580 LIGHT AND LIFE 



TABLE 1 

 Failure of CMU to Inhibit Anaerobic Pyocyanine-catalyzed Phosphorylation 



Control rate was 325 ^tmoles phosphate esterified/mg chlorophyll/hour either in air 

 or under nitrogen. Flasks contained, in 1.5 ml volume, Tris buffer, /)H 8.0, 20 Atmoles, 

 NaCl 35 Minoles, pyocyanine .045 /umoles, ADP 2.5 /xmoles, phosphate 2.5 /xmoles, 

 MgClo 5 ^moles, chloroplasts containing .040 mg of chlorophyll, and either .05 ml of 

 95% alcohol or .05 ml of 3 X lO^^ M CMU dissolved in 95'/^, alcohol. Incubation 

 time was 5 minutes at 1 5°C, light intensity 4000 foot candles ; residual phosphate was 

 determined colorimetrically. 



Phrasing it another way, these data show a combined inhibition by 

 oxygen and CMU where neither alone would be inhibitory. This fact 

 is now easily explainable, if one is aware that pyocyanine is a very 

 readily autoxidizable dye. Thus when a small amount of oxygen is 

 available, any pyocyanine reduced in reaction 2 will be re-oxidized 

 in reaction 5. The existence of reaction 5, autoxidation, will compete 

 with and essentially eliminate reaction ■/. With reaction 4 inhibited 

 by lack of a substrate and reaction 3 inhibited by CMU, the two 

 alternative ways for YO to be reduced are both blocked, and so re- 

 action / will stop for lack of the acceptor form Y. 



Since the reaction conditions in Fig. 1 included a small amount 

 of oxygen, it becomes a question why PMS did not show partial in- 

 hibition by CMU the way pyocyanine did. In Fig. 2 it can be seen 

 that the resistance of the PMS-catalyzed phosphorylation was actually 

 dependent on the color of light that was used; the reaction was re- 

 sistant in white light but not in red. The explanation here may be 

 found in our observation (14) that white light under anaerobic con- 

 ditions causes PMS to be reduced non-enzymatically. Red light is 

 not absorbed by PMS, and so does not reduce it. We could also ob- 

 serve non-enzymatic reduction of PMS by ascorbate; and adding ascor- 

 bate, even in red light, causes the reaction to be resistant to CMU 

 (Fig. 2) . Thus wlien PMS is reduced non-enzymatically, the over- 

 all reaction is highly resistant to the combination of CMU and a little 

 oxygen; when it is not being reduced non-enzymatically, then the re- 

 action is just like pyocyanine in being sensitive to the combined 

 inhibition. 



