Epr of Cu +2 binding to apo-yeast enolase

Abstract

We have studied the electron paramagnetic resonance (epr) spectra of complexes of apo-yeast enolase with 65Cu +2 in the presence and absence of substrate and magnesium ion. An unusual epr spectrum with large g , large g and A rhombicity and very narrow line-widths (10 G) is seen for the first two 65Cu +2 bound in the presence of substrate 2-phosphoglycerate (2PGA). The epr parameters, consistent with rhombic and tetragonal distortion of an octahedral geometry of the coordination sphere of the Cu +2 are g = (2.123, 2.042, 2.405) and A = (2.58, 4.19, 12.0) mK. The high g  and absence of superhyperfine splitting are strong evidence for absence of nitrogen ligands. In the presence of Mg +2 and 2PGA, the Cu +2-enolase solutions exhibit a complex epr spectrum reflecting exchange and dipolar interaction between the first two Cu +2 ions bound. The spectra of Cu +2 plus enolase in the presence and absence of Mg +2 without 2PGA are distinct but not unambiguous, each reflecting at least two inequivalent binding sites. In addition to providing information on the geometry and location of the divalent cation binding sites, the data show unequivolcally that imidazole residues, previously found to have a role in catalysis, do not participate in Cu +2 binding. Although Cu +2 does not activate the enzyme, direct binding measurements show that Cu +2 competes stoichiometrically with the activating ion, Mg +2. A reinterpretation of earlier Mn +2 enolase studies is proposed to reconcile the Cu +2 and Mn +2 data. The observations can be schematically summarized as in Figure 7, where a change in geometric symbol indicates a change in the magnetic properties of the Cu +2 at a given site. If one adds Cu +2 to apo-enolase one sees the same epr spectrum over the range 0 to 4 Cu +2 per dimer. As discussed above, the complexity of the spectrum suggests two types of sites, designated by circles and squares in the figure. It should be emphasized that we have little data from the epr on the second site, depicted by squares, so the square notation is continued throughout the scheme for simplicity. The evidence for Cu-Cu interaction here is ambiguous, although it is suggested by the excess of lines in epr. Addition of 2PGA dramatically changes the tight site to a clearly defined rhombic epr site that is unchanged by addition of subsequent copper ions. The binding site of copper on enolase apparently has exclusively oxygen ligands and there is interaction between the two first bound Cu +2 ions. The first bound Cu +2 ions assume a unique geometry when substrate is present. The epr parameters fit neither type 2 nor type 1 cases and are characterized by a rhombic g tensor and extremely sharp lines. These parameters are characteristic of rhombic and tetragonally elongated octahedral coordination of the Cu +2. Addition of Mg +2 alone to the Cu-enolase complex causes some small change in the tight site indicated by wavy circle and square. Subsequent addition of 2PGA causes a dramatic interaction between the first two sites as evidenced by exchange and dipolar interactions in the epr spectrum.