842 LIGHT AND LIFE 



molecule. The mononucleotide is excited in this region of the spec- 

 trum even more strongly than the dinucleotide, and it may well be 

 that all the emission from FAD comes from a small proportion in 

 the open form which is in equilibrium with the non-emitting closed 

 form of the molecule. 



Unlike the pyridine nucleotides, FAIN and FAD have strong ab- 

 sorption bands in the visible and near ultraviolet when they are in 

 the oxidized rather than the reduced form, and it is the oxidized, not 

 the reduced, forms that fluoresce. During the course of reduction, 

 two very weak and very broad bands centered at 565 mjx and 880- 

 900 nifx, make their appearance, and Helmut Beinert, in his contribu- 

 tion to the Symposium, attributes these respectively to a semiquinone 

 or free radical and to the dimer of the semiquinone. The band in 

 the near infrared does not occur in flavoproteins. The 560 ni/^i band, 

 which occurs in several flavoproteins as well as in free flavins, is 

 clearly indicated to be a semiquinone by the presence of an electron 

 spin resonance signal, as well as by the transient nature of the band. 

 In several flavoproteins, however, the 560 ni/i, band is persistent rather 

 than transient. These cases Beinert interprets as involving a very 

 strong binding of substrate to enzyme, with interaction of neighbor- 

 ing substrate radicals to make semiquinones which are undetectable 

 by electron spin resonance s2:)ectroscopy. Or there might be inter- 

 action between adjacent flavins bound to the same enzyme molecule, 

 with formation of an undetectable flavin biradical. Additional evi- 

 dence to support the semiquinone theory is provided by R. L. Searls 

 and D. R. Sanadi, in this Symposium. They have studied the reac- 

 tion catalyzed by dihydrothioctyl (dihydrolipoic) deliydrogenase of 

 hog heart muscle. This reaction is a reversible oxidation of DPNH 

 by thioctamide or thioctate. The reduction of thioctate is inhibited 

 by agents (arsenite, cadmium ions) known to inhibit the reactivity 

 of sulfhydryl groups near the active site on the enzyme. This enzyme 

 also catalyzes the diaphorase reaction — oxidation of DPNH by 2, 

 6-dichlorophenol indophenol and ferricyanide — which is activated by 

 arsenite or cadmium ions several fold. Searls and Sanadi postulate 

 that the thioctate reaction involves a nearby disulfhydryl group on 

 the protein which may be oxidized to a disulfide bridge and which 

 is not necessary for the diaphorase reaction. A 535 m/x peak in the 

 absorption spectrum of the Havoprotein enzyme after reduction with 

 DPNH, or with thioctate bound to a-ketoglutaric dehydrogenase, dis- 

 appears instantly upon addition of arsenite or cadmium ions. Evi- 



