HILL REACTION .\ND ITS RELATIONSHIP TO PHOTOPHOSPHORYLATION 417 



with a catalytic amount of cofactor does not involve reoxidation of the 

 reduced cofactor bv OX, but reoxidation by molecular oxygen, as shown in 

 Scheme 5 [31, 33, 35]. Such systems cause a rapid isotopic exchange 



//.' 



Chi 



;/- P \ 



R OX >iO. 



Hill oxidant 



reduced, and 



reoxidized 



byO^ 

 Scheme 5 



between ^^Oo and water, and the rate of exchange is as fast or faster than 

 the rate of phosphorylation [36]. The reoxidation of reduced cofactor is 

 partly enzymic and partly non-enzymic. In the latter case H2O2 is formed 

 [31]. The proportion of non-enzymic auto-oxidation increases with 

 increasing O., tension as evidenced by increased hydrogen peroxide 

 production, without necessarily changing the yield of ATP [35]. This may 

 be taken as additional evidence that the phosphorylation does not occur 

 during the oxidation of reduced cofactor. Warburg et al. [31] have 

 described these phenomena for naphthoquinone sulphate as a cofactor, 

 and we have studied them with riboflavin monophosphate [37] and with 

 menadione [35, 38]. In all these cases there is photophosphorylation 

 associated with a Alehler reaction [39]. 



An exception to the behaviour of the cofactors just described is 

 provided by the N-methylphenazonium salts and the related substance 

 pyocyanine [11, 12, 40 44]. With these compounds as cofactors for photo- 

 phosphorylation, the predominant, though not the only, mode of cycling 

 appears to be genuinely anaerobic; that is, OX is diverted to oxidize the 

 reduced cofactor. Such phosphorylation is characterized by a low sensitivity 

 to inhibitors of the oxygen-evolving process such as orthophenanthroline, 

 chlorophenyldimethylurea and related compounds, or high concentrations 

 of tris buffer [37, 41, 45, 46]. In the presence of these inhibitors, oxygen 

 becomes strongly inhibitory, as would be expected from the fact that the 

 reduced cofactors are auto-oxidizable, and that if oxygen excretion is not 

 possible the cofactor system must be poised so as to discharge equivalent 

 amounts of R and OX. The oxygen destroys this poising. In many respects, 

 chloroplasts with inhibited oxygen evolution show a behaviour toward 

 cofactors reminiscent of that of bacterial chromatophores. The occurrence 

 of the anaerobic cycling confirms the conclusion drawn from the data with 

 chromatophores, that O, evolution is not a necessary accompaniment for 

 phosphor}dation, 



VOL. n. 2E 



