Electronic and Geometric Structure-function Correlations of the Coupled Binuclear Copper Active Site

Chemical—spectroscopic studies of the coupled bimuclear copper active site are presented which interpret the unique spectral features associated with oxyhemocyamim. These features are them used to generate a 'spectroscopically effective' model for this active site. First the met apo [Cu(II) — I and half met [Cu(II)Cu(I)J derivatives are spectro— scopically compared; they demonstrate that exogenous ligands bridge the two coppers in the mixed valent form providing a pathway for electron de— localization. Detailed spectral studies are presented which probe the ground state wavefunction. Next, the dimer and met [Cu(II)Cu(II)] deny— atives are compared to demonstrate that there is also am endogenous bridge between the two coppers. It provides a superexchamge pathway for the antiferromagnetic coupling and is responsible for the EPR—mondetect— able mature of the coupled binuclear cupnic site. Finally, the unique optical features of oxyhemocyanin are interpreted as charge transfer transitions from a p—l,2 peroxide bridge. These chemical—spectroscopic studies are then extended oven the metallo— proteins which contain a coupled bimuclear copper active site and are involved in a variety of biological functions. Spectral studies of hemo— cyanin derivatives from the arthropod and mollusc phyla indicate that there is a structural distortion present at the arthropod site. This distortion interferes with exogenous ligand binding and correlates with the lack of catalase activity. A "spectral probe" derivative which contains a small fraction of EPR—detectable half met sites in the hemocyamin biopolymer is reported. This derivative is used to probe changes at the active site induced by shifts from relaxed to tensed protein quaternary structure. Parallel chemical—spectroscopic studies on tyrosinase are presented which indicate that its active site is extremely similar to that of the hemocyanins but highly accessible for coordination of organic substrates. An active site structural mechanism for the monooxygenation reaction is then proposed. Finally, these studies are extended to the coupled binuclear copper active site in the multicopper oxidase, laccase. Deoxy [Cu(I)Cu(I)J, met [Cu(II)Cu(II)] and half met [Cu(II)Cu(I)] derivatives of the type—2—depleted enzyme form are studied; these allow correlation to the hemocyanins and tyrosinase. While the site is found to be similar with respect to the presence of an endogenous protein bridge and N and 0 ligation of the coppers, significant differences are observed which indicate that exogenous ligands do not bridge the two coppers. This appears to correlate to irreversible binding of the peroxide intermediate produced in the four—electron reduction of dioxygen to water. INTRODUCTION A coupled binuclear copper active site [1] exists in a variety of proteins and enzymes which perform different biological functions (Table 1). The hemocyanins reversibly bind dioxygen, exhibiting cooperative interactions between active sites in the protein biopolymer. The mol— lusc hemocyanins also exhibit catalase activity. Tyrosinase, in addition to reversibly binding dioxygen and dismutating peroxide, is also a monooxygenase capable of oxygenating a monophenol to an o—diphenol and further oxidizing this diphenol to the o—quinone. Finally, in the multicopper oxidases laccase, ceruloplasmin and ascorbic acid oxidase, there exists a coupled binuclear copper active site in addition to other types of copper centers. Together these sites couple four one—electron oxidations of substrate to the four—electron reduction of dioxygen to water. The theme of this talk will be to first develop our chemical—spectroscopic approach [1,2,3] 1069