FIGURE 5.

Oxidation-induced destabilization of helices in EF-hand by oxidized methionine (not shown) results in altered conformation of EF-loop that affects Ca²⁺ liganding. Alterations in Ca²⁺ liganding involve modifications in hydrogen bonding (dashed lines) and repositioning of a water molecule (red arrow) that displaces hydrogen-bonding interaction between Asn⁹ and the chelating water ligand. Changes in binding involve diminished hydrogen bonding between Asp¹ and the backbone (CaM_ox, thin dashed line), apparent as an upshift in monodentate Asy COO⁻ absorbance from 1593(−) cm⁻¹ to 1602(+) cm⁻¹ (Fig. 4 B), which may result from destabilization of the EF-loop (not shown). This also permits the formation of a bidentate ligation of Ca²⁺ by Asp³ (CaM_ox, yellow line), and a pseudobridging interaction between the available noncoordinating oxygen atom of Asp⁵ (CaM_ox, thick dashed line), apparent as a downshift from 1580(−) cm⁻¹ to 1549(+) cm⁻¹ and 1580(−) cm⁻¹ to 1570(+) cm⁻¹, respectively (Fig. 4 B). Model depicts Ca²⁺ binding site IV (1exr.pdb) (59), with generic numbering of Ca²⁺ ligands (Fig. 1) before (CaM) and after (CaM_ox) Met oxidation.