Abstract
will not [ I] . In spite of a great deal of investigation of the magnetic properties of these centres, mainly with the aid of low temperature electron paramagnetic resonance (e.p.r.) spectroscopy [2], there are still uncertainties about the spin states of the heme components. In the fully oxidised form of the enzyme about 50% of the heme can be detected in the e.p.r. as the low spin ferric form. A minor component, about 2.3% of the total heme, is detectable as the typical g = 6 high spin ferric signal [3]. Furthermore, although both copper ions are believed to be cupric in the fully oxidised state, only one can be detected in the e.p.r. experiments [3]. It has now been demonstrated that variable temperature magnetic circular dichroism (m.c.d.) spectroscopy can distinguish the two spin states of both ferro and ferri hemoproteins [4,5]. Thus cyto- chrome c oxidase is an excellent subject to study with this technique. In this work we have made use of the variable temperature studies to identify the spin states of the heme components, using as a guide wherever possible, our low temperature data on met- and ferro- myoglobin derivatives [.5]. We have recently become aware of an apparently similar study carried out with this enzyme by Babcock et al. [6,7]. 2. Materials and methods Cytochrome oxidase (EC 1.9.3.1. ferrocytochrome 94 c - oxygen oxidoreductase) was prepared by a modifica- tion of the method due to Yonetani [S] , in which 10 pm EDTA was incorporated as a chelating agent, into the last fractionation steps to ensure the removal of adventitiously bound copper. The concentration of the cytochrome oxidase samples (in terms of heme
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