Soluble methane monooxygenase (sMMO) catalyzes the O₂-dependent oxidation of methane to methanol in methanotrophs. sMMO is a multiprotein complex composed of a hydroxylase protein (MMOH) harboring a diiron active site, a cofactorless regulatory protein (MMOB), and a FAD and [2Fe2S] cluster-containing reductase (MMOR). Transient complexes are formed between these three proteins for fully coupling O₂ activation with methane oxidation. Key to this regulation is electron delivery from MMOR to MMOH exclusively at the start of catalysis. A mid-cycle transfer would reductively quench the diiron(IV) intermediate Q generated within MMOH to break the strong methane C-H bond. Many regulatory effects of MMOB in complex with MMOH have also been identified, but perhaps the most important is to open a route into the closed active site to allow O₂ binding. In order to probe this network of sMMO protein binding events, a Protein-Observed ¹⁹F-NMR (PrOF) study was pursued by selectively replacing the native tryptophans of MMOB and MMOR with fluoro-tryptophan analogues. This avoids artifacts that arise from large fluorophore labels perturbing native binding affinities in typical fluorescence studies. The ability to track the formation of a large 275 kDa complex at protein concentrations as low as 5 μM depicts the sensitivity of the PrOF methodology. This study demonstrates that MMOB and MMOR bind in a competitive manner to the same site on MMOH with similar affinities that do not change with the different oxidation states of the MMOH iron atoms. A regulatory model is proposed where binary MMOH:MMOB and MMOH:MMOR complexes exist in equilibrium with catalysis being driven forward by irreversible steps of MMOR-mediated iron reduction and MMOB-mediated O₂ activation (Model). The observation that the catalytic cycle is completed during the dwell time of MMOB on MMOH prevents MMOR from binding MMOH mid-catalysis and quenching Q. This dynamic equilibrium model is perhaps used by many other enzymes that use electron transfer partner and regulatory proteins. The ability of PrOF ¹⁹F-NMR to study high-affinity protein-protein interactions in large complexes in a non-perturbative manner highlights the broad-applicability of this technique.

Model

Hideki Aihara

Appearances