Characterization of the partially reduced cyanide-inhibited derivative of cytochrome c oxidase by optical, electron-paramagnetic-resonance and magnetic-circular-dichroism spectroscopy

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Abstract
Optical, EPR and near IR low-temperature m.c.d. (magnetic-circular-dichroism) spectroscopy were used to characterize the partially reduced cyanide-inhibited derivative of [bovine heart] cytochrome c oxidase produced by anaerobic reductive titration with dithionite. The reductions of cytochrome a3+ and .**GRAPHIC**. were followed by observation of the EPR signals at g = 3.03, 2.21 and 1.5 and at g = 2.18, 2.03 and 1.99. As reduction proceeds new EPR signals (g = 3.58 and 1.56) appear that quantify to give 1 heme per enzyme unit when a small excess of dithionite has been titrated in. The EPR signal of the .**GRAPHIC**. titrates in parallel with the disappearance of the band at 820 nm in the optical absorption spectrum. The near-IR m.c.d. spectrum shows the presence of the low-spin ferric heme, a3+, in the oxidized state of the enzyme, as a well-resolved positive peak at 1650 nm. As reduction proceeds this band is replaced by one at 1550 nm due to heme .**GRAPHIC**. in the partially reduced state. Hence as heme .**GRAPHIC**. becomes EPR detectable it also shows a near IR m.c.d. spectrum characteristic of a low-spin ferric heme. It is concluded that the partially reduced state of cyanide-inhibited cytochrome c oxidase contains .**GRAPHIC**.

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