36 Research Frontiers 2022 Research Frontiers 2022 Life Science Non-heme iron enzymes play a key role in the activation of dioxygen in Nature [1]. Among the strategies for the four-electron activation of O 2 , some mononuclear enzymes (e.g., syringomycin halogenase SyrB2) use two electrons from the Fe II center and two electrons from the cofactor a -ketoglutarate, producing a 5-coordinate Fe IV = O intermediate that cleaves the C–H bond of activated substrates (Fig. 1(a)). Alternatively, the two Fe II centers of the binuclear enzyme soluble methane monooxygenase (sMMO) provide all four electrons, producing an Fe IV 2 site able to cleave the strong C-H bond of methane to produce methanol. This high valent intermediate Q has been long studied as its structure informs the design of catalysts for methane oxygenation. Early results assigned Q as a closed core Fe IV 2 ( m -O) 2 , later supported by resonance Raman (rR) spectroscopy [2]. In 2010, an open core complex O=Fe IV –O–Fe IV –OH was found to oxidize dihydroanthracene at a rate 100 × higher than its closed core Fe IV 2 ( m -O) 2 complex [3] and subsequent high energy resolution fluorescence detected X-ray absorption spectroscopy (HERFD- XAS) studies favored an open core structure [4]. Our study [5] utilized 57 Fe Nuclear Resonance Vibrational Spectroscopy (NRVS) to characterize the geometric and electronic structure of intermediate Q, providing insight into the closed vs. open core debate (Fig. 1(b)). 57 Fe NRVS is a synchrotron-based technique that detects vibrational modes containing iron motion. The NRVS data are interpreted using Density Functional Theory (DFT) to interrogate the structure of Q. 57 Fe NRVS was performed at SPring-8 BL09XU , Nuclear Resonant Scattering beamline, using a nuclear resonance energy of 14.4 keV, monochromated to 1.0 meV using both a high-heat-load and high- resolution monochromator. Vibrational sidebands were collected 100 meV above the elastic peak. The NRVS spectrum of Q exhibited time-dependent changes in the beam. Using Mössbauer spectroscopy, we assigned this decay to cryoreduction of Q. Selective binning of scans from the first 10 hours and after 30 hours of measurement gave the vibrational spectra of Q (early) and cryoreduced Q (late). Figure 2 presents the NRVS spectra of early and late 16 O 2 Q and late 18 O 2 Q. The early Q spectrum shows features at 605, 420, 375, 330, 300 and 275 cm –1 , where late Q shows loss of the 605, 375 and 275 cm –1 features and growth of new features at 580, 365, 240 and 220 cm –1 . Further, two decay features 580 and 420 cm –1 show isotope sensitivity (Fig. 2(b) inset). 90 closed and open core DFT optimized structures of Q were generated and used to simulate the NRVS and prior spectroscopic data. The closed core models contained two bridging O(H) x and one terminal ligand, while the open core models all contained one oxo bridge, with differing levels of protonation of carboxylate and oxygenic ligands. Comparison of the simulated vibrational spectra of each model to past rR data [2] showed that both open and closed core models could reproduce the experimental data, but eliminated some models. Further, time-dependent DFT analysis investigated which models would show rR enhancement of a 690 cm –1 feature by excitation at 351 nm. This analysis excluded all open core models except for those with two terminal hydroxides (OH– Fe IV –O–Fe IV –OH) or both a terminal hydroxide and an oxo (O = Fe IV – O – Fe IV – OH). For the remaining structural models, the full set of NRVS data (early and late 16 O 2 , late 18 O 2 ) were simulated and compared to experimental NRVS data. Figure 3 shows the NRVS simulations of open (a) and closed (b) core models that were the best simulations of the experimental data. The top panels display the experimental data, the middle panels show simulations of the early and late 16 O 2 data, and the bottom panel compares simulations of the 16 O 2 and 18 O 2 late data. Figure 3(a) shows simulations for the closed core model containing a bis- m -oxo core and a terminal hydroxide (bis( m O)–OH-12). Simulations to the low energy region (<450 cm –1 ) are in good agreement with the experimental data. Further, the small 16/18 O 2 isotope sensitivity of the experimental decay data is well reproduced due to the longer Fe-OH bond which dictates the magnitude of this isotope shift. Figure 3(b) displays simulations for the best open core model, O=Fe IV –O–Fe IV –OH model (OC16). Comparison of the low energy region again shows good agreement to experiment, however, the simulations fail to reproduce Nuclear resonance vibrational spectroscopy definition of intermediate Q in methane monooxygenase Fig. 1. Reactivity of mononuclear and binuclear non-heme iron enzymes. (b) (a) or closed core open core Mononuclear non-heme iron enzyme (SyrB2) Binuclear non-heme iron enzyme (sMMO) 37 Research Frontiers 2022 Research Frontiers 2022 the small magnitude of the isotope shift in the late data and this and other open core structural models were eliminated. Thus, the NRVS data on Q from SPring-8 show that the closed but not the open core structure is supported by the experiment. Experimentally, intermediate Q in the binuclear sMMO enzyme performs hydrogen atom abstraction (HAA) from methane with a Δ G ǂ of 10.3 kcal/mol, while the Fe IV O intermediate of the mononuclear enzyme SyrB2 has a Δ G ǂ of 17.7 kcal/mol for its substrate threonine. Correcting for the difference in bond strength of the substrates, SyrB2’s Δ G ǂ for methane would be 24 kcal/mol. DFT calculations on the Q models give a barrier for a closed core of 12.4 kcal/mol (close to experiment), while the open core O=Fe IV –O– Fe IV –OH and mononuclear Fe IV O sites are calculated to be 25.0 and 22.1 kcal/mol respectively. The low barrier for the closed core is due to the greater thermodynamic driving force associated with the reorganization of the resulting Fe III Fe IV ( m -O)( m -OH) closed core, enabling efficient reactivity of sMMO in the conversion of methane to methanol. References [1] E. I. Solomon et al. : Chem. Rev. 100 (2000) 235. [2] R. Banerjee et al. : Nature 518 (2015) 431. [3] G. Xue et al. : Nat. Chem. 2 (2010) 400. [4] G. E. Cutsail et al. : J. Am. Chem. Soc. 140 (2018) 16807. [5] A. B. Jacobs, R. Banerjee, D. E. Deweese, A. Braun, J. T. Babicz Jr., L. B. Gee, K. D. Sutherlin, L. H. Böttger, Y. Yoda, M. Saito, S. Kitao, Y. Kobayashi, M. Seto, K. Tamasaku, J. D. Lipscomb, K. Park and E. I. Solomon: J. Am. Chem. Soc. 143 (2021) 16007. Dory E. DeWeese, Augustin Braun and Edward I. Solomon* Department of Chemistry Stanford University, USA *Email: edward.solomon@stanford.edu Fig. 2. NRVS spectra of Q: ( a top) 16 O 2 early (red), late (black) and Fe II Fe II reactant species (yellow), ( a bottom) spectra corrected for residual Fe II Fe II . ( b ) 16 O 2 (black) and 18 O 2 (blue) late Q with isotope sensitive features (inset). Fig. 3. Model simulations: top panel: experimental data, middle panels: simulations of the early (red) and late (black) 16 O 2 data, bottom panel: simulations of the 16 O 2 and 18 O 2 late (blue) data ( a ) representative closed core model (bis( m O) – OH-12) ( b ) Best open core model (OC16). (a) (b) Energy (cm –1 ) PVDOS (cm) × 10 – 3 PVDOS (cm) × 10 – 3 Energy (cm –1 ) 0 0 0 5 10 15 20 25 30 20 0 10 20 30 100 200 300 400 500 600 700 0 100 200 300 400 500 600 400 500 600 700 0 100 220 240 275 300 330 365 375 420 605 Early - Late 560-580 200 300 400 500 600 700 Late Q Scans ( 16 O 2 ) Late Q Scans ( 18 O 2 ) 12-15 cm –1 4-7 cm –1 (a) (b) Energy (cm –1 ) Energy (cm –1 ) PVDOS (cm) × 10 –3 PVDOS (cm) × 10 –3 0 0 5 10 15 0 0 5 10 15 0 10 20 30 5 10 15 200 400 600 800 0 200 400 600 800 0 5 10 0 5 10 10 20 30 0 0 200 400 600 800 0 200 400 600 800 220-240 560-580 220-240 260 590 288 357 550 612 685-690 275 300 300 336 381 428 467 560-583 –16 –22 –35 –10 –22 0 180-215 529 589 604-621 692 270 ν ν (Fe-N) ν ν (Fe-Coo – ) ν ν (Fe 2 -OH) & ν ν 2 ν ν (Fe 2 -OH) & ν ν 4 ν ν (Fe2 -OH) ν ν (Fe 2 -OH) & ν ν (Fe-O) & ν ν s (Fe-OH-Fe) Localized ν ν (Fe-O) ν ν (Fe-N) Bis( μ μ O)–OH-12 Bis( μ μ O)–OH-12 Bis( μ μ O)–OH-12 Decay Bis( μ μ O)–OH-12 Decay 18 O 2 Bis( μ μ O)–OH-12 Decay ν ν (Fe-Coo – ) ν ν 4 ± ν ν (Fe-Coo – ) ν ν 5 ν ν 6 ν ν 5 ν ν 6 ν ν 3 ν ν 3 ν ν 3 ν ν 4 ν ν 1 & ν ν 3 ν ν 2 ν ν 1 330 375 420 605 Early - Early Q-MMOH red Late Q-MMOH red Early Q-MMOH red OC16 OC16 Decay OC16 Decay 18 O OC16 Decay Late Q-MMOH red Late Early - Late 560-580
home
- JP
- EN