B. VENNESLAND, T. NAKAMOTO, AND B. STERN Gil 



rates. It can be seen that in the presence of catalase and ethanol with 

 FiNfN, the amount of orthophosphate esterified is no more than that 

 which is ccpiivalent stoichiometrically to the FMN added. If the 

 FMN is photoreduced prior to orthophosphate addition, there is no 

 detectable phosphorylation whatever. With K3 there is also a marked 

 inhibitory effect of catalase-ethanol, though the inhibition is not so 

 striking as with FMN. (With phenazine methosulfate and pyocyanine, 

 the inhibitory effect of catalase-ethanol is small or absent under 

 anaerobic conditions.) Separate experiments show that though 

 ethanol has a small inhibitory effect, neither catalase alone nor ethanol 

 alone is responsible for the marked inhibitions observed here. Both 

 reagents must be added together. When these results were first 

 obtained, we felt uncertain about whether the inhibition by catalase- 

 ethanol should be attributed to a consumption of H^Oo formed by 

 reduction of photochemically generated Oo, or whether the peroxide 

 might perhaps be an oxygen precursor. Our present views favor 

 the former alternative, i.e., that the system is actually forming O2 

 and reutilizing it. The HoO. may be regarded as a product of the 

 auto-oxidation of the reduced cofactor (Equation 3) . Since reaction 

 3 consumes more O^ than is formed in reaction 1, cycling of the sys- 

 tem is prevented when the HoOs is removed with catalase-ethanol, 

 unless an external oxidation is provided. 



reduced cofactor + Oo — > cofactor + H2O2 {3) 



The question arose whether the auto-oxidation of the added co- 

 factor can account for all of the oxygen consumption which can be 

 demonstrated with O^^. Our first estimates indicated that this was 

 not the case (5) . That is, reactions 2 and 3 might very well be pro- 

 ceeding simultaneously. In order to determine what proportion of 

 the reoxidation involves H.2O2 formation, Nakamoto has made a 

 set of measurements from which representative data have been as- 

 sembled in Tables 3 and 4. These measurements were carried out 

 with different amounts of O2 in the gas phase. Catalase-ethanol does 

 not inhibit photophosphorylation with FMN or K.., under aerobic 

 conditions, but the amount of peroxide formed can be measured by 

 determining the oxygen consumption manometrically. Alternatively, 

 acetaldehyde formation can be measured, since this procedure is more 

 sensitive, and can be shown to give equivalent information, as pointed 

 out by Dr. Krogmann in the following paper. The FMN concentra- 

 tions employed in the experiment sunnnarized in Table 3 were suffi- 

 cient to give optimal rates at the oxygen tensions employed. The 



