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 IV 2 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 IV 2 intermediate. Previous 57 Fe 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. Graphical abstract

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