skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Characterization of the Particulate Methane Monooxygenase Metal Centers in Multiple Redox States by X-ray Absorption Spectroscopy

Abstract

The integral membrane enzyme particulate methane monooxygenase (pMMO) converts methane, the most inert hydrocarbon, to methanol under ambient conditions. The 2.8-{angstrom} resolution pMMO crystal structure revealed three metal sites: a mononuclear copper center, a dinuclear copper center, and a nonphysiological mononuclear zinc center. Although not found in the crystal structure, solution samples of pMMO also contain iron. We have used X-ray absorption spectroscopy to analyze the oxidation states and coordination environments of the pMMO metal centers in as-isolated (pMMO{sub iso}), chemically reduced (pMMO{sub red}), and chemically oxidized (pMMO{sub ox}) samples. X-ray absorption near-edge spectra (XANES) indicate that pMMO{sub iso} contains both Cu{sup I} and Cu{sup II} and that the pMMO Cu centers can undergo redox chemistry. Extended X-ray absorption fine structure (EXAFS) analysis reveals a Cu-Cu interaction in all redox forms of the enzyme. The Cu-Cu distance increases from 2.51 to 2.65 {angstrom} upon reduction, concomitant with an increase in the average Cu-O/N bond lengths. Appropriate Cu2 model complexes were used to refine and validate the EXAFS fitting protocols for pMMO{sub iso}. Analysis of Fe EXAFS data combined with electron paramagnetic resonance (EPR) spectra indicates that Fe, present as Fe{sup III}, is consistent with heme impurities. These findings are complementarymore » to the crystallographic data and provide new insight into the oxidation states and possible electronic structures of the pMMO Cu ions.« less

Authors:
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
929827
Report Number(s):
BNL-80384-2008-JA
Journal ID: ISSN 0020-1669; INOCAJ; TRN: US200822%%1034
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Inorganic Chemistry; Journal Volume: 45
Country of Publication:
United States
Language:
English
Subject:
03 NATURAL GAS; 36 MATERIALS SCIENCE; ABSORPTION; ABSORPTION SPECTROSCOPY; BOND LENGTHS; CRYSTAL STRUCTURE; ELECTRON SPIN RESONANCE; ELECTRONIC STRUCTURE; FINE STRUCTURE; METALS; METHANE; PARTICULATES; national synchrotron light source

Citation Formats

Lieberman,R. Characterization of the Particulate Methane Monooxygenase Metal Centers in Multiple Redox States by X-ray Absorption Spectroscopy. United States: N. p., 2006. Web. doi:10.1021/ic060739v.
Lieberman,R. Characterization of the Particulate Methane Monooxygenase Metal Centers in Multiple Redox States by X-ray Absorption Spectroscopy. United States. doi:10.1021/ic060739v.
Lieberman,R. Sun . "Characterization of the Particulate Methane Monooxygenase Metal Centers in Multiple Redox States by X-ray Absorption Spectroscopy". United States. doi:10.1021/ic060739v.
@article{osti_929827,
title = {Characterization of the Particulate Methane Monooxygenase Metal Centers in Multiple Redox States by X-ray Absorption Spectroscopy},
author = {Lieberman,R.},
abstractNote = {The integral membrane enzyme particulate methane monooxygenase (pMMO) converts methane, the most inert hydrocarbon, to methanol under ambient conditions. The 2.8-{angstrom} resolution pMMO crystal structure revealed three metal sites: a mononuclear copper center, a dinuclear copper center, and a nonphysiological mononuclear zinc center. Although not found in the crystal structure, solution samples of pMMO also contain iron. We have used X-ray absorption spectroscopy to analyze the oxidation states and coordination environments of the pMMO metal centers in as-isolated (pMMO{sub iso}), chemically reduced (pMMO{sub red}), and chemically oxidized (pMMO{sub ox}) samples. X-ray absorption near-edge spectra (XANES) indicate that pMMO{sub iso} contains both Cu{sup I} and Cu{sup II} and that the pMMO Cu centers can undergo redox chemistry. Extended X-ray absorption fine structure (EXAFS) analysis reveals a Cu-Cu interaction in all redox forms of the enzyme. The Cu-Cu distance increases from 2.51 to 2.65 {angstrom} upon reduction, concomitant with an increase in the average Cu-O/N bond lengths. Appropriate Cu2 model complexes were used to refine and validate the EXAFS fitting protocols for pMMO{sub iso}. Analysis of Fe EXAFS data combined with electron paramagnetic resonance (EPR) spectra indicates that Fe, present as Fe{sup III}, is consistent with heme impurities. These findings are complementary to the crystallographic data and provide new insight into the oxidation states and possible electronic structures of the pMMO Cu ions.},
doi = {10.1021/ic060739v},
journal = {Inorganic Chemistry},
number = ,
volume = 45,
place = {United States},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}
  • Parallel X-ray absorption edge and EPR studies of the particulate methane monooxygenase in situ reveal that the enzyme contains unusually high levels of copper ions with a significant portion of the copper ions existing as Cu(I) in the `as-isolated` form (70-80%). The observation of high levels of reduced copper in a monooxygenase is surprising considering that the natural cosubstrate of the enzyme is dioxygen. Toward clarifying the roles of the various copper ions in the enzyme, we have successfully prepared different states of the protein in the membrane-bound form at various levels of reduction using dithionite, dioxygen, and ferricyanide. EPRmore » intensity analysis of the fully-oxidized preparations indicates that the bulk of copper ions are arranged in cluster units. The fully-reduced protein obtained by reduction by dithionite has been used to initiate the single turnover of the enzyme in the presence of dioxygen. Differential reactivity toward dioxygen was observed upon analyzing the copper reduction levels in these synchronized preparations. The enzyme is capable of supporting turnover in the absence of external electron donors in the highly reduced states. These results suggest the presence of at least two classes of copper ions in the particulate methane monoxygenase. As a working hypothesis, we have referred to these classes of copper ions as (1) the catalytic (C) clusters, and (2) the electron transfer (E) clusters. 56 refs., 4 figs., 1 tab.« less
  • Here, we report the use of time-resolved X-ray absorption spectroscopy in the ns–μs time scale to track the light induced two electron transfer processes in a multi-component photocatalytic system, consisting of [Ru(bpy) 3] 2+/ a diiron(III,III) model/triethylamine. EXAFS analysis with DFT calculations confirms the structural configurations of the diiron(III,III) and reduced diiron(II,II) states.
  • Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. The nature of the pMMO active site and the overall metal content are controversial, with spectroscopic and crystallographic data suggesting the presence of a mononuclear copper center, a dinuclear copper center, a trinuclear center, and a diiron center or combinations thereof. Most studies have focused on pMMO from Methylococcus capsulatus (Bath). pMMO from a second organism, Methylosinus trichosporium OB3b, has been purified and characterized by spectroscopic and crystallographic methods. Purified M. trichosporium OB3b pMMO contains 2 copper ions per 100 kDa protomer. Electron paramagneticmore » resonance (EPR) spectroscopic parameters indicate that type 2 Cu(II) is present as two distinct species. Extended X-ray absorption fine structure (EXAFS) data are best fit with oxygen/nitrogen ligands and reveal a Cu-Cu interaction at 2.52 Angstroms. Correspondingly, X-ray crystallography of M. trichosporium OB3b pMMO shows a dinuclear copper center, similar to that observed previously in the crystal structure of M. capsulatus (Bath) pMMO. There are, however, significant differences between the pMMO structures from the two organisms. A mononuclear copper center present in M. capsulatus (Bath) pMMO is absent in M. trichosporium OB3b pMMO, whereas a metal center occupied by zinc in the M. capsulatus (Bath) pMMO structure is occupied by copper in M. trichosporium OB3b pMMO. These findings extend previous work on pMMO from M. capsulatus (Bath) and provide new insight into the functional importance of the different metal centers.« less