title: Expression and characterization of ferredoxin and flavin adenine dinucleotide binding domains of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath).
abstract: Soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath) catalyzes the selective oxidation of methane to methanol, the first step in the primary catabolic pathway of methanotrophic bacteria. A reductase (MMOR) mediates electron transfer from NADH through its FAD and [2Fe-2S] cofactors to the dinuclear non-heme iron sites housed in a hydroxylase (MMOH). The structurally distinct [2Fe-2S], FAD, and NADH binding domains of MMOR facilitated division of the protein into its functional ferredoxin (MMOR-Fd) and FAD/NADH (MMOR-FAD) component domains. The 10.9 kDa MMOR-Fd (MMOR residues 1-98) and 27.6 kDa MMOR-FAD (MMOR residues 99-348) were expressed and purified from recombinant Escherichia coli systems. The Fd and FAD domains have absorbance spectral features identical to those of the [2Fe-2S] and flavin components, respectively, of MMOR. Redox potentials, determined by reductive titrations that included indicator dyes, for the [2Fe-2S] and FAD cofactors in the domains are as follows: -205.2 +/- 1.3 mV for [2Fe-2S](ox/red), -172.4 +/- 2.0 mV for FAD(ox/sq), and -266.4 +/- 3.5 mV for FAD(sq/hq). Kinetic and spectral properties of intermediates observed in the reaction of oxidized MMOR-FAD (FAD(ox)) with NADH at 4 degrees C were established with stopped-flow UV-visible spectroscopy. Analysis of the influence of pH on MMOR-FAD optical spectra, redox potentials, and NADH reaction kinetics afforded pK(a) values for the semiquinone (FAD(sq)) and hydroquinone (FAD(hq)) MMOR-FAD species and two protonatable groups near the flavin cofactor. Electron transfer from MMOR-FAD(hq) to oxidized MMOR-Fd is extremely slow (k = 1500 M(-1) s(-1) at 25 degrees C, compared to 90 s(-1) at 4 degrees C for internal electron transfer between cofactors in MMOR), indicating that cofactor proximity is essential for efficient interdomain electron transfer.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Blazyk
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Lippard
mesh_terms: D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport / Q000235:genetics; D005287:Ferredoxin-NADP Reductase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D005288:Ferredoxins / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry*; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry*; D007700:Kinetics; D020619:Methylococcus capsulatus / Q000201:enzymology* / Q000235:genetics; D009243:NAD / Q000737:chemistry; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet
chemical_list: D005288:Ferredoxins; D011994:Recombinant Proteins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D010105:Oxygenases; C021555:methane monooxygenase; D005287:Ferredoxin-NADP Reductase