COBALT(II)-SUBSTITUTED LIMULUS-POLYPHEMUS HEMOCYANIN - COBALT EQUILIBRIA, LIGAND-BINDING, AND OXYGENATION CHEMISTRY

Co(II)-substituted Limulus polyphemus hemocyanin has been prepared and characterized. The apoprotein binds up to seven Co(II)’s per molecule; two of these are in the active site, and up to five are adventitiously bound in peripheral sites. The binding of Co(II) in the active site is cooperative with an equilibrium constant of 109.34 M−2. The adventitious site equilibrium constant is 104.13 M−1 for each of five independent binding sites. While a Co(II)-substituted hemocyanin containing only active-site cobalt was not prepared, the active-site cobalt is associated with intense visible absorption with a maximum at 566 nm (є = 470 M−1 cm−1), and the adventitiously bound cobalts contribute insignificantly to any visible absorption. The optical spectrum of the active-site Co(II) is consistent with each Co(II) bound to three imidazole nitrogens and a fourth, exchangeable ligand, in approximate C3v symmetry. The active-site Co(II)’s bind a single hydroxide, chloride, or azide, bridging between the cobalts. Co(II)-substituted hemocyanin reversibly binds dioxygen, generating peroxide-to-cobalt charge-transfer transitions at 319 and 404 nm. The preliminary kinetics of oxygenation of Co(II)-substituted hemocyanin show that the rate is dependent upon [OH−] to the first order. The chemistry, kinetics, and spectroscopy of the oxygenated derivative are very similar to μ-peroxo, μ-hydroxo dibridged Co(III) complexes. The evidence from the study of Co(II)-substituted L. polyphemus hemocyanin supports the hypothesis that native oxyhemocyanin has a μ-hydroxo endogenous bridging ligand. © 1990, American Chemical Society. All rights reserved.