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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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journal contribution
posted on 05.12.2006, 00:00 by Steven O. Mansoorabadi, Olafur Th. Magnusson, Russell R. Poyner, Perry A. Frey, George H. Reed
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) analogue 5‘-deoxy-3‘,4‘-anhydroadenosylcobalamin (anAdoCbl). Different spectra are observed in the presence and absence of the substrate (R,S)-1,2-propanediol. In both cases, the spectra include a prominent half-field transition (ΔMS = 2) that is a hallmark of strongly coupled triplet spin systems. The appearance of 59Co hyperfine splitting in the EPR signals and the positions (g values) of the signals in the spectra show that half of the triplet spin is contributed by the low-spin Co2+ of cob(II)alamin. Line width effects from isotopic labeling (13C and 2H) in the 5‘-deoxy-3‘,4‘-anhydroribosyl ring demonstrate that the other half of the 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 spins of the triplets have rhombic symmetry because of electron spin delocalization within the organic radical component and the proximity of the radical to the low-spin Co2+. The dipole−dipole interaction was modeled as a summation of point−dipole interactions involving the spin-bearing orbitals of the anhydroadenosyl radical and cob(II)alamin. Geometries which are consistent with the ZFS tensors in the presence and absence of the substrate position the 5‘-carbon of the anhydroadenosyl radical 3.5 and 4.1 Å from Co2+, respectively. Homolytic cleavage of the cobalt−carbon bond of the analogue in the absence of the substrate indicates that, in diol dehydrase, binding of the coenzyme to the protein weakens the bond prior to binding of the substrate.

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