Cytochrome oxidase and its derivatives. I. Carbon monoxide and cyanide compounds

Abstract

1. Cytochrome oxidase has been prepared from bovine heart muscle by simplifying modifications of the methods of Okunuki and Yonetani (without the use of trypsin), and of that of Smith & Stotz (with the use of trypsin). The latter method gave a preparation almost as pure as the first, but less stable. The absorption spectra in cholate-phosphate and Emasolphosphate have been studied in a Perkin-Elmer Recording Spectrophotometer. The spectra of the ferrous and ferric oxidase and the differential spectrum (ferrous minus ferric) were very similar to those reported by Griffiths & Wharton and by Yonetani and indicated that these preparations were somewhat purer spectroscopically than those of Smith and Wainio. 2. The gradual inhibition of the enzyme by repeated treatment with cholate during its purification and its reactivation by the non-ionic detergent Emasol have been confirmed. In Emasol the activity measured by the method of Smith at pH 5·8 is almost 10 times as high as at pH 7·0; in cholate the difference is far smaller. 3. The spectrum of the carbon monoxide compound and the differential spectrum (FeCO minus Fe 2+ ) are the same in Emasol and cholate. The relevance of these observations on theories of the nature of cytochrome a 3 is discussed. 4. The claim of Yonetani that the ferric cyanide compound of cytochrome a3 cannot be reduced by dithionite or p -phenylenediamine + ascorbate could not be confirmed. However, the rate of reduction is so slow, that his method of deducing the differential spectra (ferrous minus ferric) of cytochrome a and cytochrome a 3 separately can be used as approximation if certain precautions are applied. 5. Ferric cytochrome a 3 , in the absence of cyanide, is also reduced by dithionite much more slowly than cytochrome a . This method gives a γ/α ratio for the differential spectra (ferrous minus ferric) of 3·5 for cytochrome a , and of 14 to 26 for cytochrome a 3 . The difference is thus even greater than that found by Yonetani. 6. Increasing the concentration of cyanide by a factor of 50 does not alter the absorption curve of the ferrous cyanide oxidase. The comparatively minor and very similar alteration of the spectrum of the ferrous oxidase by cyanide and carbon monoxide in the visible part of the spectrum (shoulder at 590 nm) cannot therefore be ascribed to incomplete reaction which has sometimes been used to explain the carbon monoxide curve. The findings under 4-6 strongly support the existence of two different cytochromes a and a 3 in both the SmithStotz and the Okunuki-Yonetani preparation.