Studies on the ferricytochrome a-ferrocytochrome a3-carbon monoxide complex of mammalian cytochrome oxidase. Conditions for preparation and some properties.

Abstract

The conditions for the preparation of the ferricytochrome a-ferrocytochrome a3-carbon monoxide complex (a3+, a3(2)+CO) of cytochrome oxidase [EC 1.9.3.1] by the ferricyanide-reoxidation method and some properties of the prepared complex were studied. The addition of a small volume of concentrated ferricyanide solution to the dithionite-reduced and carbon monoxide-treated cytochrome oxidase preparation was required to obtain the (a3+, a3(2)+CO) spectrum showing absorption maxima at 590, 545, and 429 nm. The addition of larger volumes of ferricyanide solution, thus introducing larger amounts of oxygen into the preparation, caused decomposition of the carbon monoxide complex. A part of the added ferricyanide was immediately reduced by dithionite whereas the remainder was gradually reduced by partial oxidation product(s) of dithionite. The (a3+, a3(2)+CO) complex was stable only when excess ferricyanide remained in the reaction mixture. The formation of the (a3+, a3(2)+CO) spectrum was observed when sodium citrate, phosphate or borate buffer containing either cholate or a non-ionic detergent was employed as the solvent buffer, but not with the buffers containing sodium dodecyl sulfate (SDS) or cetyltrimethyl-ammonium bromide (CETAB). The formation was considerably inhibited by trishydroxymethyl-aminomethane(Tris)-HCl buffer. The (a3+, a3(2)+CO) spectrum appeared with maximal intensity at around pH 7. The pH-dependency of the intensity of the spectrum was not in parallel with the pH-dependent change of the polymerization state of the cytochrome oxidase preparation. On freezing to liquid nitrogen temperature, the (a3+, a3(2)+CO) complex prepared in usual solvent buffers was mostly converted to the oxidized form of cytochrome oxidase (a3+, a3(3)+. However, when prepared in the phosphate buffer, pH 8.0, containing 1.2% (w/v) sodium cholate and with 20% saturation with ammonium sulfate, the complex mostly remained unchanged after the freezing. Based on the results obtained, the stability of the juxta-heme structure of cytochrome a3 was also discussed.