Abstract
The enzymatic conversion of the greenhouse gas, methane, to a liquid fuel, methanol, is performed by methane monooxygenases (MMOs) under mild conditions. The copper stoichiometry of particulate MMO (pMMO) has been long debated, with a dicopper site previously proposed on the basis of a 2.51 Å Cu-Cu feature in extended X-ray absorption fine structure (EXAFS) data. However, recent crystallographic data and advanced electron paramagnetic resonance (EPR) characterization support the presence of only mononuclear copper sites. To reconcile these data, we have collected high-energy resolution fluorescence detected (HERFD) and partial fluorescence yield (PFY) EXAFS spectra of Methylococcus (M.) capsulatus (Bath) pMMO. Both methods reveal only monocopper sites. These data were compared to previously published pMMO PFY-EXAFS data from M. capsulatus (Bath) and Methylomicrobium alcaliphilum 20Z, supporting dicopper and monocopper sites, respectively. The FT-EXAFS feature previously attributed to a dicopper site can be reproduced by the inclusion of a metallic copper background signal. The exact position of this feature is dependent on the nature of the sample and the percentage of background contamination, indicating that visual inspection is not sufficient for identifying background metallic contributions. Additionally, an undamaged X-ray absorption spectrum was obtained, consistent with the copper oxidation-state speciation determined by EPR quantification. X-ray photodamage studies suggest that the previously observed Cu(i) XAS features are in part attributable to photodamage. This study illustrates the complex array of factors involved in EXAFS measurement and modeling of pMMO and more generally, dilute metalloproteins with multiple metal centers.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 6194-6209 |
| Number of pages | 16 |
| Journal | Chemical Science |
| Volume | 12 |
| Issue number | 17 |
| DOIs | |
| State | Published - May 7 2021 |
Funding
The authors gratefully acknowledge Timothy Stemmler (Wayne State University) for providing previously published EXAFS data and for helpful discussion. GEC and SD acknowledge the Max Planck Gesellscha\uE09D for support. SD acknowledges funding from the European Research Council (ERC), under the Horizon 2020 research and innovation programme (grant agreement No 856446). ACR acknowledges the National Institutes of Health (GM118035). MOR acknowledges support from Brian M. Hoffman (Northwestern University) and the National Institutes of Health (GM111097) and the National Science Foundation (MCB-1908587). Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The staff at SSRL (Matthew Latimer and Erik Nelson at BL9-3; Dimosthenis Sokaras and Thomas Kroll at BL6-2) are thanked for their assistance at the beamlines. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (P41GM103393).
ASJC Scopus subject areas
- General Chemistry