Determination of the iron(IV) local spin states of the Q intermediate of soluble methane monooxygenase by Kβ X-ray emission spectroscopy
George E. Cutsail, R. Banerjee, Derek B. Rice, Olivia McCubbin Stepanic, John D. Lipscomb, Serena DeBeer
Abstract
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