Angular dependences of perpendicular and parallel mode electron paramagnetic resonance of oxidized beef heart cytochrome c oxidase

Cytochrome c oxidase catalyzes the reduction of oxygen to water with a conc omitant conservation of energy in the form of a transmembrane proton gradie nt. The enzyme has a catalytic site consisting of a binuclear center of a c opper ion and a heme group. The spectroscopic parameters of this center are unusual. The origin of broad electron paramagnetic resonance (EPR) signals in the oxidized state at rather low resonant field, the so-called g' = 12 signal, has been a matter of debate for over 30 years, We have studied the angular dependence of this resonance in both parallel and perpendicular mod e X-band EPR in oriented multilayers containing cytochrome c oxidase to res olve the assignment. The "slow" form and compounds formed by the addition o f formate and fluoride to the oxidized enzyme display these resonances, whi ch result from transitions between states of an integer-spin multiplet aris ing from magnetic exchange coupling between the five unpaired electrons of high spin Fe(III) heme a(3) and the single unpaired electron of Cu-B. The f irst successful simulation of similar signals observed in both perpendicula r and parallel mode X-band EPR spectra in frozen aqueous solution of the fl uoride compound of the closely related enzyme, quinol oxidase or cytochrome bo(3), has been reported recently (Oganesyan et at., 1998, J. Am. Chem. Se c. 120:4232-4233). This suggested that the exchange interaction between the two metal ions of the binuclear center is very weak (\J\ approximate to 1 cm(-1)), with the axial zero-field splitting (D approximate to 5 cm(-1)) of the high-spin heme dominating the form of the ground state. We show that t his model accounts well for the angular dependences of the X-band EPR spect ra in both perpendicular and parallel modes of oriented multilayers of cyto chrome c oxidase derivatives and that the experimental results are inconsis tent with earlier schemes that use exchange coupling parameters of several hundred wavenumbers.