142 LIGHT AND LIFE 



40. Weber, C, this symposium. 



41. Weber, G., and Teale, F. J. W .. Discussions Faraday Society, 27, 134 (I9r)9). 



42. Winer, A., and Schwert, G., ,/. Biol. Chetn.. 234. 11,-).-) (19r)9). 



43. Winer, A., and Theorell, H., Acta. Cheiu. Scand., 13, 1038 (1959). 



DISCUSSION 



Dr. (;o\[\[().\kr: Since Dr. X'click has Ijrought up the matter of the evidence 

 lor free radical formation in IJavoprotein oxidation-reduction, it might be 

 pertinent to mention the relevant data. 



ESR signals due to free radicals in flavoprotein enzyme systems have been 

 published by Commoner et al. (Science, 126, bl , 1957) , Ehrenberg and 

 Ludwig (Science, 127, 1177, 1958), Kubo et al. {Bull. Soc. Chem. Biol, 41, 

 981, 1958), and Bray et al. {Bioche?n. J., 73, 193, 1958). In these cases, 

 kinetic data were lacking, so that no conclusions could be drawn regarding 

 the precise origin of the free radical in the enzyme reaction mechanism. 

 More recently it has been pos.sible to carry out quantitative kinetic studies 

 of the ESR signals observed in two flavoprotein enzyme systems, lactic 

 oxidative decarboxylase (Commoner, Lippincott and Passonneau, Proc. Nat. 

 Acad. Sci. U. S., 44, 1099, 1958) and succinic dehydrogenase (Commoner and 

 Hollocher, Proc. Nat. Acad. Sci. U. S., 46, 405, 1960; and Hollocher and Com- 

 moner, Proc. Nat. Acad. Sci. U. S., 46, 416, 1960). The latter investigations 

 show unequivocally that the ESR signals arise from free radical intermediates 

 in the oxidation-reduction of the flavoprotein. 



Most detailed data are a\ailable in the case of succinic dehydrogenase. 

 ESR studies of mitochondrial jjarticles from pig heart muscle reveal a signal 

 which, from the effects of inhibitors, can be siirjwn to arise from succinic 

 dehydrogenase. The intensity of tlie signal bears simple relationships to 

 temperature, enzyme concentration, the concentration of funiaratc + 

 succinate, tiic fumarate/succinate ratio, and the concentration of malonate. 



These data pro\icle a test of various possiljle reaction mechanisms. From 

 these considerations it can lie concluded that the observed ESR signal is 

 clue to a free radical form ol the en/yme-substrate complex, which is in 

 ecjuilii^riimi witii the; non-lree radical form of the complex. The free radical 

 ionn ol the complex occurs in the ecjiu'Iibriinn established among succinate, 

 IJavoprotein and fumarate. It does not occm- when the reduced flavoprotein 

 is reoxidized by cytochrome b. 



liie most recent iinestigatioiis of tiiis system (Hollocher and Cioinmoner, 

 Federation Proc. 19, 28, 1960) have been carried out with purified succinic 

 dehydrogenase. The results are identical wiili those obtained from heart 

 |jartick' |}rej)arations aiid lead lo c|uantitati\e estimates of the various reac- 

 tion constaius. Ilie ec|uilibriinn constant relating the non-free radical form 

 of the enzyme substrate complex to the free radical form is about .3. 



It appears. thereh)re. that there is considerable e\ idence which shows 

 thai free radical lornis ol llaxoprotein en/ymes play specific roles in the 

 enzymatic reaction mechanisms. 



