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 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 spectroscopic studies have shown that MMOHQ employs antiferromagnetic coupling of the two FeIV sites to yield a diamagnetic cluster. Unfortunately, this lack of net spin prevents the determination of the local spin state (Sloc) 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 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 Mössbauer quantification. Comparisons of the MMOHQ spectrum with those of known Sloc = 1 and Sloc = 2 FeIV sites in chemical and biological models reveal that MMOHQ possesses Sloc = 2 iron sites. This experimental determination of the local spin state will help guide future computational and mechanistic studies of sMMO catalysis.Graphical abstract