282 W. W. Wainio 



could be attributed to its copper content. Our evidence in support of this view 

 was that the 605 m/n absorption in the reduced state was related to the copper 

 content in several fractions made from the heart mitochondrial fragment 

 (Eichel, Wainio, Person and Cooperstein, 1950). Because the activity could 

 not be related to the copper content, and because later analysis of the pyridine 

 haemochrome of the prosthetic group of the soluble enzyme revealed the 

 presence of iron (Person, Wainio and Eichel, 1953), we were forced to abandon 

 this view. Our position then became that the enzyme is a tetrapolymer of 

 haemo-protein submolecules, as suggested by the statement of Warburg 

 (1949), 'I almost regret that we cannot . . . assume that the enzyme is a four- 

 fold polymerized haem compound', and by the experiments of Ball, Stritt- 

 matter and Cooper (1951) with carbon monoxide. It was thought that the 

 apparent reactivity of two components could equally well be explained in 

 terms of a differential sensitivity of the parts of a single polymeric molecule. 

 When it became possible by the addition of a lipid activator to relate the 

 activity to the copper content (Wainio, Vander Wende and Shimp, 1959), our 

 hypothesis was altered so that the enzyme is now considered to be a single 

 complex molecule containing both copper and haem. By virtue of its two 

 dissimilar parts the enzyme could exhibit the properties demanded by 

 Keilin and Hartree and others for the cytochromes a and a^ and by Warburg 

 for the oxygen-transporting enzyme. 



The advocates of two enzymes present the following evidence in support 

 of their position. 



(1) Morrison and Stotz (1955) and Morrison, Connelly and Stotz (1958) 

 have claimed the existence of two cytochrome fl-haems, an a^ and an a^. 

 They challenge the position of Lemberg (1953), who states that crypto- 

 porphyrin a, which Morrison, Connelly and Stotz (1958) find is closely 

 related to the porphyrin of haem a^, is an artifact derived from the single 

 haem a. 



(2) Smith (1955) found that in the steady state after the addition of cyto- 

 chrome c to the system ascorbate-cytochromes a + a^-oxygen the 605 m/u 

 peak was 59 % of the totally reduced value, whereas the 445 m/u peak was 

 only 24 % reduced. It was concluded that the two peaks could not be those 

 of one enzyme. 



(3) Lundegardh (1957) has made a careful study of the effects of carbon 

 monoxide and cyanide on the spectrum of yeast and by optical subtraction 

 has obtained the spectra of the two components. The ratios of the maxima 

 of the absorption peaks were given for cytochrome a, y:y,= 1-0, and for 

 cytochrome a^, y:a = 20-8. 



The failure by us (Eichel, Wainio, Person and Cooperstein, 1950; Wainio, 

 Eichel and Cooperstein, 1952) and by others to separate the two enzymes by 

 techniques that have separated the other cytochromes may be taken as 

 presumptive evidence in support of the one-enzyme concept. It must be 



