Heme—Heme interaction in cytochrome c oxidase in situ as measured by EPR spectroscopy

Abstract

The electron paramagnetic resonance (epr) properties of the components of cytochrome oxidase in mitochondria and phosphorylating submitochondrial particles has been examined in samples at liquid helium temperature. When the preparations are aerobic the g 6 signal is very small but in anaerobic samples a g 6 signal appears with an apparent half-reduction potential at pH 7 ( E m7 ) of 380 mV and disappears with an E m7 of 230 mV (cytochrome a). This g 6 signal represents an equilibrium mixture of two high-spin heme components with different symmetries for the heme environment. The g 3 signal is maximal in aerobic samples and disappears as the potential is lowered. The g 3 signal consists of at least two components, one with E m7 of 380 mV (cytochrome 3) and the other 230 mV (cytochrome c). The addition of CO to reduced cytochrome a 3 changes oxidized cytochrome a from high spin ( g 6) to low spin ( g 3). The addition of azide changes the oxidized cytochrome a from high spin to low spin ( g 2.9) when cytochrome a 3 is reduced but not when cytochrome a 3 is oxidized. The epr detectable copper has an n value of 1.0 and an E m7 of 250 mV while the high potential iron-sulfur protein (not a component of cytochrome oxidase) has an n value of 1.0 and an E m7 of 280 mV.