BENTLEY GLASS 843 



dence that the 535 ni/x peak is attributable to a seniiquinoid free 

 radical FADH* S* SH was louml in a model reaction: the forma- 



tion from dihydrothioctate (a disuHhydryl compound) and riboflavin 

 phosphate (FMN) which had an absorption maximum of 535 m/x 

 that disappeared upon standing and was replaced by broad peaks 

 at 565 ni/x and 880 ni/x, corresponding to the flavin semiquinone and 

 its dimer. There is also evidence that FMN will form a complex 

 possessing a 535 m^ absorption band, by reacting with tryptophan or 

 other electron acceptors; and electron spin resonance measurements 

 showed no signal immediately after mixing dihydrothioctate and 

 FMN; but a free radical signal appeared as it turned green, some 20 

 minutes later. FMN plus enzyme alone turned green but gave no 

 ESR signal. The 535 m^ band is thought by Searls and Sanadi to 

 indicate the presence of a charge transfer complex, heretofore un- 

 demonstrated in biological systems. This "diaphorase" enzyme is 

 unusual among flavoproteins in that the fluorescence of the bound 

 flavin is enhanced rather than quenched. Velick and Beinert describe 

 a number of other flavoprotein enzyme-coenzyme complexes, all of 

 which seem to be characterized by the formation of a flavin semiqui- 

 none intermediate. (1) the "old yellow enzyme," the first flavopro- 

 tein ever purified, resolved, and reconstituted (Theorell, 1935) , does 

 not dissociate significantly under functional conditions. It oxidizes 

 TPNH with various hydrogen acceptors, and binds one molecule of 

 the resulting TPN per molecule of flavin, the latter seemingly also 

 oxidized. In the absence of oxygen, an electron spin resonance signal 

 gives notice of the formation of a flavin semiquinone intermediate. 

 (2) Butyryl dehydrogenase, in its green form (oxidized) has a broad 

 absorption band, with a maximum at about 720 ni/x, so far found 

 in no other flavoprotein except the "old yellow enzyme" after treat- 

 ment with ammonium sulfate at pH 10, a treatment which also turns 

 it green. This band probably comes from an interaction of enzyme 

 and flavin, but its interpretation is still unclear. (3) Acyl-coenzyme-A 

 dehydrogenases reversibly dehydrogenate fatty acyl groups linked 

 with coenzyme A. A semiquinone type of absorption band at 570 m/x 

 has been observed during the course of reduction by these FAD 

 enzymes, although no electron spin resonance signal could be detected. 

 (4) Lactate oxidase, by a first reaction step, forms pyruvate and per- 

 oxide which remain bound to the enzyme until they react to form 

 acetate and CO2. There is a weak electron spin resonance signal. (5) 



