346 RIBOFLAVIN 



a stable product by lyophilization which was 98 % pure, and had eight-fold 

 better yield. This enzyme reacted 10" times faster with cytochrome than 

 with oxygen, so it was concluded that the direct action of the reductase 

 with oxygen was of no physiological importance. 



b. Mechanism of Action of Cytochrome C Reductase 



Reduced cytochrome c reductase reacts with oxidized cytochrome c 

 (CyFe+++) according to the equation. 



Reduced Cy c reductase + 2CyFe+++ — > 



Cy c reductase + 2CyFe++ + 2H+ 



When hexose monophosphate (Robison ester), Robison ester dehydrogen- 

 ase (zwischenferment), and coenzyme II (TPN) are added to a solution 

 containing cytochrome c and cytochrome c reductase, 



Glucose-6-phosphate + TPN + H.O zwischenferment ^ 



H2-TPN + phosphogluconic acid 



H2-TPN + cytochrome c reductase -^ 



TPN + reduced cytochrome c reductase 



which in turn will reduce cytochrome c. 



7. Cytochrome C Reductase of Liver 



Proof of the reduction of cytochrome c in animal tissue by reduced coen- 

 zyme II (H2-TPN) was not reported until 1949, when Horecker^^ isolated 

 TPN cytochrome c reductase from pig liver. This flavoprotein has flavin 

 adenine dinucleotide as a prosthetic group but otherwise is quite similar 

 to the cytochrome c reductase obtained from yeast by Haas, Horecker, and 

 Hogness. 



a. Preparation 



The details of the preparation of acetone liver powder and its subsequent 

 extraction, trypsin digestion, salt precipitations, gel adsorptions, and fur- 

 ther purifications are described by Horecker. ^^ 



b. Properties 



The enzyme can easily be split into the protein and flavin adenine di- 

 nucleotide prosthetic groups. The protein fraction can be reactivated by 

 either flavin adenine dinucleotide or riboflavin phosphate. Of interest is the 

 fact the apoenzymes of both yeast and liver cytochrome c reductases form 



63 B. L. Horecker, J. Biol. Chem. 183, 593 (1950). 



