Cobalt(II) substituted derivatives of Carcinus maenas hemocyanin: Magnetic characterization, magnetooptic, and kinetic studies regarding the geometry of the active site

Mononuclear (CoII-Hc) and dinuclear (CoII2-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 CoII-Hc posseses a S ) 3/2 ground state, demonstrating that the optical properties are consistent with a distorted tetrahedral coordination geometry. The CoII 2-Hc, CoII 2-N3-Hc, and CoII-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 CoII 2-Hc and CoII 2-N3-Hc reveal strong antiferromagnetic coupling with jJj > 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 CoII2-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 CoII 2-Hc to form a CoIII-O2--CoIII complex and hence dioxygen-binding to CoII 2-Hc. Kinetic investigation of the reaction of dinuclear CoII2-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.