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Determination of the iron(IV) local spin states of the Q intermediate of soluble methane monooxygenase by Kβ X-ray emission spectroscopy.

ABSTRACT: Soluble methane monooxygenase (sMMO) facilitates the conversion of methane to methanol at a non-heme Fe^IV₂ intermediate MMOH_Q, which is formed in the active site of the sMMO hydroxylase component (MMOH) during the catalytic cycle. Other biological systems also employ high-valent Fe^IV sites in catalysis; however, MMOH_Q is unique as Nature's only identified Fe^IV₂ intermediate. Previous ⁵⁷Fe Mössbauer spectroscopic studies have shown that MMOH_Q employs antiferromagnetic coupling of the two Fe^IV sites to yield a diamagnetic cluster. Unfortunately, this lack of net spin prevents the determination of the local spin state (S_loc) of each of the irons by most spectroscopic techniques. Here, we use Fe Kβ X-ray emission spectroscopy (XES) to characterize the local spin states of the key intermediates of the sMMO catalytic cycle, including MMOH_Q trapped by rapid-freeze-quench techniques. A pure XES spectrum of MMOH_Q is obtained by subtraction of the contributions from other reaction cycle intermediates with the aid of Mössbauer quantification. Comparisons of the MMOH_Q spectrum with those of known S_loc = 1 and S_loc = 2 Fe^IV sites in chemical and biological models reveal that MMOH_Q possesses S_loc = 2 iron sites. This experimental determination of the local spin state will help guide future computational and mechanistic studies of sMMO catalysis.

SUBMITTER: Cutsail GE

PROVIDER: S-EPMC9470658 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Determination of the iron(IV) local spin states of the Q intermediate of soluble methane monooxygenase by Kβ X-ray emission spectroscopy.

Cutsail George E GE Banerjee Rahul R Rice Derek B DB McCubbin Stepanic Olivia O Lipscomb John D JD DeBeer Serena S

Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 20220821 6

Soluble methane monooxygenase (sMMO) facilitates the conversion of methane to methanol at a non-heme FeIV2 intermediate MMOHQ, which is formed in the active site of the sMMO hydroxylase component (MMOH) during the catalytic cycle. Other biological systems also employ high-valent FeIV sites in catalysis; however, MMOHQ is unique as Nature's only identified FeIV2 intermediate. Previous 57Fe Mössbauer spectrosco ...[more]

PMID: 35988092

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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.

Project description:The controlled oxidation of methane to methanol is a chemical transformation of great value, particularly in the pursuit of alternative fuels, but the reaction remains underutilized industrially because of inefficient and costly synthetic procedures. In contrast, methane monooxygenase enzymes (MMOs) from methanotrophic bacteria achieve this chemistry efficiently under ambient conditions. In this Account, we discuss the first observable step in the oxidation of methane at the carboxylate-bridged diiron active site of the soluble MMO (sMMO), namely, the reductive activation of atmospheric O(2). The results provide benchmarks against which the dioxygen activation mechanisms of other bacterial multicomponent monooxygenases can be measured. Molecular oxygen reacts rapidly with the reduced diiron(II) cen-ter of the hydroxylase component of sMMO (MMOH). The first spectroscopically characterized intermediate that results from this process is a peroxodiiron(III) species, P*, in which the iron atoms have identical environments. P* converts to a second peroxodiiron(III) unit, H(peroxo), in a process accompanied by the transfer of a proton, probably with the assistance of a residue near the active site. Proton-promoted O-O bond scission and rearrangement of the diiron core then leads to a diiron(IV) unit, termed Q, that is directly responsible for the oxidation of methane to methanol. In one section of this Account, we provide a detailed discussion of these processes, with particular emphasis on possible structures of the intermediates. The geometries of P* and H(peroxo) are currently unknown, and recent synthetic modeling chemistry has highlighted the need for further structural characterization of Q, currently assigned as a di(μ-oxo)diiron(IV) "diamond core." In another section of the Account, we discuss in detail proton transfer during the O(2) activation events. The role of protons in promoting O-O bond cleavage, thereby initiating the conversion of H(peroxo) to Q, was previously a controversial topic. Recent studies of the mechanism, covering a range of pH values and in D(2)O instead of H(2)O, confirmed conclusively that the transfer of protons, possibly at or near the active site, is necessary for both P*-to-H(peroxo) and H(peroxo)-to-Q conversions. Specific mechanistic insights into these processes are provided. In the final section of the Account, we present our view of experiments that need to be done to further define crucial aspects of sMMO chemistry. Here our goal is to detail the challenges that we and others face in this research, particularly with respect to some long-standing questions about the system, as well as approaches that might be used to solve them.

Dataset Information

Determination of the iron(IV) local spin states of the Q intermediate of soluble methane monooxygenase by Kβ X-ray emission spectroscopy.

Publications

Determination of the iron(IV) local spin states of the Q intermediate of soluble methane monooxygenase by Kβ X-ray emission spectroscopy.

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