In these experiments the apo-form of reduced BsSCO was mixed with a CuCl2
solution in a 1:1 molar ratio. Although this reactant mixture is not ideal for kinetic analysis, that is not our primary concern here. Rather we kept to a low Cu(II):BsSCO ratio so as to minimize the contribution of “free Cu(II)” to the EPR spectrum. The EPR spectra observed at 10–140 ms under our experimental conditions has contributions from two spectral forms of Cu(II) in complex with BsSCO and this is consistent with our previous stopped-flow, transient absorption studies on the kinetics of Cu(II) binding to BsSCO [14
]. There is also a small amount of the equilibrium copper bound species that is present in the sample before mixing. This species is hard to avoid during handling of the sample due to the extremely high affinity of reduced, apo-BsSCO for Cu(II) [12
]. This equilibrium species is characterized by a superhyperfine splitting in the g
region that could be very well modeled as being due to a single equatorially coordinated nitrogen atom, and that is presumably derived from the coordinated histidine residue (i.e
., H135). Peisach and Blumberg analysis is entirely consistent with this conclusion, and also strongly suggests two equatorial coordinated sulfurs; there are two coordinated cysteines in BsSCO. Hence, the EPR spectroscopy of the equilibrium species of BsSCO faithfully predicts the salient features of the Cu(II) coordination sphere.
The second and more strongly contributing species is a new form. Although the difference spectrum that corresponds to the intermediate species exhibits poorly resolved structure in the g
region that hints at coordinated nitrogen, that region is complicated by extensive overlap with strong features from the other two species contributing to the experimental spectrum. In addition, even small Cu quadrupolar couplings manifest themselves as small features in this region when included in simulations. This structure cannot, therefore, be confidently assumed to indicate nitrogen coupling. On the other hand, the Peisach and Blumberg analysis is unambiguous in identifying that the Cu(II) ion in the intermediate species is equatorially coordinated by at least two nitrogens and not at all by sulfur. This coordination environment is markedly different from both CuCl2
(aq.) and the BsSCO equilibrium species.
It appears that Cu(II) is initially bound by nitrogens derived from BsSCO, most likely backbone peptide nitrogens, such as is observed at high Cu(II):protein ratios with the prion protein [22
]. This initial complex then evolves to the final form with two sulfur atoms, from the two conserved cysteine residues, as inner sphere ligands. We propose that the initial BsSCO:Cu complex with nitrogen ligation serves to prevent copper catalyzed oxidation of the thiol-containing residues of BsSCO. Such oxidation is observed for thiol-based reagents [8
] and for cysteine residues in other proteins [23
]. Spontaneous conversion of the BsSCO–Cu(II) complex to oxidized BsSCO plus Cu(I) has been observed at high ionic strength with the native protein [6
]. Redox conversion of the BsSCO–Cu(II) complex has also been observed for the H135A mutant of BsSCO at low ionic strength [24
BsSCO binds copper(II) in a thermodynamically tight (KD
~3 pM) and kinetically inert (koff
) complex. Progression to the equilibrium form takes only a couple of seconds at neutral pH with the isomerisation step occurring at a first order rate of 1.5 s−1
]. The thermodynamic stability is largely conferred by the energetics of the two-step binding process and slow kinetic exchange is determined by the off rate of the stable complex. At the observed formation rates if reduced BsSCO is exposed to Cu(II) in vivo the equilibrium species is likely to accumulate. One could view the equilibrium copper bound form of BsSCO as a potential trap and, if copper exchange is to occur, this trap must be escaped. If the rule of microscopic reversibility is applied then the intermediate characterized here represents a potential form of BsSCO that could be involved in metal exchange. However, conversion from the equilibrium, thiol-ligated form to the intermediate, nitrogen-ligated form would require an energetic input, as might be derived from the interaction of BsSCO with its target protein. The nitrogen-ligated intermediate does not have the stability of the final, thiol-ligated form, and yet does not undergo premature thiol oxidation as reported for the H135A mutant of BsSCO [24
]. The initial interaction between BsSCO and copper via nitrogen ligation may be critical to maintain the reduced, functional state of BsSCO.