162 E. R. Waygood and G. A. Maclachlan 



cofactor, e.g., resorcinol or DCP, etc., and presumably riboflavin 

 oxidizes resorcinol, for example, to its semiquinol which can in turn 

 oxidize Mn+-' to Mn^^ by the Kenten-Mann reaction (4). Quite apart 

 from this work, Andreae (2) has demonstrated that light-activated 

 riboflavin generates Mn^^ from Mw- in the presence of monohydric 

 and polyhydric phenols which do not readily form quinones on oxi- 

 dation, and he has postulated a similar reaction. The ease with 

 which riboflavin forms free radicals w'hen illuminated suggests that 

 this would occur not too infrequently in the presence of metal ions 

 in darkness or in the dilfuse light of the Warburg bath. 



In the absence of lAA, standard Mn-phenol-peroxidase systems 

 containing 6 ixM of riboflavin consumed oxygen in the dark, a 

 phenomenon that was accelerated by illumination. The oxygen con- 

 sumed by these systems could not have been due to irreversible co- 

 factor oxidation entirely since it appeared to be capable of contin- 

 uing indefinitely and by 400 min. had exceeded the molar oxygen 

 equivalence of the cofactors. Since no oxygen uptake occurred in 

 the absence of a cofactor or manganese it is suggested that the prod- 

 uct of the reaction in light or darkness must be oxidized manganese 

 formed by reversible cofactor oxidation. From the work of Kenten 

 and Mann (4) it is known that manganic ions are to some extent 

 stable in orthophosphate, and there is also the possibility that MnO^ 

 could be produced. A mechanism whereby riboflavin (Rb), a redox 

 catalyst (ROH), and peroxidase could interact to oxidize manganese 

 is summarized as follows: 



Rb + 2ROH -^ Rb-2H + 2RO- (A) 



Rb-2H + Oo -^ Rb 4- HoOo (B) 



peroxidase 



HoO., + 2R0H' > 2H..O + 2RO- (C) 



or catalase 



RO- + Mn-2 ^ H- ;?:± ROH + Mn-^ ) 



) (D) 



RO- + Mn*=^ -f H^ -^ ROH + Mn^^ ) 



Reaction (B) is a well-known spontaneous reaction, (C) is normal 

 peroxidation, and (D) the Kenten-Mann reaction. This reaction 

 sequence is similar to that proposed by Andreae (2) with the excep- 

 tion that peroxidase and catalase are considered here to perform a 

 peroxidatic reaction (C) and thus dispose of hydrogen peroxide 

 which would otherwise speed the decomposition of oxidized man- 

 ganese. In the absence of enzyme some oxygen uptake occurred (6) 

 and manganic ions were formed (2), though to a lesser extent. 



It is important to note that light absorbed by riboflavin is not 

 an absolute requirement, but merely an accelerator of these reac- 

 tions. In darkness, manganic ions were still produced by Andreae's 



