High-Resolution X-ray Absorption Spectroscopy of Soluble Methane Monooxygenase: Room Temperature and Cryogenic Insights into Active Site Electronic Structure Modulation.
6 affiliations
- 1Max Planck Institute for Chemical Energy Conversion, Mülheim an der Ruhr45470, Germany.
- 2Max-Planck-Institut für Kohlenforschung, Mülheim an der Ruhr45470, Germany.
- 3Cornell High Energy Synchrotron Source, Wilson Laboratory, Cornell University, Ithaca, New York14853, United States.
- 4School of Applied Engineering and Physics, Cornell University, Ithaca, New York14853, United States.
- 5Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota55455, United States.
- 6Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama36849, United States.
Abstract
Room temperature HERFD XAS (High Energy Resolution Fluorescence Detected X-ray Absorption Spectroscopy) data collected on the stable Fe(III)Fe(III) (ox) and Fe(II)Fe(II) (red) states of the soluble methane monooxygenase hydroxylase (sMMOH) from Methylosinus trichosporium (Mt) OB3b using laminar flow cells reveal structural differences with varied temperature, pH, and presence of regulatory protein MMOB. High quality room-temperature data on the X-ray radiation sensitive MMOH-ox:MMOB state shows two distinct radiative reduction events, confirming the presence of a so-called semimet (Fe(II)Fe(III)) state in Mt sMMO, and for the first time presents the undamaged HERFD XAS spectra of MMOH-ox and MMOH-ox:MMOB. Clear differences in the whiteline and pre-edge of MMOH-red:MMOB appear between room temperature and cryogenic data, indicating the presence of cryogenic artifacts. In light of this new room temperature data, TD-DFT calculations based on optimized QM/MM models have been used to assign the structures of MMOH-red and ox states. MMOH-red and MMOH-red:MMOB have been assigned as 6,6-coordinate on the basis of pre-edge intensities, with minor perturbations in the ligand field seen upon MMOB binding. MMOH-ox:MMOB and MMOH-ox have been assigned as 6,6-coordinate with different protonation states at pH 7 on the basis of calculated models and changes in the experimental pre-edge energies and intensities for MMOH-ox:MMOB at pH 7.5. Together these results establish a revised structural and mechanistic framework for sMMO and demonstrate the potential of microfluidic HERFD XAS for capturing authentic enzymatic states.
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BibTeX
@article{stepanic2026highresolution,
title = {{High-Resolution X-ray Absorption Spectroscopy of Soluble Methane Monooxygenase: Room Temperature and Cryogenic Insights into Active Site Electronic Structure Modulation.}},
author = {Olivia McCubbin Stepanic and Sinjini Bhattacharjee and Christopher J Pollock and Kara A Zielinski and Rahul Banerjee and Derek B Rice and Kushal Sengupta and John D Lipscomb and Dimitrios A Pantazis and Frank Neese and Lois Pollack and Serena DeBeer},
journal = {Journal of the American Chemical Society},
year = {2026},
month = oct,
doi = {10.1021/jacs.6c13395},
url = {https://europepmc.org/article/MED/42954554},
note = {CC BY},
}
RIS
TY - JOUR
TI - High-Resolution X-ray Absorption Spectroscopy of Soluble Methane Monooxygenase: Room Temperature and Cryogenic Insights into Active Site Electronic Structure Modulation.
AU - Olivia McCubbin Stepanic
AU - Sinjini Bhattacharjee
AU - Christopher J Pollock
AU - Kara A Zielinski
AU - Rahul Banerjee
AU - Derek B Rice
AU - Kushal Sengupta
AU - John D Lipscomb
AU - Dimitrios A Pantazis
AU - Frank Neese
AU - Lois Pollack
AU - Serena DeBeer
PY - 2026
DA - 2026/10/01
JO - Journal of the American Chemical Society
DO - 10.1021/jacs.6c13395
UR - https://europepmc.org/article/MED/42954554
AB - Room temperature HERFD XAS (High Energy Resolution Fluorescence Detected X-ray Absorption Spectroscopy) data collected on the stable Fe(III)Fe(III) (ox) and Fe(II)Fe(II) (red) states of the soluble methane monooxygenase hydroxylase (sMMOH) from Methylosinus trichosporium (Mt) OB3b using laminar flow cells reveal structural differences with varied temperature, pH, and presence of regulatory protein MMOB. High quality room-temperature data on the X-ray radiation sensitive MMOH-ox:MMOB state shows two distinct radiative reduction events, confirming the presence of a so-called semimet (Fe(II)Fe(III)) state in Mt sMMO, and for the first time presents the undamaged HERFD XAS spectra of MMOH-ox and MMOH-ox:MMOB. Clear differences in the whiteline and pre-edge of MMOH-red:MMOB appear between room temperature and cryogenic data, indicating the presence of cryogenic artifacts. In light of this new room temperature data, TD-DFT calculations based on optimized QM/MM models have been used to assign the structures of MMOH-red and ox states. MMOH-red and MMOH-red:MMOB have been assigned as 6,6-coordinate on the basis of pre-edge intensities, with minor perturbations in the ligand field seen upon MMOB binding. MMOH-ox:MMOB and MMOH-ox have been assigned as 6,6-coordinate with different protonation states at pH 7 on the basis of calculated models and changes in the experimental pre-edge energies and intensities for MMOH-ox:MMOB at pH 7.5. Together these results establish a revised structural and mechanistic framework for sMMO and demonstrate the potential of microfluidic HERFD XAS for capturing authentic enzymatic states.
SN - 0002-7863
SN - 1520-5126
ER -