Copper X-ray absorption spectra of cytochrome oxidase as a means of characterizing the preparation

Two preparations are used in these studies: 1) intact beef heart mitochondria as isolated from the tissue and 2) ''submitochondrial particles'' (SMP) obtained by sonication of the beef heart mitochondria accompanied by differential centrification. Beamline I-5 providing approximately 1010 photons per second and using Si-220 monochromator crystals were used together with a high efficiency X-ray fluorescence detection system. These samples were studied at -1000C in order to minimize the mobility of hydrated electrons and radical intermediates produced. The redox state was assayed periodically and it was found that the rate of cytochrome c reduction in these systems was reduced over 103 in the low temperature technique. Comparison of the copper edge as well as the EXAFS data for preparations using the Yonetani method show that these preparations are identical within the error to these data for SMF and mitochondria preparations. On reduction with CO these similarities remain. Preparations using the Hartzell-Beinert method showed significant differences in both the copper edge and EXAFS data. Thus, the Yonetani method produces a purified preparation which is homogeneous (greater than 85%) and structurally identical to cytochrome oxidase in submitochrondria particles and mitochrondria. The determination of the copper edge spectrum is adequate to characterize the preparation and determine whether the preparation is appropriate for further study. Supported in part by NIH Grants GM 33165, GM31992, HL31909