278 



Discussion 



Thus the ratio of cation to a hypothetical active site for reduced cytochrome c- 

 cytochrome oxidase interaction appears to be critical, that is, as the enzyme prepar- 

 ation is diluted at a fixed cation concentration more cytochrome c is required to obtain 

 half maximal activity. With cytochrome c oxidase activities as currently determined 

 spectrophotometrically the ratio of cation to hypothetical active site on the protein 

 would be very large indeed introducing a serious source of inhibition of the type 

 described above and would make the interpretation of the results of such assays most 

 difficult. This would also explain in part the inability to saturate the cytochrome 

 oxidase with cytochrome c as determined by such measurements. 



A second point bearing on the problem of cytochrome a and cytochrome 03 is the 

 unexplained observation concerning the steady-state of these pigments during succinate 



0-04 



o 



Ml 



370 400 430 460 490 



Wavelength, 

 Fig. 3. 



550 580 



m// 



oxidation by a cytochrome c deficient heart muscle preparation. Figure 3 shows the 

 difference spectra obtained when succinate is added to such a preparation. The dotted 

 line represents the steady-state obtained in the presence of succinate while the solid 

 hne is that recorded when a sample is anaerobic. As one would expect, the pigments 

 cytochrome b and c^, as determined from the magnitude of the absorption bands at 

 563 and 553 m/< respectively, are about 80 to 90 % reduced in the steady-state. Un- 

 explained is the large reduction at 605 m/f , a value almost 35 % of that observed on 

 anaerobiosis. If one considers a respiratory chain as represented by the scheme: 



succinate ->- dehydrogenase -^ b -> c^-^ c -^ a ^i- a^^^i- O2 



then the removal of cytochrome c should result in the reduction of cytochromes b 

 and Ci by succinate, but little or no reduction of cytochromes a or 03. This is especially 

 true in the light of the observation that the rate of cytochrome 03-oxygen reaction 

 and that of cytochromes a and 03 interaction (Chance) is extremely rapid. The 

 presence of a small amount of endogenous cytochrome c would be offset by the large 

 velocity of these reactions and would not tend to invalidate the observations presented 

 here. It should be noted, also, that the absorption at 444 m/t is only about 10% of 

 that obtained on anaerobiosis. In agreement with previous measurements on the 

 steady-state reduction of cytochromes a and a^, in a purified preparation (Smith), one 

 must conclude that the 444 and 605 m/t absorption bands are not due to a single 

 pigment, as based on their differences in steady-state values. One must also conclude 

 that the linear chain representation as presented above is not truly representative in 

 this instance and that one may have to place cytochrome a to a side path, much in 

 the manner that Chance has put cytochrome /> in a side path in such modified heart 

 preparations. 



