348 Enzymes: Kinetics of Oxidations /I8:4 



state of the iron; they do not show the charge on the molecule. The pN 

 and FAD reactions are two electron changes, whereas the cytochromes 

 undergo one electron change. Thus, two cytochrome b'" molecules must 

 be reduced for every FADH 2 oxidized. Likewise, 4 cyt a,' 3 are oxidized 

 by one 2 molecule. 



In the reactions of the cytochrome chain in intact mitochondria, no 

 spectroscopically observable intermediate complexes have been found 

 in the sense that a complex is formed between the hydroperoxidases and 

 hydrogen peroxide. This is similar to the absence of a spectrophoto- 

 metrically detectable complex between the intermediate complex of 

 peroxidase E-S u and reduced cytochrome c. However, the various 

 members of the cytochrome chain do change spectroscopically from the 

 reduced to the oxidized form. 



If the structural integrity of the functional unit within the mitochon- 

 drion is not maintained, the reaction chain is altered. Kinetic experi- 

 ments show that the types and the order of the enzymes involved in 

 oxidation, as well as the active ones, are a function of their relatively 

 fixed positions within the mitochondria. The reactions of the enzymes 

 in intact mitochondria are also qualitatively different from those in 

 mitochondria whose functional groups are disarranged. In damaged 

 mitochondria, the energy liberated by the oxidation is converted to 

 heat. By contrast, in intact mitochondria, the energy of oxidation may 

 be converted to another form of chemical energy through oxidative 

 phosphorylation. 



The cytochrome chain in intact mitochondria will react slowly in the 

 absence of ADP and (F) . The rate of oxidation is speeded manyfold 

 when oxidative phosphorylation can occur. There are also other 

 chemical substances such as dinitrophenol which will accelerate the 

 reaction, although these do not conserve the chemical energy in a form 

 useful to the cell. This indicates that the cytochrome chain, in the 

 intact mitochondria, is in some sense inhibited at various points. For 

 instance, in the absence of either phosphate or ADP, cyt c tends to 

 accumulate in the reduced form and cyt a in the oxidized form. This 

 inhibition can be accounted for with various models, the difficult thing 

 being to find a real basis for distinguishing between the various models. 



It is possible that oxidation represents carrying electrons in a semi- 

 conductor-like fashion through the proteins along the chain. Oxidation 

 might equally represent reactions between molecules, free to rotate and 

 vibrate, about restricted centers. It is conceivable that the inhibition 

 in the absence of phosphorylation is due to the presence of an unknown 

 chemical inhibitor, or that it is a steric inhibition. A major limitation 

 of testing the various hypotheses is the inability to vary the relative con- 

 centrations of most of the members of the chain without destroying the 

 ability to phosphorylate ADP. Under these circumstances, it is not 

 surprising that data obtained by spectrophotometric studies have as yet 



