Fully oxidized cytochrome bo(3) from Escherichia coli has been studied in its oxidized and several ligand-bound forms using electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopies. In each form, the spin-coupled high-spin Fe(III) heme o(3) and CUB(II) ion at the active site give rise to similar fast-relaxing broad features in the dual-mode X-band EPR spectra. Simulations of dual-mode spectra are presented which show that this EPR can arise only from a dinuclear site in which the metal ions are weakly coupled by an anisotropic exchange interaction of \J\ approximate to 1 cm(-1). A variable-temperature and magnetic field (VTVF) MCD study is also presented for the cytochrome bo(3) fluoride and azide derivatives. New methods are used to extract the contribution to the MCD of the spin-coupled active site in the presence of strong transitions from low-spin Fe(III) heme b. Analysis of the MCD data, independent of the EPR study, also shows that the spin-coupling within the active site is weak with \J\ approximate to 1 cm(-1). These conclusions overturn a long-held view that such EPR signals in bovine cytochrome c oxidase arise from an S = 2 ground state resulting from strong exchange coupling (\J\ > 10(2) cm(-1)) within the active site.