Nancy C. Eickman

Preparation and characterization of met apo hemocyanin: A single copper(II) active site

Abstract A new derivative of Busycon canaliculatum hemocyanin has been prepared for which one copper has been removed from the binuclear active site of the holoprotein and the remaining copper has been oxidized with a variety of small molecule oxidizing agents. This met apo derivative [( )…Cu(II)] binds a number of ligands; EPR spectra of several forms are reported and compared to those obtained for a singly oxidized (half met-L) derivative [Cu(I)…Cu(II)L]. The site of the oxidized copper for both forms is found to be quite similar in structure but shows large differences in ligand binding ability.

Reactions and interconversion of met and dimer hemocyanin

Abstract The regenerable methemocyanin of Busycon canaliculatum is shown to be EPR-nondetectable. The small EPR signal present in met preparations is a nonregenerable binuclear cupric unit accounting for ∼ 6% of the active sites. A variety of anions are found to bind to met with the reactions being complicated by both reduction and competitive binding of buffer. The metastable dimer hemocyanin is shown to rearrange either directly to EPR-nondectable met or through an EPR-detectable regenerable binuclear cupric form obtained on reaction of dimer hemocyanin with azide. These results combined with previous results on half met derivatives are used to support the presence of an endogenous protein bridge between the two coppers in hemocyanin.

Spectroscopic studies of ligand perturbation effects on the half oxidized active site of busycon canaliculatum hemocyanin

We report the preparation and characterization of an important new series of hemocyanin derivatives containing a variety of anions bound to the cupric ion in a singly oxidized active site. Spectroscopic studies on these half met-L derivatives, Cu(I)…Cu(II)-L, where L = CN−, NO2−, CH3CO2−, and N3− reveal that the unpaired electron is localized on one copper in an approximately tetragonal site with a dx2 − y2 ground state. Ligand variation is used as a perturbation on the spectral features to show that the small molecule (and by analogy, oxygen in oxyhemocyanin) is bound in the equatorial plane of the copper.

Ligand displacement reactions of oxyhemocyanin: comparison of reactivities of arthropods and molluscs.

Abstract The ligand displacement reactions of oxyhemocyanin have been compared over a series of arthropods and molluscs. The arthropods (with the exception of Limulus ) are found to be more reactive than the molluscs, (kcancer = .04 hr−1, kbusycon = .002 hr−1, klimulus ⪡ 10−4hr−1 N3− reactions). Correlation of the spectral properties of the oxy sites require these to be extremely similar with small differences being associated with shifts in the dd transition energies. The met produced by ligand displacement contains variable amounts of EPR detectable, group 2 exogenous ligand damaged sites (arthropods 25–35%, molluscs 3–9%, Limulus mollusc ∼ Limulus ∼ 0) group 2 ligand damaged site is also reported for the half met derivatives.

Chemical and spectroscopic conformation of an exogenous ligand bridge in half met hemocyanin

Abstract Half met-N 3 − hemocyanin is shown to undergo a unique change at the Cu(II)⋯Cu(I) active site with temperature, exhibiting class II mixed valent properties at low temperature (The appearance of an intense near IR intervalence-transfer transition and a delocalized EPR spectrum). This requires a Cu(II)ue5f8Nue5fbNue5fbNue5f8Cu(I) bridging geometry. The effects of CO coordination to half met-N 3 − , combined with the presence of a low energy N 3 − → Cu(II) charge transfer transition, demonstrate that azide is also bridging at room temperature. Finally, half met-N 3 − is found to be capable of coordination of a second N 3 − at the copper(II) site.