of Phe side-chains as an internal intensity standard. (b) Room-temperature RR spectra of Fe(ii)-azurin (grey trace) and its {Fe(NO)₂}⁹ complexes formed with ¹⁴NO (top black trace) and ¹⁵NO (top red trace). Lower traces correspond to the nitrosyl complexes minus Fe(ii)-azurin (¹⁴NO, black trace; ¹⁵NO red trace) and the double difference spectrum (green trace).

Fig. 7. Room-temperature FT-IR spectra of the reaction of Fe(ii)-Az with excess DEA-NONOate. Successive accumulations are overlapped in the center of the graph and difference spectra for maximum accumulation of the {FeNO}⁷ and {Fe(NO)₂}⁹ species as black and red traces at the bottom of the graph; the inset plots the intensities of the 1799 and 1724 cm⁻¹ bands as a function of time.

Fig. 8. 4.2 K 45 mT Mössbauer spectra of the NO treated Fe(ii)-M121H/H46EAz complex before and after cryoreduction. Left panel: top, the spectrum measured on NO treated Fe(ii)-M121H/H46EAz before (black dashed line) cryoreduction and the spectral components of the axial (blue line) and the rhombic (purple line) {FeNO}⁷ species; bottom, the difference spectrum (black dashed line) after subtracting the {FeNO}⁷ species simulations from the experimental spectrum and the spectral simulation of the {FeNO}⁸ (species 3, green line). Right panel: top, the spectrum of the same sample shown in the left panel measured before (orange dashed line) and after (cyan line) cryoreduction; bottom, the difference spectrum obtained by subtracting the after-cryoreduction spectrum from the before-cryoreduction spectrum (black dashed line) and the simulations for the decreased axial (blue dashed line) {FeNO}⁷ species and the increased new {FeNO}⁸ species, species 3′ (green dashed line). The sample was prepared by anaerobically adding 1 eq. of Proli NONOate (from 50 mM Proli NONOate stock solution in 10 mM NaOH) into 600 μl 2 mM ⁵⁷Fe(ii)-M121H/H46EAz solution under stirring and then freezing in liquid nitrogen for 5 min. See the main text and the ESI for the simulation parameters.

Fig. 9. Optimized active site structures (A–O). Color scheme: C – cyan, Fe – black, N – blue, O – red, S – yellow, H – grey.

Fig. 10. Proposed reaction pathway for {Fe(NO)₂}⁹ formation. Color scheme: C – cyan, Fe – black, N – blue, O – red, S – yellow, H – grey.