164 E. R. Way good and G. A. Maclaclilan 



probably the manganic ion. This is supported by the toUowing evi- 

 dence. Riboflavin suppresses oxygen uptake and carbon dioxide evo- 

 lution equally; therefore, it must compete ^\ith an oxidized inter- 

 mediate in the initiation reactions or in the first step of propaga- 

 tion. Reduced riboflavin decolorizes manganiversene instantaneous- 

 ly, and kinetic evidence suggests that no reaction occurs with other 

 oxidized intermediates. For example, any reaction of reduced ribo- 

 flavin with oxidized cofactor radical (reverse of reaction A) could 

 occur only at the expense of the normal reaction of the cofactor 

 radical with Mn+2 since an increase in Mn^^ concentration failed to 

 counteract the retardation by riboflavin in darkness, then manganese 

 and riboflavin probably do not compete for the cofactor radical. 

 Similarly riboflavin does not compete with the cofactor for the 

 skatole peroxy-radical; otherwise different cofactors would have a 

 significant effect on the degree of retardation and an increased con 

 centration of cofactor by itself should counteract the retardation. 



The alternative explanation is that reduced riboflavin competes 

 with lAA for Mn+*^, but increased concentrations of lAA alone do 

 not overcome the retardation. However, because the critical optimum 

 concentration of the cofactor is dependent on the concentration of 

 lAA, the unbalance caused by varying the concentration of lAA in- 

 dependently of the cofactor may be the cause of the ineffectiveness 

 of lAA in overcoming the retardation by itself (Table 2). \\lien the 

 concentration ratio lAA/DCP was maintained constant at 4.4, in- 

 creasing concentrations of lAA were able to overcome the inhibi- 

 tion. Such evidence makes it appear most probable that Mn^-' is 

 implicated in a reaction with reduced riboflavin. 



Accordingly it is possible to formulate a hypothesis for the 

 mechanism of retardation as follows: 



In darkness (retardation). 



Rb + 2ROH -^ Rb-2H + 2RO- 

 RO- + Mn-2 _^ H- ;e± ROH + Mn^^ 

 Rb-2H -f 2Mn^3 _^ Rb _|- 2Mn*2 ^ 2H- 



This series of slow reactions would be substituted in part for the 

 normal sequence of reactions. Some Rb-2H possibly cannot escape 

 reaction with oxygen, but it would more likely be preferentially oxi- 

 dized by Mn^3 



In light. The retardation is overcome since Rb-2H reacts prefer- 

 entially with Oo (light activated) and the HoOo produced supjjorts 

 the production of cofactor radicals for manganigcnesis in tlic Ken- 

 ten-Mann reaction as follows: 



