Circular Dichroism and Magnetic Circular Dichroism Spectroscopic Studies of the Non-Heme Ferrous Active Site in Clavaminate Synthase and Its Interaction with 
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- 作者：Elizabeth G. Pavel ; Jing Zhou ; Robert W. Busby ; Michele Gunsior ; Craig A. Townsend ; and Edward I. Solomon
- 刊名：Journal of the American Chemical Society
- 出版年：1998
- 出版时间：February 4, 1998
- 年：1998
- 卷：120
- 期：4
- 页码：743 - 753
- 全文大小：227K
- 年卷期：v.120,no.4(February 4, 1998)
- ISSN：1520-5126
文摘
Clavaminate synthase (CS) is one member of a large class ofnon-heme iron enzymes that require
-ketoglutarate (-KG) as a cosubstrate. While the majority ofthis class catalyzes the hydroxylation ofunactivated C-H bonds, CS is unusual in that in addition toperforming hydroxylation chemistry, it alsocatalyzes the key oxidative ring closure and desaturation steps in thebiosynthetic pathway to the potent
-lactamase inhibitor clavulanic acid. A single non-hemeFe2+ site is responsible for all three of thesereactions(hydroxylation, oxidative ring closure, and desaturation), during which1 equiv of -KG per reaction isdecarboxylated into succinate and CO2. We have appliedcircular dichroism (CD), magnetic circular dichroism(MCD), and variable-temperature, variable-field (VTVH) MCDspectroscopies to probe the geometric andelectronic structure of the ferrous active site in the isozyme CS2 andits interaction with -KG. CD titrationexperiments show stoichiometric binding of Fe2+ to theapoenzyme, either with or without -KG, as well asstoichiometric binding of -KG to the iron-containing enzyme.However, in the absence of the metal, the-KG binding constant is reduced, indicating that Fe2+facilitates cosubstrate binding at the active site.Ligandfield CD and MCD data show that resting CS2 contains a six-coordinateferrous center (10Dq = 10 050cm-1,
5Eg = 1690 cm-1)and that addition of -KG perturbs the site to produce a differentsix-coordinate center(10Dq = 9500 cm-1,5Eg = 1630cm-1). VTVH MCD analysis finds aground-state splitting for restingCS2 (5T2g 
-400cm-1) that is fairly typical ofsix-coordinate ferrous sites, but a much larger splittingforCS2 + -KG (5T2g -1000cm-1), indicative ofFe2+--KG 
interactions. UV/vis absorption,CD, andMCD spectroscopies have been applied to further probe the interactionof the cosubstrate with the metalloenzyme.These data show the appearance of low-lying metal-to-ligandcharge-transfer transitions which demonstratethat -KG binds directly to the iron. Furthermore, analysis andcomparison to model complex data supporta bidentate binding mode of -KG, indicating that cosubstratedisplaces two ligands from the six-coordinateresting active site to form a new six-coordinate -KG-boundFe2+ site. These results provide the firstdirectspectroscopic information about the nature of the CS2 ferrous activesite and its interaction with -KG andlend insight into the mechanism of this multifunctionalenzyme.
-ketoglutarate (-KG) as a cosubstrate. While the majority ofthis class catalyzes the hydroxylation ofunactivated C-H bonds, CS is unusual in that in addition toperforming hydroxylation chemistry, it alsocatalyzes the key oxidative ring closure and desaturation steps in thebiosynthetic pathway to the potent-lactamase inhibitor clavulanic acid. A single non-hemeFe2+ site is responsible for all three of thesereactions(hydroxylation, oxidative ring closure, and desaturation), during which1 equiv of -KG per reaction isdecarboxylated into succinate and CO2. We have appliedcircular dichroism (CD), magnetic circular dichroism(MCD), and variable-temperature, variable-field (VTVH) MCDspectroscopies to probe the geometric andelectronic structure of the ferrous active site in the isozyme CS2 andits interaction with -KG. CD titrationexperiments show stoichiometric binding of Fe2+ to theapoenzyme, either with or without -KG, as well asstoichiometric binding of -KG to the iron-containing enzyme.However, in the absence of the metal, the-KG binding constant is reduced, indicating that Fe2+facilitates cosubstrate binding at the active site.Ligandfield CD and MCD data show that resting CS2 contains a six-coordinateferrous center (10Dq = 10 050cm-1,5Eg = 1690 cm-1)and that addition of -KG perturbs the site to produce a differentsix-coordinate center(10Dq = 9500 cm-1,5Eg = 1630cm-1). VTVH MCD analysis finds aground-state splitting for restingCS2 (5T2g -400cm-1) that is fairly typical ofsix-coordinate ferrous sites, but a much larger splittingforCS2 + -KG (5T2g -1000cm-1), indicative ofFe2+--KG interactions. UV/vis absorption,CD, andMCD spectroscopies have been applied to further probe the interactionof the cosubstrate with the metalloenzyme.These data show the appearance of low-lying metal-to-ligandcharge-transfer transitions which demonstratethat -KG binds directly to the iron. Furthermore, analysis andcomparison to model complex data supporta bidentate binding mode of -KG, indicating that cosubstratedisplaces two ligands from the six-coordinateresting active site to form a new six-coordinate -KG-boundFe2+ site. These results provide the firstdirectspectroscopic information about the nature of the CS2 ferrous activesite and its interaction with -KG andlend insight into the mechanism of this multifunctionalenzyme.