Methane monooxygenase and compound Q: lessons in oxygen activation

Abstract

Methane monooxygenase (MMO) catalyzes the NADH and O 2 coupled oxidation of CH 4 to yield CH 3OH. The enzyme consists of three components: MMOH (hydroxylase), MMOB (effector), and MMOR (reductase). We have structurally characterized MMOH and MMOB. The active site is located in MMOH and contains a bis-μ-hydroxo-bridged diiron cluster that is responsible for O 2 activation and substrate oxidation. Transient kinetic studies have revealed many reaction cycle intermediates and allowed some of them to be characterized. The unique intermediate Q contains a bis-μ-oxo-Fe(IV)Fe(IV) cluster and can directly oxidize CH 4. Large deuterium isotope effects are observed for the reaction of Q with methane but, remarkably, not ethane. Arrhenius plots are nonlinear for the CH 4 reaction with Q but not for other substrates. We propose that the Q reaction with substrates occurs in two steps, substrate binding and CH bond cleavage, with the latter rate limiting only for CH 4. Mutagenesis of MMOB shows that it affects the kinetics throughout the reaction cycle. One mutant dramatically alters the rates of Q decay and product release according to substrate size. It is proposed that MMOB serves to gate substrates into the active site of MMOH, thereby allowing it to select methane from more readily oxidized substrates.