Vanadyl(IV) conalbumin. I. Metal binding site configurations

Abstract

Electron paramagnetic resonance (epr) and ultraviolet difference spectroscopy of vanadyl conalbumin indicate a binding capacity of two vanadyl ions, VO2+, per protein molecule in the pH 8–11 range; the binding capacity drops in the pH 6–8 range with an apparent pKa′ = 6.6. Iron-saturated conalbumin does not bind vanadyl ions, which suggests common binding sites for iron and vanadium. Ultraviolet difference spectroscopy indicates 2–3 tyrosines are involved in the binding of each metal ion; pH titrations show that three protons are released per vanadyl ion bound by conalbumin. Room and liquid nitrogen temperature X-band (ca. 9.2–9.5 gHz) epr spectra show that the vanadyl ion binds in three magnetically distinct environments (A, B, and C) that arise from interconvertible metal site configurations. These configurations are probably examples of conformational substrates of the protein. Q-band (ca 34 gHz) epr spectra resolve the spectral features more clearly and show that two configurations (A and B) have axially symmetric epr parameters but angles of noncoincidence of 12° and 8°, respectively, between the z components of the g and nuclear hyperfine tensors. The third (C) configuration has rhombic magnetic symmetry and a 6° angle of noncoincidence. These observations demonstrate that the metal sites are of low symmetry and are flexible in their geometry about the metal.The isotropic g and nuclear hyperfine tensor values and the line widths used in computer-simulated epr spectra are consistent with four oxygen or three oxygen and one nitrogen donor atoms binding equatorially to the VO2+ group. The apparent stability constant indicates that vanadyl ion binds to conalbumin approximately twelve orders of magnitude more weakly than iron to human serotransferrin but still sufficiently strongly to overcome hydrolysis.