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^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.