Analysis of the Cob(II)alamin-5'-Deoxy-3',4'-anhydroadenosyl Radical Triplet Spin System in the Active Site of Diol Dehydrase
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- 作者：Steven O. Mansoorabadi ; Olafur Th. Magnusson ; Russell R. Poyner ; Perry A. Frey ; George H. Reed
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：December 5, 2006
- 年：2006
- 卷：45
- 期：48
- 页码：14362 - 14370
- 全文大小：297K
- 年卷期：v.45,no.48(December 5, 2006)
- ISSN：1520-4995
文摘
A triplet spin system (S = 1) is detected by low-temperature electron paramagnetic resonance(EPR) spectroscopy in samples of diol dehydrase and the functional adenosylcobalamin (AdoCbl) analogue5'-deoxy-3',4'-anhydroadenosylcobalamin (anAdoCbl). Different spectra are observed in the presence andabsence of the substrate (R,S)-1,2-propanediol. In both cases, the spectra include a prominent half-fieldtransition (
hars/Delta.gif" BORDER=0 >MS = 2) that is a hallmark of strongly coupled triplet spin systems. The appearance of 59Cohyperfine splitting in the EPR signals and the positions (g values) of the signals in the spectra show thathalf of the triplet spin is contributed by the low-spin Co2+ of cob(II)alamin. Line width effects fromisotopic labeling (13C and 2H) in the 5'-deoxy-3',4'-anhydroribosyl ring demonstrate that the other half ofthe spin triplet is from an allylic 5'-deoxy-3',4'-anhydroadenosyl (anhydroadenosyl) radical. The zero-field splitting (ZFS) tensors describing the magnetic dipole-dipole interactions of the component spinsof the triplets have rhombic symmetry because of electron spin delocalization within the organic radicalcomponent and the proximity of the radical to the low-spin Co2+. The dipole-dipole interaction wasmodeled as a summation of point-dipole interactions involving the spin-bearing orbitals of theanhydroadenosyl radical and cob(II)alamin. Geometries which are consistent with the ZFS tensors in thepresence and absence of the substrate position the 5'-carbon of the anhydroadenosyl radical 3.5 and 4.1Å from Co2+, respectively. Homolytic cleavage of the cobalt-carbon bond of the analogue in the absenceof the substrate indicates that, in diol dehydrase, binding of the coenzyme to the protein weakens thebond prior to binding of the substrate.
hars/Delta.gif" BORDER=0 >MS = 2) that is a hallmark of strongly coupled triplet spin systems. The appearance of 59Cohyperfine splitting in the EPR signals and the positions (g values) of the signals in the spectra show thathalf of the triplet spin is contributed by the low-spin Co2+ of cob(II)alamin. Line width effects fromisotopic labeling (13C and 2H) in the 5'-deoxy-3',4'-anhydroribosyl ring demonstrate that the other half ofthe spin triplet is from an allylic 5'-deoxy-3',4'-anhydroadenosyl (anhydroadenosyl) radical. The zero-field splitting (ZFS) tensors describing the magnetic dipole-dipole interactions of the component spinsof the triplets have rhombic symmetry because of electron spin delocalization within the organic radicalcomponent and the proximity of the radical to the low-spin Co2+. The dipole-dipole interaction wasmodeled as a summation of point-dipole interactions involving the spin-bearing orbitals of theanhydroadenosyl radical and cob(II)alamin. Geometries which are consistent with the ZFS tensors in thepresence and absence of the substrate position the 5'-carbon of the anhydroadenosyl radical 3.5 and 4.1Å from Co2+, respectively. Homolytic cleavage of the cobalt-carbon bond of the analogue in the absenceof the substrate indicates that, in diol dehydrase, binding of the coenzyme to the protein weakens thebond prior to binding of the substrate.