Cobalt(II) Substituted Derivatives ofCarcinus maenasHemocyanin:  Magnetic Characterization, Magnetooptic, and Kinetic Studies Regarding the Geometry of the Active Site

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Mononuclear (Co II −Hc) and dinuclear (Co II 2 −Hc) Co(II) substituted derivatives of Carcinus maenas hemocyanin (Hc) are characterized by magnetic susceptibility measurements, magnetic circular dichroism spectroscopy, and kinetic experiments with cyanide. Magnetization measurements up to 8 T reveal that the mononuclear Co II −Hc posseses a S = 3 / 2 ground state, demonstrating that the optical properties are consistent with a distorted tetrahedral coordination geometry. The Co II 2 −Hc, Co II 2 −N 3 −Hc, and Co II −Hc derivatives exhibit visible and near-infrared magnetic circular dichroism (MCD) signals and magnetic characteristics which also reflect tetrahedral coordination geometry. Magnetic susceptibility measurements of dinuclear Co II 2 −Hc and Co II 2 −N 3 −Hc reveal strong antiferromagnetic coupling with | J | > 100 cm -1, consistent with the existence of a ligand bridging the metal centers. The results of the magnetic susceptibility measurements together with the MCD data imply that the putative bridging ligand of the dinuclear Co II 2 −Hc can be substituted by small anions like azide. Otherwise a coordination number higher than 4 would have been achieved. No evidence for an octahedrally coordinated Co(III) species was obtained from magnetic or magnetooptical studies, ruling out the oxidation of Co II 2 −Hc to form a Co III −O 2 - −Co III complex and hence dioxygen-binding to Co II 2 −Hc. Kinetic investigation of the reaction of dinuclear Co II 2 −Hc with excess cyanide by stopped-flow spectrophotometry reveals that the protein adds cyanide in a fast initial equilibrium reaction to form an adduct. This is followed by slow cyanide-assisted removal of the metal ions from the active site in two steps.