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. 2011 Jun 7;50(22):5067-76.
doi: 10.1021/bi101668x. Epub 2011 May 11.

X-ray absorption spectroscopy structural investigation of early intermediates in the mechanism of DNA repair by human ABH2

Nitai Charan Giri  1 , Hong Sun, Haobin Chen, Max Costa, Michael J Maroney
Affiliations

X-ray absorption spectroscopy structural investigation of early intermediates in the mechanism of DNA repair by human ABH2

Nitai Charan Giri et al. Biochemistry. .

Abstract

Human ABH2 repairs DNA lesions by using an Fe(II)- and αKG-dependent oxidative demethylation mechanism. The structure of the active site features the facial triad of protein ligands consisting of the side chains of two histidine residues and one aspartate residue that is common to many non-heme Fe(II) oxygenases. X-ray absorption spectroscopy (XAS) of metallated (Fe and Ni) samples of ABH2 was used to investigate the mechanism of ABH2 and its inhibition by Ni(II) ions. The data are consistent with a sequential mechanism that features a five-coordinate metal center in the presence and absence of the α-ketoglutarate cofactor. This aspect is not altered in the Ni(II)-substituted enzyme, and both metals are shown to bind the cofactor. When the substrate is bound to the native Fe(II) complex with α-ketoglutarate bound, a five-coordinate Fe(II) center is retained that features an open coordination position for O(2) binding. However, in the case of the Ni(II)-substituted enzyme, the complex that forms in the presence of the cofactor and substrate is six-coordinate and, therefore, features no open coordination site for oxygen activation at the metal.

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Figures

Figure 1
Figure 1
Demethylation of 3- methylcytosine showing the step catalyzed by ABH2.
Figure 2
Figure 2
Top: Fe K-Edge XANES spectra; Fe(ABH2) (brown), Fe(ABH2)-αKG (red) and Fe(ABH2)- αKG + substrate (orange). Bottom: Ni K-Edge XANES spectra; Ni(ABH2) (blue), Ni(ABH2)-αKG (green) and Ni(ABH2)-αKG + substrate (purple). Inserts: Expansions of the pre-edge XANES region showing peaks associated with 1s → 3d electronic transitions.
Figure 3
Figure 3
EXAFS analysis. Left: Unfiltered, k3-weighted EXAFS spectra (colored lines) and fits (black lines, from Table 1). Right: Fourier-transformed EXAFS data and fits.
Figure 4
Figure 4
Comparison of the complexes of ascorbate and α-ketoglutarate with the Fe(II) site in ABH2.
Figure 5
Figure 5
Proposed structures of the Fe and Ni sites of ABH2. M-ABH2 (left), in presence of αKG (middle), and in presence of both αKG and substrate (right). Iron is brown, Nickel cyan, carbon gray, oxygen red and nitrogen blue.
Scheme 1
Scheme 1
Consensus mechanism of nonheme Fe(II) oxygenases.
Scheme 2
Scheme 2
Variation of consensus mechanism for ABH2.
See this image and copyright information in PMC

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