title: Domain engineering of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath).
abstract: Soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath) is a three-component enzyme system that catalyzes the conversion of methane to methanol. A reductase (MMOR), which contains [2Fe-2S] and FAD cofactors, facilitates electron transfer from NADH to the hydroxylase diiron active sites where dioxygen activation and substrate hydroxylation take place. By separately expressing the ferredoxin (MMORFd, MMOR residues 1-98) and FAD/NADH (MMOR-FAD, MMOR residues 99-348) domains of the reductase, nearly all biochemical properties of full-length MMOR are retained, except for interdomain electron transfer rates. To investigate the extent to which rapid electron transfer between domains might be restored and further to explore the modularity of MMOR, MMOR-Fd and MMOR-FAD were connected in a non-native fashion. Four different linker sequences were employed to create MMOR reversed-domain (MMOR-RD) constructs, MMOR(99-342)-linker-MMOR(2-98), with a domain connectivity observed in other homologous oxidoreductases. The optical, redox, and electron transfer properties of the four MMOR-RD proteins were characterized and compared with those of wild-type MMOR. The linker sequence plays a key role in controlling solvent accessibility to the FAD cofactor, as evidenced by perturbed flavin optical spectra, decreased FADox/FADsq redox potentials, and increased steady-state oxidase activities in three of the constructs. Stopped-flow optical spectroscopy revealed slow interdomain electron transfer (k < 0.04 s(-1) at 4 degrees C, compared with 90 s(-1) for wild-type MMOR) for all three MMOR-RD proteins with 7-residue linkers. A long (14-residue), flexible linker afforded much faster electron transfer between the FAD and [2Fe-2S] cofactors (k = 0.9 s(-1) at 4 degrees C).
authors: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors: Jessica L
authors: JL
authors: Blazyk
authors: Stephen J
authors: SJ
authors: Lippard
mesh_terms: D002384:Catalysis; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry; D004579:Electron Transport; D004583:Electrons; D005415:Flavins / Q000737:chemistry; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D020619:Methylococcus capsulatus / Q000201:enzymology*; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry; D013696:Temperature; D014466:Ultraviolet Rays
chemical_list: D005415:Flavins; D004247:DNA; D010105:Oxygenases; C021555:methane monooxygenase; D010100:Oxygen