Revisiting the Mechanism of Dioxygen Activation in Soluble Methane Monooxygenase from M. capsulatus (Bath): Evidence for a Multi-Step Proton-Dependent Reaction Pathway
Stopped-flow kinetic investigations of soluble methane monooxygenase (sMMO) from M. capsulatus (Bath) have clarified discrepancies that exist in the literature regarding several aspects of catalysis by this enzyme. The development of thorough kinetic analytical techniques has led to the discovery of...
| Main Authors: | , |
|---|---|
| Other Authors: | |
| Format: | Article |
| Language: | en_US |
| Published: |
American Chemical Society (ACS)
2013
|
| Online Access: | http://hdl.handle.net/1721.1/82145 https://orcid.org/0000-0002-2693-4982 |
| _version_ | 1826195175181910016 |
|---|---|
| author | Tinberg, Christine E. Lippard, Stephen J. |
| author2 | Massachusetts Institute of Technology. Department of Chemistry |
| author_facet | Massachusetts Institute of Technology. Department of Chemistry Tinberg, Christine E. Lippard, Stephen J. |
| author_sort | Tinberg, Christine E. |
| collection | MIT |
| description | Stopped-flow kinetic investigations of soluble methane monooxygenase (sMMO) from M. capsulatus (Bath) have clarified discrepancies that exist in the literature regarding several aspects of catalysis by this enzyme. The development of thorough kinetic analytical techniques has led to the discovery of two novel oxygenated iron species that accumulate in addition to the well-established intermediates H[subscript peroxo] and Q. The first intermediate, P*, is a precursor to H[subscript peroxo] and was identified when the reaction of reduced MMOH and MMOB with O[subscript 2] was carried out in the presence of ≥540 μM methane to suppress the dominating absorbance signal due to Q. The optical properties of P* are similar to those of H[subscript peroxo], with ε[subscript 420] = 3500 M[superscript −1] cm[superscript −1] and ε[subscript 720] = 1250 M[superscript −1] cm[superscript −1]. These values are suggestive of a peroxo-to-iron(III) charge-transfer transition and resemble those of peroxodiiron(III) intermediates characterized in other carboxylate-bridged diiron proteins and synthetic model complexes. The second identified intermediate, Q*, forms on the pathway of Q decay when reactions are performed in the absence of hydrocarbon substrate. Q* does not react with methane, forms independently of buffer composition, and displays a unique shoulder at 455 nm in its optical spectrum. Studies conducted at different pH values reveal that rate constants corresponding to P* decay/H[subscript peroxo] formation and H[subscript peroxo] decay/Q formation are both significantly retarded at high pH and indicate that both events require proton transfer. The processes exhibit normal kinetic solvent isotope effects (KSIEs) of 2.0 and 1.8, respectively, when the reactions are performed in D[subscript 2]O. Mechanisms are proposed to account for the observations of these novel intermediates and the proton dependencies of P* to H[subscript peroxo] and H[subscript peroxo] to Q conversion. |
| first_indexed | 2024-09-23T10:08:22Z |
| format | Article |
| id | mit-1721.1/82145 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T10:08:22Z |
| publishDate | 2013 |
| publisher | American Chemical Society (ACS) |
| record_format | dspace |
| spelling | mit-1721.1/821452022-09-26T15:57:31Z Revisiting the Mechanism of Dioxygen Activation in Soluble Methane Monooxygenase from M. capsulatus (Bath): Evidence for a Multi-Step Proton-Dependent Reaction Pathway Tinberg, Christine E. Lippard, Stephen J. Massachusetts Institute of Technology. Department of Chemistry Tinberg, Christine E. Lippard, Stephen J. Stopped-flow kinetic investigations of soluble methane monooxygenase (sMMO) from M. capsulatus (Bath) have clarified discrepancies that exist in the literature regarding several aspects of catalysis by this enzyme. The development of thorough kinetic analytical techniques has led to the discovery of two novel oxygenated iron species that accumulate in addition to the well-established intermediates H[subscript peroxo] and Q. The first intermediate, P*, is a precursor to H[subscript peroxo] and was identified when the reaction of reduced MMOH and MMOB with O[subscript 2] was carried out in the presence of ≥540 μM methane to suppress the dominating absorbance signal due to Q. The optical properties of P* are similar to those of H[subscript peroxo], with ε[subscript 420] = 3500 M[superscript −1] cm[superscript −1] and ε[subscript 720] = 1250 M[superscript −1] cm[superscript −1]. These values are suggestive of a peroxo-to-iron(III) charge-transfer transition and resemble those of peroxodiiron(III) intermediates characterized in other carboxylate-bridged diiron proteins and synthetic model complexes. The second identified intermediate, Q*, forms on the pathway of Q decay when reactions are performed in the absence of hydrocarbon substrate. Q* does not react with methane, forms independently of buffer composition, and displays a unique shoulder at 455 nm in its optical spectrum. Studies conducted at different pH values reveal that rate constants corresponding to P* decay/H[subscript peroxo] formation and H[subscript peroxo] decay/Q formation are both significantly retarded at high pH and indicate that both events require proton transfer. The processes exhibit normal kinetic solvent isotope effects (KSIEs) of 2.0 and 1.8, respectively, when the reactions are performed in D[subscript 2]O. Mechanisms are proposed to account for the observations of these novel intermediates and the proton dependencies of P* to H[subscript peroxo] and H[subscript peroxo] to Q conversion. National Institute of General Medical Sciences (U.S.) (Grant GM032134) National Institutes of Health (U.S.) (Interdepartmental Biotechnology Training Grant T32 GM08334) 2013-11-15T20:08:13Z 2013-11-15T20:08:13Z 2009-11 2009-11 Article http://purl.org/eprint/type/JournalArticle 0006-2960 1520-4995 http://hdl.handle.net/1721.1/82145 Tinberg, Christine E., and Stephen J. Lippard. “Revisiting the Mechanism of Dioxygen Activation in Soluble Methane Monooxygenase from M. capsulatus (Bath): Evidence for a Multi-Step, Proton-Dependent Reaction Pathway.” Biochemistry 48, no. 51 (December 29, 2009): 12145-12158. https://orcid.org/0000-0002-2693-4982 en_US http://dx.doi.org/10.1021/bi901672n Biochemistry Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf American Chemical Society (ACS) PMC |
| spellingShingle | Tinberg, Christine E. Lippard, Stephen J. Revisiting the Mechanism of Dioxygen Activation in Soluble Methane Monooxygenase from M. capsulatus (Bath): Evidence for a Multi-Step Proton-Dependent Reaction Pathway |
| title | Revisiting the Mechanism of Dioxygen Activation in Soluble Methane Monooxygenase from M. capsulatus (Bath): Evidence for a Multi-Step Proton-Dependent Reaction Pathway |
| title_full | Revisiting the Mechanism of Dioxygen Activation in Soluble Methane Monooxygenase from M. capsulatus (Bath): Evidence for a Multi-Step Proton-Dependent Reaction Pathway |
| title_fullStr | Revisiting the Mechanism of Dioxygen Activation in Soluble Methane Monooxygenase from M. capsulatus (Bath): Evidence for a Multi-Step Proton-Dependent Reaction Pathway |
| title_full_unstemmed | Revisiting the Mechanism of Dioxygen Activation in Soluble Methane Monooxygenase from M. capsulatus (Bath): Evidence for a Multi-Step Proton-Dependent Reaction Pathway |
| title_short | Revisiting the Mechanism of Dioxygen Activation in Soluble Methane Monooxygenase from M. capsulatus (Bath): Evidence for a Multi-Step Proton-Dependent Reaction Pathway |
| title_sort | revisiting the mechanism of dioxygen activation in soluble methane monooxygenase from m capsulatus bath evidence for a multi step proton dependent reaction pathway |
| url | http://hdl.handle.net/1721.1/82145 https://orcid.org/0000-0002-2693-4982 |
| work_keys_str_mv | AT tinbergchristinee revisitingthemechanismofdioxygenactivationinsolublemethanemonooxygenasefrommcapsulatusbathevidenceforamultistepprotondependentreactionpathway AT lippardstephenj revisitingthemechanismofdioxygenactivationinsolublemethanemonooxygenasefrommcapsulatusbathevidenceforamultistepprotondependentreactionpathway |