292 W. W. Wainio 



the reduced enzyme by bubbling oxygen through the solution reduced with 

 dithionite. The 444 mju and 605 m/i peaks are diminished in height and the 

 maxima shift to 426-28 mfi and 603 m/<, respectively. The further addition of 

 ferricyanide shifts the maxima to 424 m/n and 600 m/n. These last values are 

 about 5 m/z higher than those reported by other investigators for the oxidized 

 enzyme (see p. 347 of Wainio and Cooperstein, 1956) and seem to suggest 

 that even with ferricyanide the preparation cannot become fully oxidized. 

 As pointed out by Chance in the discussion of the paper of Okunuki, 

 Hagihara, Sekuzu and Horio (1958), it is possible that the oxygenated inter- 

 mediate compound is a mixture of the oxidized and reduced forms of an 

 altered or damaged preparation. The maxima are very reminiscent of the 

 partially reduced solution of our enzyme which we produced with ferrocyanide 

 (Wainio, 1955c). This mixture had maxima at 425 m// and 603 mfi. 



C. Reactions with Carbon Monoxide and Nitric Oxide 



Warburg (1926) was the first to suggest that a reduced iron-containing 

 enzyme should be the point of attachment of carbon monoxide. He and his 

 associates went on to determine the photochemical action spectrum of the 

 carbon monoxide compound of cytochrome c oxidase in yeast (Warburg and 

 Negelein, 1928, 1929; Warburg, 1932; Kubowitz and Haas, 1932). Their 

 method was based on the earlier observation that the inhibition by carbon 

 monoxide is light-reversible and that the degree of reversibility is dependent 

 on the wavelength of the incident Hght (Warburg, 1926). The maxima for 

 the absorption by the components which absorbed the energy of the light to 

 cause the reversal were found at 283 m/j, and 430 m/i, at about 520 m/u, and 

 at 540 m^ and 590 m^. Chance (1953) has since shown that the photo- 

 chemical dissociation spectrum of the carbon monoxide compound of the 

 enzyme is the same in mammalian heart muscle. 



The actual combination of the reduced cytochrome c oxidase with carbon 

 monoxide was demonstrated by Keilin and Hartree (1939), who found a 

 partial shift in the spectrum of a heart muscle preparation with new bands at 

 432 m^ and 590 m/n. These bands, which correspond to two of the principal 

 peaks found by Warburg for the yeast enzyme, and the partial shift in the 

 spectrum have been confirmed by Ball, Strittmatter and Cooper (1951), 

 Dannenberg and Kiese (1952), Wainio (1955c) and Sekuzu et al. (1959) with 

 soluble preparations of cytochrome c oxidase. The effects noted by Wainio 

 (1955c) are presented in Fig. 1 where it can be seen that the y-peak shifted 

 from 443 m// to 430 m/f, while the a-peak shifted from 605 m/^ to 603 m/« 

 and became asymmetrical on its lower wavelength side. It is to be observed 

 that a considerable absorption still persists at 443 m/< and 605 m/^i. The 

 difference spectrum has maxima for the carbon monoxide compound of the 

 reduced enzyme (downward peaks in Fig. 1) at 428 m/f, 545 m// and 590 m/<. 



The efi"ect of nitric oxide on the reduced enzyme (Wainio, 1955c) is shown 



