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A non-radical mechanism for methane hydroxylation at the diiron active site of soluble methane monooxygenase
ips between T~t~arne~ Hem
4 ber~~~.~egulated ~~~~~s~~~~~es~e~ase (PDE) from Escherichia coli (ECDOS) is rebwxxic. Each Xloil(jrIc? is zolnposed of
an ~,~-ter~~i~al sensor domain (amino acids: l-201) containing two PAS domains (PAS-A: 2 i- 4 and PASB: 144201) and a catalytic domain (336407). Heme is bound to the PAS-A domain, and the redox state ofthe heme iron regulates FIX activity. We examined tile relationships between tetramer formation, beme binding and FIX activi:y using deletion mutants. Deletion ofthe PAS-B domain resu!ted in loss of heme bin ing affinity :o PAS-A. However9 the mutant protein was &ill tetrameric and significantly active compared to its wild-type counterpart, suggesting that the ?AS-R domain plays an Important role in modulating the appropriate structure of the PAS-A domain to maintain binding to ihe heme and that heme 9inding is not essential for catalysis. An N-terminal truncated mutant Qamii3o acids: 14-807) was active, c~rI~r~9jI3g that rhe ~~~r~~-bou~~ PAS domain is not essential for catalysis. C-Terminal truncated mutants (amino aci s: i -490 or 1-7&q were moreomeric, indicating that specitic amino acids located within the deleted regions are responsible for ie:zamer formation. in view ofthe finding that the isolated PAS-A domain is dimeric, these amino acids in the C-terminal region appear to play 3 major role in enzyme architecture. Qne C-terminal truncated mutant (I-490) did not display any PIX activity, whereas anoihei mutant (l-605) wa.s active, indicating that the catalytic site is located between positions 491 a*?d605. In addition, :etrmer fmmdion appears to be important in ECDOS catalysis. C-termirral
In tnis ~~rese~ta~~o~ we propose a non-radical mechanism for the conversion of rne~~a~eto and ay soluble ~me~~arre rnQ~QQxygel3ase (5 O), the active site of which involves a diiron active center. We assume the active site ofthe MM ~nt~~ediate that has a direct reactivity to substrate methane to e a bis(~-oxo)diiro~~I~~ hare one of-the iron s coordinat~vely unsaturated (five-coordinate). Is such a di on complex seasonablyfo ? The answer to this important question is positive from the viewpoint of energeti calculations. Thus, our model has a vacant coordination site for substrate me~ba~e.If involves a c~o~di~ati~~~y ~~~sat~ratediron atom at the active center, methane is effectively conveyed into metbano~ in n-sy singlet state in a non-radical mechanism; in the first step a C-H bond ~fmetbane is dissociated via a ered cm state {T’S1) resulting in an important intermediate that involves a hydroxo iigand and a met the second step the binding of the methyl ligand and the hydroxo ligand via a three-centered transition state (TS2) results in the ~~~rnat~~~ of a “metba~o~complex. This mechanism is essentially identical to t at of the metbane-methanol co~v~rsi~~ by the bare F lex and relevant transition-metal-ox0 complexes in the gas p ase.~eit~~~ radical speciesnor ionic speciesare invoa in this mecba~ism. We look in detail at kinetic isotope effects (ICI&) for the straction from methane on the basis ~~ft~a~s~tio~ state theory with Wigner tunneling corrections.
4 ber~~~.~egulated ~~~~~s~~~~~es~e~ase (PDE) from Escherichia coli (ECDOS) is rebwxxic. Each Xloil(jrIc? is zolnposed of
an ~,~-ter~~i~al sensor domain (amino acids: l-201) containing two PAS domains (PAS-A: 2 i- 4 and PASB: 144201) and a catalytic domain (336407). Heme is bound to the PAS-A domain, and the redox state ofthe heme iron regulates FIX activity. We examined tile relationships between tetramer formation, beme binding and FIX activi:y using deletion mutants. Deletion ofthe PAS-B domain resu!ted in loss of heme bin ing affinity :o PAS-A. However9 the mutant protein was &ill tetrameric and significantly active compared to its wild-type counterpart, suggesting that the ?AS-R domain plays an Important role in modulating the appropriate structure of the PAS-A domain to maintain binding to ihe heme and that heme 9inding is not essential for catalysis. An N-terminal truncated mutant Qamii3o acids: 14-807) was active, c~rI~r~9jI3g that rhe ~~~r~~-bou~~ PAS domain is not essential for catalysis. C-Terminal truncated mutants (amino aci s: i -490 or 1-7&q were moreomeric, indicating that specitic amino acids located within the deleted regions are responsible for ie:zamer formation. in view ofthe finding that the isolated PAS-A domain is dimeric, these amino acids in the C-terminal region appear to play 3 major role in enzyme architecture. Qne C-terminal truncated mutant (I-490) did not display any PIX activity, whereas anoihei mutant (l-605) wa.s active, indicating that the catalytic site is located between positions 491 a*?d605. In addition, :etrmer fmmdion appears to be important in ECDOS catalysis. C-termirral
In tnis ~~rese~ta~~o~ we propose a non-radical mechanism for the conversion of rne~~a~eto and ay soluble ~me~~arre rnQ~QQxygel3ase (5 O), the active site of which involves a diiron active center. We assume the active site ofthe MM ~nt~~ediate that has a direct reactivity to substrate methane to e a bis(~-oxo)diiro~~I~~ hare one of-the iron s coordinat~vely unsaturated (five-coordinate). Is such a di on complex seasonablyfo ? The answer to this important question is positive from the viewpoint of energeti calculations. Thus, our model has a vacant coordination site for substrate me~ba~e.If involves a c~o~di~ati~~~y ~~~sat~ratediron atom at the active center, methane is effectively conveyed into metbano~ in n-sy singlet state in a non-radical mechanism; in the first step a C-H bond ~fmetbane is dissociated via a ered cm state {T’S1) resulting in an important intermediate that involves a hydroxo iigand and a met the second step the binding of the methyl ligand and the hydroxo ligand via a three-centered transition state (TS2) results in the ~~~rnat~~~ of a “metba~o~complex. This mechanism is essentially identical to t at of the metbane-methanol co~v~rsi~~ by the bare F lex and relevant transition-metal-ox0 complexes in the gas p ase.~eit~~~ radical speciesnor ionic speciesare invoa in this mecba~ism. We look in detail at kinetic isotope effects (ICI&) for the straction from methane on the basis ~~ft~a~s~tio~ state theory with Wigner tunneling corrections.