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

Mononuclear (Co-II-Hc) and dinuclear (Co-2(II)-Hc) Co(II) substituted derivatives of Carcinus maenas hemocyanin (He) 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-2(II)-Hc, Co-2(II)-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-2(II)-Hc and Co-2(II)-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-2(II)-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-2(II)-Hc to form a Co-III-O-2(-)-Co-III complex and hence dioxygen-binding to Co-2(II)-Hc. Kinetic investigation of the reaction of dinuclear Co-2(II)-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.