Intermediate P* from soluble methane monooxygenase contains a diferrous cluster

Rahul Banerjee, Katlyn K. Meier, Eckard Münck, John D Lipscomb

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

During a single turnover of the hydroxylase component (MMOH) of soluble methane monooxygenase from Methylosinus trichosporium OB3b, several discrete intermediates are formed. The diiron cluster of MMOH is first reduced to the FeIIFeII state (Hred). O2 binds rapidly at a site away from the cluster to form the FeIIFe II intermediate O, which converts to an FeIIIFe III-peroxo intermediate P and finally to the FeIVFe IV intermediate Q. Q binds and reacts with methane to yield methanol and water. The rate constants for these steps are increased by a regulatory protein, MMOB. Previously reported transient kinetic studies have suggested that an intermediate P* forms between O and P in which the g = 16 EPR signal characteristic of the reduced diiron cluster of Hred and O is lost. This was interpreted as signaling oxidation of the cluster, but a low level of accumulation of P* prevented further characterization. In this study, three methods for directly detecting and trapping P* are applied together to allow its spectroscopic and kinetic characterization. First, the MMOB mutant His33Ala is used to specifically slow the decay of P* without affecting its formation rate, leading to its nearly quantitative accumulation. Second, spectra-kinetic data collection is used to provide a sensitive measure of the formation and decay rate constants of intermediates as well as their optical spectra. Finally, the substrate furan is included to react with Q and quench its strong chromophore. The optical spectrum of P* closely mimics those of Hred and O, but it is distinctly different from that of P. The reaction cycle rate constants allowed prediction of the times for maximal accumulation of the intermediates. Mössbauer spectra of rapid freeze-quench samples at these times show that the intermediates are formed at almost exactly the predicted levels. The Mössbauer spectra show that the diiron cluster of P*, quite unexpectedly, is in the FeIIFeII state. Thus, the loss of the g = 16 EPR signal results from a change in the electronic structure of the FeIIFeII center rather than oxidation. The similarity of the optical and Mössbauer spectra of Hred, O, and P* suggests that only subtle changes occur in the electronic and physical structure of the diiron cluster as P* forms. Nevertheless, the changes that do occur are necessary for O2 to be activated for hydrocarbon oxidation.

Original languageEnglish (US)
Pages (from-to)4331-4342
Number of pages12
JournalBiochemistry
Volume52
Issue number25
DOIs
StatePublished - Jun 25 2013

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methane monooxygenase
Rate constants
Oxidation
Kinetics
Paramagnetic resonance
Methylosinus trichosporium
Methane
Chromophores
Hydrocarbons
Mixed Function Oxygenases
Electronic structure
Methanol
Water
Substrates
Proteins

Cite this

Intermediate P* from soluble methane monooxygenase contains a diferrous cluster. / Banerjee, Rahul; Meier, Katlyn K.; Münck, Eckard; Lipscomb, John D.

In: Biochemistry, Vol. 52, No. 25, 25.06.2013, p. 4331-4342.

Research output: Contribution to journalArticle

Banerjee, Rahul ; Meier, Katlyn K. ; Münck, Eckard ; Lipscomb, John D. / Intermediate P* from soluble methane monooxygenase contains a diferrous cluster. In: Biochemistry. 2013 ; Vol. 52, No. 25. pp. 4331-4342.
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abstract = "During a single turnover of the hydroxylase component (MMOH) of soluble methane monooxygenase from Methylosinus trichosporium OB3b, several discrete intermediates are formed. The diiron cluster of MMOH is first reduced to the FeIIFeII state (Hred). O2 binds rapidly at a site away from the cluster to form the FeIIFe II intermediate O, which converts to an FeIIIFe III-peroxo intermediate P and finally to the FeIVFe IV intermediate Q. Q binds and reacts with methane to yield methanol and water. The rate constants for these steps are increased by a regulatory protein, MMOB. Previously reported transient kinetic studies have suggested that an intermediate P* forms between O and P in which the g = 16 EPR signal characteristic of the reduced diiron cluster of Hred and O is lost. This was interpreted as signaling oxidation of the cluster, but a low level of accumulation of P* prevented further characterization. In this study, three methods for directly detecting and trapping P* are applied together to allow its spectroscopic and kinetic characterization. First, the MMOB mutant His33Ala is used to specifically slow the decay of P* without affecting its formation rate, leading to its nearly quantitative accumulation. Second, spectra-kinetic data collection is used to provide a sensitive measure of the formation and decay rate constants of intermediates as well as their optical spectra. Finally, the substrate furan is included to react with Q and quench its strong chromophore. The optical spectrum of P* closely mimics those of Hred and O, but it is distinctly different from that of P. The reaction cycle rate constants allowed prediction of the times for maximal accumulation of the intermediates. M{\"o}ssbauer spectra of rapid freeze-quench samples at these times show that the intermediates are formed at almost exactly the predicted levels. The M{\"o}ssbauer spectra show that the diiron cluster of P*, quite unexpectedly, is in the FeIIFeII state. Thus, the loss of the g = 16 EPR signal results from a change in the electronic structure of the FeIIFeII center rather than oxidation. The similarity of the optical and M{\"o}ssbauer spectra of Hred, O, and P* suggests that only subtle changes occur in the electronic and physical structure of the diiron cluster as P* forms. Nevertheless, the changes that do occur are necessary for O2 to be activated for hydrocarbon oxidation.",
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T1 - Intermediate P* from soluble methane monooxygenase contains a diferrous cluster

AU - Banerjee, Rahul

AU - Meier, Katlyn K.

AU - Münck, Eckard

AU - Lipscomb, John D

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N2 - During a single turnover of the hydroxylase component (MMOH) of soluble methane monooxygenase from Methylosinus trichosporium OB3b, several discrete intermediates are formed. The diiron cluster of MMOH is first reduced to the FeIIFeII state (Hred). O2 binds rapidly at a site away from the cluster to form the FeIIFe II intermediate O, which converts to an FeIIIFe III-peroxo intermediate P and finally to the FeIVFe IV intermediate Q. Q binds and reacts with methane to yield methanol and water. The rate constants for these steps are increased by a regulatory protein, MMOB. Previously reported transient kinetic studies have suggested that an intermediate P* forms between O and P in which the g = 16 EPR signal characteristic of the reduced diiron cluster of Hred and O is lost. This was interpreted as signaling oxidation of the cluster, but a low level of accumulation of P* prevented further characterization. In this study, three methods for directly detecting and trapping P* are applied together to allow its spectroscopic and kinetic characterization. First, the MMOB mutant His33Ala is used to specifically slow the decay of P* without affecting its formation rate, leading to its nearly quantitative accumulation. Second, spectra-kinetic data collection is used to provide a sensitive measure of the formation and decay rate constants of intermediates as well as their optical spectra. Finally, the substrate furan is included to react with Q and quench its strong chromophore. The optical spectrum of P* closely mimics those of Hred and O, but it is distinctly different from that of P. The reaction cycle rate constants allowed prediction of the times for maximal accumulation of the intermediates. Mössbauer spectra of rapid freeze-quench samples at these times show that the intermediates are formed at almost exactly the predicted levels. The Mössbauer spectra show that the diiron cluster of P*, quite unexpectedly, is in the FeIIFeII state. Thus, the loss of the g = 16 EPR signal results from a change in the electronic structure of the FeIIFeII center rather than oxidation. The similarity of the optical and Mössbauer spectra of Hred, O, and P* suggests that only subtle changes occur in the electronic and physical structure of the diiron cluster as P* forms. Nevertheless, the changes that do occur are necessary for O2 to be activated for hydrocarbon oxidation.

AB - During a single turnover of the hydroxylase component (MMOH) of soluble methane monooxygenase from Methylosinus trichosporium OB3b, several discrete intermediates are formed. The diiron cluster of MMOH is first reduced to the FeIIFeII state (Hred). O2 binds rapidly at a site away from the cluster to form the FeIIFe II intermediate O, which converts to an FeIIIFe III-peroxo intermediate P and finally to the FeIVFe IV intermediate Q. Q binds and reacts with methane to yield methanol and water. The rate constants for these steps are increased by a regulatory protein, MMOB. Previously reported transient kinetic studies have suggested that an intermediate P* forms between O and P in which the g = 16 EPR signal characteristic of the reduced diiron cluster of Hred and O is lost. This was interpreted as signaling oxidation of the cluster, but a low level of accumulation of P* prevented further characterization. In this study, three methods for directly detecting and trapping P* are applied together to allow its spectroscopic and kinetic characterization. First, the MMOB mutant His33Ala is used to specifically slow the decay of P* without affecting its formation rate, leading to its nearly quantitative accumulation. Second, spectra-kinetic data collection is used to provide a sensitive measure of the formation and decay rate constants of intermediates as well as their optical spectra. Finally, the substrate furan is included to react with Q and quench its strong chromophore. The optical spectrum of P* closely mimics those of Hred and O, but it is distinctly different from that of P. The reaction cycle rate constants allowed prediction of the times for maximal accumulation of the intermediates. Mössbauer spectra of rapid freeze-quench samples at these times show that the intermediates are formed at almost exactly the predicted levels. The Mössbauer spectra show that the diiron cluster of P*, quite unexpectedly, is in the FeIIFeII state. Thus, the loss of the g = 16 EPR signal results from a change in the electronic structure of the FeIIFeII center rather than oxidation. The similarity of the optical and Mössbauer spectra of Hred, O, and P* suggests that only subtle changes occur in the electronic and physical structure of the diiron cluster as P* forms. Nevertheless, the changes that do occur are necessary for O2 to be activated for hydrocarbon oxidation.

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