Literature summary for 1.14.13.25 extracted from

Jones, J.C.; Banerjee, R.; Shi, K.; Semonis, M.M.; Aihara, H.; Pomerantz, W.C.K.; Lipscomb, J.D.
Soluble methane monooxygenase component interactions monitored by 19F NMR (2021), Biochemistry, 60, 1995-2010 .

Search for more literature for 1.14.13.25

Cloned(Commentary)

Cloned (Comment)	Organism
plasmids encoding wild-type MMOB and W77F and K15C MMOB are transformed into Escherichia coli BL21(DE3) chemically competent cells	Methylosinus trichosporium

Crystallization (Commentary)

Crystallization (Comment)	Organism
analysis of the cyrstal structure of the sMMOH:5FWMMOB complex (PDB ID 7M8Q) showing the interface region containing 5FW76 and 5FW77, and of the structure of the sMMOH:BTFA-K15C-5FW-MMOB complex (PDB ID 7M8R) interface region showing the relative position of the BTFA and 5FW 19F-labels	Methylosinus trichosporium

Protein Variants

Protein Variants	Comment	Organism
K15C	site-directed mutagenesis in the MMOB component	Methylosinus trichosporium
additional information	the two tryptophan residues in MMOB and the single tryptophan residue in MMOR are converted to 5-fluorotryptophan (5FW) by expression in defined media containing 5-fluoroindole. In addition, the mechanistically significant N-terminal region of MMOB is 19F-labeled by reaction of the K15C variant with 3-bromo-1,1,1-trifluoroacetone (BTFA). The 5FW and BTFA modifications cause minimal structural perturbation. Resonances from the 275 kDa complexes of sMMOH with 5FW-MMOB and BTFAK15C-5FW-MMOB are readily detected at 5 microM labeled protein concentration. This approach shows directly that MMOR and MMOB competitively bind to sMMOH with similar KD values, independent of the oxidation state of the sMMOH diiron cluster	Methylosinus trichosporium
W77F	site-directed mutagenesis in the MMOB component	Methylosinus trichosporium

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	impact of the incorporation of 5-fluorotryptophan and BTFA on sMMO steady state and single-turnover kinetics	Methylosinus trichosporium

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Fe	the sMMOH active site contains a dinuclear iron cluster, which serves to activate molecular oxygen for insertion into the C-H bond of methane	Methylosinus trichosporium

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
245000	-	sMMO, (alphabetagamma)2	Methylosinus trichosporium

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
methane + NAD(P)H + H+ + O2	Methylosinus trichosporium	-	methanol + NAD(P)+ + H2O	-	?

Organism

Organism	UniProt	Comment	Textmining
Methylosinus trichosporium	A0A2D2D5X0 AND A0A2D2D0T8 AND Q53563 AND A0A2D2D0X7	MMOH, MMOR, MMOB, and MMOD	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
methane + NAD(P)H + H+ + O2	-	Methylosinus trichosporium	methanol + NAD(P)+ + H2O	-	?

Subunits

Subunits	Comment	Organism
trimer	soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme, all three sMMO protein components are: hydroxylase (MMOH), reductase (MMOR), and regulatory protein (MMOB), (alphabetagamma)2, 1 * 245000, hydroxylase (sMMOH), + 1 * 38000, flavin adenine dinucleotide (FAD) and 2Fe-2S cluster-containing reductase (MMOR) + 1 * 1000 cofactorless regulatory component (MMOB)	Methylosinus trichosporium

Synonyms

Synonyms	Comment	Organism
sMMO	-	Methylosinus trichosporium
soluble methane monooxygenase	-	Methylosinus trichosporium

Cofactor

Cofactor	Comment	Organism	Structure
FAD	in the 2Fe-2S cluster-containing reductase (MMOR)	Methylosinus trichosporium
NAD(P)H	-	Methylosinus trichosporium
[2Fe-2S]-center	in the 2Fe-2S cluster-containing reductase (MMOR)	Methylosinus trichosporium

General Information

General Information	Comment	Organism
additional information	soluble methane monooxygenase component interactions monitored by 19F NMR spectroscopy. Modeling for regulation in which the dynamic equilibration of MMOR and MMOB with sMMOH allows a transient formation of key reactive complexes that irreversibly pull the reaction cycle forward. The slow kinetics of exchange of the sMMOH:MMOB complex is proposed to prevent MMOR-mediated reductive quenching of the high-valent reaction cycle intermediate Q before it can react with methane	Methylosinus trichosporium
physiological function	soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme capable of catalyzing the conversion of methane to methanol at ambient temperature and pressure. The enzyme consists of three protein components: a 245 kDa (alphabetagamma)2 hydroxylase (sMMOH), a 38 kDa flavin adenine dinucleotide (FAD) and 2Fe-2S cluster-containing reductase (MMOR), and a 15 kDa cofactorless regulatory component (MMOB). The sMMOH active site contains a dinuclear iron cluster, which serves to activate molecular oxygen for insertion into the C-H bond of methane. The resting state of sMMOH contains a diferric cluster (Fe3+Fe3+, sMMOHox) in which the irons are bridged by two solvent (OH- or H2O) molecules in addition to the carboxylate of Glu144. sMMOHox can form a complex with MMOR and receive two electrons to form the diferrous cluster (Fe2+Fe2+, sMMOHred) in which Glu243 shifts to bridge the irons via one carboxylate oxygen, one bridging solvent is lost, and the bond to the second solvent is weakened. In this new configuration, the diiron cluster can bind O2 between the irons upon dissociation of the weakly bound solvent. But O2 binding is observed to be very slow in the absence of the regulatory component MMOB. Binding of MMOB effects a 1000fold increase in the rate constant for the O2 binding to the diiron cluster to form the first spectroscopically distinct intermediate of the reaction cycle, termed P. One cause of the decreased rate of O2 binding in the sMMOH active site in the absence of MMOB is the near closure of the molecular tunnel that mediates the transit of O2 from the solvent. This bottleneck is relieved by conformational changes in both MMOB and sMMOHred when the sMMOHred:MMOB complex forms. A second cause of the low reactivity of O2 with sMMOHred is the position of the Glu209 ligand to the diiron cluster, which blocks the approach to the open iron coordination site. An angle change of this residue in the sMMOHred:MMOB complex exposes the site for O2 binding. The formation of intermediate P is followed by a spontaneous formation of a peroxo-intermediate P, and finally, O-O bond cleavage to yield the reactive dinuclear Fe4+ intermediate Q. Q can react directly with methane to form methanol with the incorporation of one atom of oxygen sourced from O2. Intermediate Q is generated and stabilized by precisely coordinated sMMO protein component interactions. Regulation of electron transfer in the sMMO system, mechanism, modeling, detailed overview. MMOR causes both the N-terminal tail and the core region of MMOB to dissociate from sMMOH	Methylosinus trichosporium

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Release 2023.1 (January 2023)