Characterization of a Succinyl-CoA Radical-Cob(II)alamin Spin Triplet Intermediate in the Reaction Catalyzed by Adenosylcobalamin-Dependent Methylmalonyl-CoA Mutase

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• 作者：Steven O. Mansoorabadi ; Rugmini Padmakumar ; Nisso Fazliddinova ; Monica Vlasie ; Ruma Banerjee ; and George H. Reed
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：March 8, 2005
• 年：2005
• 卷：44
• 期：9
• 页码：3153 - 3158
• 全文大小：203K
• 年卷期：v.44,no.9(March 8, 2005)
• ISSN：1520-4995

文摘

The electron paramagnetic resonance (EPR) spectrum of an intermediate freeze trapped duringthe steady state of the reaction catalyzed by the adenosylcobalamin (AdoCbl)-dependent enzyme,methylmalonyl-CoA mutase, has been studied. The EPR spectrum is that of a hybrid triplet spin systemcreated as a result of strong electron-electron spin coupling between an organic radical and the low-spinCo²⁺ in cob(II)alamin. The spectrum was analyzed by simulation to obtain the zero-field splitting (ZFS)parameters and Euler angles relating the radical-to-cobalt interspin vector to the g axis system of thelow-spin Co²⁺. Labeling of the substrate with ¹³C and ²H was used to probe the identity of the organicradical partner in the triplet spin system. The patterns of inhomogeneous broadening in the EPR signalsproduced by [2'-¹³C]methylmalonyl-CoA and [2-¹³C]methylmalonyl-CoA as well as line narrowing resultingfrom deuterium substitution in the substrate were consistent with those expected for a succinyl-CoA radicalwherein the unpaired electron was centered on the carbon hars/alpha.gif" BORDER=0> to the free carboxyate group of the rearrangedradical. The interspin distance and the Euler angles were used to position this product radical into theactive site of the enzyme.