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Free keywords:
C-H BOND; DIAMOND CORE; RIBONUCLEOTIDE REDUCTASE; ENZYMATIC OXIDATION;
ESCHERICHIA-COLI; CATALYTIC CYCLE; PHOTOSYSTEM-II; ACTIVE-SITE; FE-IV=O;
ACTIVATIONBiochemistry & Molecular Biology; Chemistry; Methane monooxygenase; Non-heme iron; Spin state; X-ray emission
spectroscopy; Rapid freeze-quench;
Abstract:
Soluble methane monooxygenase (sMMO) facilitates the conversion of methane to methanol at a non-heme Fe-2(IV) 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 Fe-IV sites in catalysis; however, MMOHQ is unique as Nature's only identified Fe-2(IV) intermediate. Previous Fe-57 Mossbauer spectroscopic studies have shown that MMOHQ 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 K beta X-ray emission spectroscopy (XES) to characterize the local spin states of the key intermediates of the sMMO catalytic cycle, including MMOHQ trapped by rapid-freeze-quench techniques. A pure XES spectrum of MMOHQ is obtained by subtraction of the contributions from other reaction cycle intermediates with the aid of Mossbauer quantification. Comparisons of the MMOHQ spectrum with those of known S-loc = 1 and S-loc = 2 Fe-IV sites in chemical and biological models reveal that MMOHQ 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.
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