Spectroscopic probes of homotropic and heterotropic interactions of the hemocyanin active site

Abstract

Under physiological conditions, both arthropod and mollusc hemocyanins are assembled into highly aggregated biopolymers with a molecular architecture that differs dramatically between the two phyla. For arthropods, a single subunit has a molecular weight of ∼70,000 and contains one binuclear copper active site. At pH below 8 and in the presence of Ca 2+ these subunits aggregate, and in the case of Limulus polyphemus which is the arthropod discussed here, eight hexamers comprise the resultant biopolymer. For molluscs, the smallest subunit contains eight binuclear copper active sites covalently linked in a single polypeptide chain of ∼400,000 daltons. At pH less than 8, Busycon canaliculatum being representative, twenty chains aggregate to form a whole molecule. When aggregated, all of the hemocyanins are highly cooperative in their oxygen binding, with Hill coefficients dependent on pH and the presence of divalent cations. The cooperative oxygen binding can be interpreted as the equilibrium between a low oxygen affinity tensed quaternary structure (deoxy) and a high oxygen affinity relaxed quaternary structure (oxy), in which the conformational change is induced by the homotropic effector (O 2) and heterotropic effectors (H +, Ca 2+). We have been able to develop a direct spectroscopic approach to study these allosteric interactions at the active site level through the preparation of Spectral Probe (SP) derivatives of the hemocyanin biopolymer [1]. These SP derivatives contain a small fraction of EPR-detectable half met [Cu(II)Cu(I)] sites homogenously dispersed among the EPR nondetectable oxy binuclear copper active sites. Our studies demonstrate that deoxygenation of the dominant oxy site (homotropic effect) induces change in the hemocyanin quaternary structure which results in significant spectral and structural changes in the half met probe sites. The extensively defined and well-understood chemistry and spectroscopy of he half met derivatives [2], suggest that the changes in half met SP sites represent elimination of the exogenous ligand (due to the steric competition of endogenous ligand, OR − and rearrangement of the geometric structure at the copper site. Furthermore, Busycon hemocyanin SP derivatives show the regulatory role of the heterotropic effectors: where Ca 2+ can induce cooperativity by stabilizing the tensed quaternary structure, proton increases the oxygen affinity and stabilizes the relaxed quaternary structure. Finally, we use the SP derivatives to study the intersubunit and intrasubunit interactions in hemocyanin. We have also been able to probe the allosteric relationship between changes in the coupled binuclear copper site and the Ca 2+ binding site [3]. Here, we substitute Eu 3+ for Ca 2+ and study its resonance emitted f–f fluorescence. Direct excitation of Eu 3+ emission is accomplished by a pulsed laser source. Transition between the nondegenerate ground ( 7F o) and excited ( 5D o) states gives a single unsplit line. Since this transition energy is very sensitive to the environment of Eu(III), we are able to differentiate the bound Eu 3+ peak from the free Eu 3+ peak. Excited state life times of bound Eu 3+ measured in D 2O and H 2O permit a direct estimation of the number molecules bound to the Eu 3+ [4], thereby providing a probe to explore the changes at the hemocyanin Ca 2+ binding site related to deoxygenation [3].