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High-Resolution Extended X-ray Absorption Fine Structure Analysis Provides Evidence for a Longer Fe···Fe Distance in the Q Intermediate of Methane Monooxygenase.

ABSTRACT: Despite decades of intense research, the core structure of the methane C-H bond breaking diiron(IV) intermediate, Q, of soluble methane monooxygenase remains controversial, with conflicting reports supporting either a "diamond" diiron core structure or an open core structure. Early extended X-ray absorption fine structure (EXAFS) data assigned a short 2.46 Å Fe-Fe distance to Q (Shu et al. Science 1997, 275, 515 ) that is inconsistent with several theoretical studies and in conflict with our recent high-resolution Fe K-edge X-ray absorption spectroscopy (XAS) studies (Castillo et al. J. Am. Chem. Soc. 2017, 139, 18024 ). Herein, we revisit the EXAFS of Q using high-energy resolution fluorescence-detected extended X-ray absorption fine structure (HERFD-EXAFS) studies. The present data show no evidence for a short Fe-Fe distance, but rather a long 3.4 Å diiron distance, as observed in open core synthetic model complexes. The previously reported 2.46 Å feature plausibly arises from a background metallic iron contribution from the experimental setup, which is eliminated in HERFD-EXAFS due to the increased selectivity. Herein, we explore the origin of the short diiron feature in partial-fluorescent yield EXAFS measurements and discuss the diagnostic features of background metallic scattering contribution to the EXAFS of dilute biological samples. Lastly, differences in sample preparation and resultant sample inhomogeneity in rapid-freeze quenched samples for EXAFS analysis are discussed. The presented approaches have broad implications for EXAFS studies of all dilute iron-containing samples.

SUBMITTER: Cutsail GE

PROVIDER: S-EPMC6470014 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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High-Resolution Extended X-ray Absorption Fine Structure Analysis Provides Evidence for a Longer Fe···Fe Distance in the Q Intermediate of Methane Monooxygenase.

Cutsail George E GE Banerjee Rahul R Zhou Ang A Que Lawrence L Lipscomb John D JD DeBeer Serena S

Journal of the American Chemical Society 20181116 48

Despite decades of intense research, the core structure of the methane C-H bond breaking diiron(IV) intermediate, Q, of soluble methane monooxygenase remains controversial, with conflicting reports supporting either a "diamond" diiron core structure or an open core structure. Early extended X-ray absorption fine structure (EXAFS) data assigned a short 2.46 Å Fe-Fe distance to Q (Shu et al. Science 1997, 275, 515 ) that is inconsistent with several theoretical studies and in conflict with our rec ...[more]

PMID: 30398343

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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

High-Resolution Extended X-ray Absorption Fine Structure Analysis Provides Evidence for a Longer Fe···Fe Distance in the Q Intermediate of Methane Monooxygenase.

Publications

High-Resolution Extended X-ray Absorption Fine Structure Analysis Provides Evidence for a Longer Fe···Fe Distance in the Q Intermediate of Methane Monooxygenase.

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