Investigation of the structural basis of heterotropic effects in hemocyanin

Abstract

Hemocyanin (Hc) is a multisubunit copper protein whose physiological function involves the reversible binding of oxygen as a {dollar}\mu{dollar}-{dollar}\eta\sp2{lcub}:{rcub}\eta\sp2{dollar} peroxo bridge at a binuclear copper active site. In this study the structural features of the active site involved in the modulation of oxygen affinity are investigated by cw and pulsed electron paramagnetic resonance spectroscopies, and by x-ray absorption spectroscopy. A chemically modified subunit containing Cu(II) sites incorporated into a multimeric native (diamagnetic) Hc was used as paramagnetic probe. An investigation of the superhyperfine coupling pattern for both the metal bound nitrogen (by cw EPR) and the remote nitrogen (by pulsed EPR) of the copper ligand imidazoles was carried out. Upon dissociation of the multisubunit aggregate into monomers, a process that produces a decrease in oxygen affinity in the native protein, the nitrogen nuclear hyperfine coupling pattern changed from three equivalent imidazoles to two weakly and one strongly coupled imidazoles.;The effects on the coordination structure of Cu(I) of heterotropic agents such as pH, L-lactate and chloride in Panulirus interruptus deoxy-Hc and subunit II of Limulus polyphemus deoxy-Hcs were studied by k-edge x-ray absorption and extended x-ray absorption fine structure (EXAFS) spectroscopies. On the basis of the relationship established between the intensity of the 1s {dollar}\to{dollar} 4p{dollar}\sb{lcub}\rm z{rcub}{dollar} transition in the k-edge absorption spectrum and the coordination geometry of Cu(I) in several model complexes, the relationship between oxygen affinity and coordination geometry of Cu(I) in deoxy-Hcs was investigated. We have shown that the average coordination geometry of Cu(I) in deoxy-Hc changes from pseudotetrahedral to more trigonal either by raising pH or by adding chloride. Since high pH increases O{dollar}\sb2{dollar} affinity but chloride produces an opposite effect, it is suggested that O{dollar}\sb2{dollar} affinity in Hc may not be uniquely governed by Cu(I) coordination geometry in deoxy-Hc. Another structural feature of the binuclear active site of Hc investigated is the metal-metal distance. EXAFS data for the outer coordination shell of the oxy- and deoxy-Hcs were fitted using amplitude and phase functions derived from model compounds and theoretical calculations. A correlation between increased metal-metal distance and decreased oxygen affinity was found.