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

Soluble methane monooxygenase (sMMO) facilitates the conversion of methane to methanol at a non-heme Fe intermediate MMOH, 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 sites in catalysis; however, MMOH is unique as Nature's only identified Fe intermediate. Previous Fe Mössbauer spectroscopic studies have shown that MMOH employs antiferromagnetic coupling of the two Fe sites to yield a diamagnetic cluster. Unfortunately, this lack of net spin prevents the determination of the local spin state (S) 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 trapped by rapid-freeze-quench techniques. A pure XES spectrum of MMOH is obtained by subtraction of the contributions from other reaction cycle intermediates with the aid of Mössbauer quantification. Comparisons of the MMOH spectrum with those of known S = 1 and S = 2 Fe sites in chemical and biological models reveal that MMOH possesses S = 2 iron sites. This experimental determination of the local spin state will help guide future computational and mechanistic studies of sMMO catalysis.