578 LIGHT AND LIFE 



change reaction sequence 1, 2, 3, and 5. Atmospheric oxygen must 

 be present; evidently reaction 3 does not produce enough oxygen to 

 satisfy the amount required in reaction 5. Nakamoto et al. have 

 demonstrated an oxygen requirement for phosphorylation when FMN 

 is the added redox cofactor (22, 23) , and we have been able to con- 

 firm this (unpulx) on using nitrogen gas more highly purified from 

 oxygen. Secondly, during the reaction sequence an oxygen exchange 

 should be demonstrable between water and the atmosphere. Naka- 

 moto et al. have observed this with FMN (23) , and the amount of ex- 

 change was sufficient to account for the ATP formed. We had pre- 

 viously published one observation (18) in which a small exchange 

 was observable, but was not sufficient to account for the phosphory- 

 lation occurring with FMN. It is now apparent that the earlier ex- 

 periments led us to an incorrect conclusion, for technical reasons: 

 the amount of exchange observed was limited under the conditions 

 then used by the rate of diffusion of oxygen between the gas and 

 water phases. Dr. Krall has recently repeated the observations under 

 conditions where diffusion was not limiting and has indeed seen a 

 light- and FMN-dej)endent oxygen exchange reaction sufficient to 

 account for the phosphorylation going on (pers. conuuun.) 



Our own experience with the variety of reaction sequences possi- 

 ble in chloroplasts centers around the inhibitor, CMU (/?-chlorophenyl 

 dimethyl urea) (12, 14). Fig. 1 shows that it was an inhibitor for 

 phosphorylation, inu the extent of its inhibition depended on the 

 nature of the redox cofactor. Reactions catalyzed by flavin mono- 

 nucleotide (FMN) were very badly inhibited; pyocyanine as co- 

 factor gave an intermediate inhibition; and PMS (phenazine metho- 

 sulfate) was very much more resistant. All the Hill reactions, how- 

 ever, no matter what the Hill oxidant was, were inhibited as much 

 as the FMN-catalyzed phosphorylation. The contrast between the 

 Hill reactions and the PMS-catalyzed phosphorylation originally led 

 us to propose reaction 3 as the site of inhibition by CMU. 



Now that the FMN reaction sequence seems established as an oxy- 

 gen exchange reaction (eq. 7, 2, 3, and 5) it is entirely consistent 

 for the whole sequence to be inhibited by blocking reaction 3 with 

 CMU. At the time that inhibition of FMN was first observed, how- 

 ever, it seemed anomalous because we thought that FMN catalyzed 

 an electron trans|)ort cycle (eq. 1, 2, and •/) . In order to account for 

 the FMN inhibition we devised a complicated scheme in which two 

 oxidants were produced in sequence, and FMN and PMS varied in 

 the site at which they were reoxidized (12). A very similar compli- 



