Preparation of a spectral probe derivative of the hemocyanin biopolymer: effects of allosteric interactions on the coupled binuclear copper active site.

Abstract

A series ofderivatives for both the arthropod and mollusc hemocyanin biopolymers has been prepared; the derivatives contain a small fraction ofelectron paramagnetic resonancedetectable half-met [Cu(fl) Cu(I)] sites dispersed among the nondetectable oxy binuclear copper active sites. Upon deoxygenation, large changes in the electron paramagnetic resonance signal of these half-met spectral probe derivatives are observed, which are further adjusted by the heterotropic effectors Ca2+ and HW. The active site structural changes indicated by these spectral changes as the hemocyanins go from a relaxed to a tensed quaternary structure are then discussed.Hemocyanins, the oxygen transport proteins in the hemolymph of molluscs and arthropods, reversibly bind dioxygen with a stoichiometry ofone dioxygen per two copper ions. Under physiological conditions, the hemocyanins are present as highly aggregated biopolymers with a molecular architecture that differs dramatically between the two phyla. For the arthropods, a single subunit has a molecular weight of "'70,000 and contains one binuclear copper active site. At pH values below 8 and in the presence of Ca2+, these subunits aggregate into one, two, four, and in the case of Limulus polyphemus, up to eight hexamers.Alternatively, the smallest subunit in the molluscs contains about eight binuclear copper active sites (domains) covalently linked in a single polypeptide chain of "'400,000 daltons. In the mollusc Busycon canaliculatum, at pH less than 8, twenty chains aggregate to form a whole molecule that contains 160 active sites and appears as a cylindrical barrel under the electron microscope. When aggregated, the hemocyanins are highly cooperative in their oxygen binding, with Hill coefficients dependent on species, pH, and the presence of divalent cations (1,2).In general, current thermodynamic and kinetic theories of oxygen binding to the hemocyanins parallel [with small modifications (3, 4)] the allosteric model of hemoglobin. Deoxyhemocyanin has a "tensed" quaternary structure with a low oxygen affinity. Initial binding of oxygen to a limited number of active sites alters the successive oxygen affinities ofthe remaining sites by a ligand-induced conformational change of the protein (5) (i.e., oxygen is a homotropic effector); this results in the oxyhemocyanin having a "relaxed" quaternary structure with a high oxygen affinity. Protons and divalent cations act as heterotropic effectors, which, depending upon the species, stabilize either the tensed or relaxed quaternary structure.For hemoglobin, through both protein crystallography (6) and the detailed study of iron prophyrin model complexes (7), significant advances have been made in the development of a structural model that accounts for the active site changes related to these changes in oxygen affinity. Two basic spectroscopic approaches have been taken to relate this mechanical model to the hemoglobin tetramer. One approach involves the preparation of valence hybrids in which either the a or the (3 chains con...

EXAFS studies of binuclear copper site of oxy-, deoxy-, and metaquo-, metfluoro-, and metazidohemocyanin from arthropods and molluscs

“…1982, 104, 3607. formation change that is implied by the cooperativity in 02 binding displayed by He.35 Indeed, EPR studies of a spectral probe derivative of He (containing a small fraction of EPR-detectable half-met set) have demonstrated ligation changes associated with the transition from the relaxed (oxy) to the tense (deoxy) quaternary structures. 36 The results are consistent with greater steric competition between exogenous liquids and the endogenous bridge in the tense structure.…”

Copper Active Sites in Biology

“…335 When the adjacent sites are oxygenated, the EPR spectrum of the half-met spectral probe in both arthropod and mollusc Hc is similar to the half-met monomer in the presence of excess azide (Figure 54). Upon deoxygenation, the spectral probe converts to a class II mixed-valent state, resembling the nonexcess azide spectra (Figure 54 deoxy, compare to Figure 49B red), which converts back to the excess azide spectra upon re-oxygenation.…”

“…Direct evidence for active site structural perturbations arising from O 2 binding to adjacent functional units has come from the preparation of an Hc derivative denoted the “spectral probe”, where a small fraction of the active sites in the quaternary structure have been converted to the EPR-active half-met state . When the adjacent sites are oxygenated, the EPR spectrum of the half-met spectral probe in both arthropod and mollusk Hc’s is similar to that of the half-met monomer in the presence of excess azide (Figure ).…”