Dataset Information

Initial investigations of C4a-(hydro)peroxyflavin intermediate formation by dibenzothiophene monooxygenase.

ABSTRACT: Dibenzothiophene monooxygenase is the initiating enzyme in the Rhodococcus 4S biodesulfurization pathway. A member of the Class D flavin monooxygenases, it uses FMN to activate molecular oxygen for oxygenation of the substrate, dibenzothiophene. Here, we have used stopped-flow spectrophotometry to show that DszC forms a peroxyflavin intermediate in the absence of substrate. Mutagenesis of Ser163 and His391 to Ala appears to decrease the binding affinity for reduced FMN and eliminates the enzyme's ability to stabilize the peroxyflavin intermediate.

SUBMITTER: Gonzalez-Osorio L

PROVIDER: S-EPMC5118162 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Initial investigations of C4a-(hydro)peroxyflavin intermediate formation by dibenzothiophene monooxygenase.

Gonzalez-Osorio Liliana L Luong Kelvin K Jirde Samatar S Palfey Bruce A BA Vey Jessica L JL

Biochemical and biophysical research communications 20161101 1-2

Dibenzothiophene monooxygenase is the initiating enzyme in the Rhodococcus 4S biodesulfurization pathway. A member of the Class D flavin monooxygenases, it uses FMN to activate molecular oxygen for oxygenation of the substrate, dibenzothiophene. Here, we have used stopped-flow spectrophotometry to show that DszC forms a peroxyflavin intermediate in the absence of substrate. Mutagenesis of Ser163 and His391 to Ala appears to decrease the binding affinity for reduced FMN and eliminates the enzyme' ...[more]

PMID: 27815073

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Project description: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 Fe(II)Fe(II) state (H(red)). O₂ binds rapidly at a site away from the cluster to form the Fe(II)Fe(II) intermediate O, which converts to an Fe(III)Fe(III)-peroxo intermediate P and finally to the Fe(IV)Fe(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 H(red) 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 H(red) 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 Fe(II)Fe(II) state. Thus, the loss of the g = 16 EPR signal results from a change in the electronic structure of the Fe(II)Fe(II) center rather than oxidation. The similarity of the optical and Mössbauer spectra of H(red), 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.

Dataset Information

Initial investigations of C4a-(hydro)peroxyflavin intermediate formation by dibenzothiophene monooxygenase.

Publications

Initial investigations of C4a-(hydro)peroxyflavin intermediate formation by dibenzothiophene monooxygenase.

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