HELMUT BEINERT 167 



ever, into a yellow form by reducing agents, substrate, and some other 

 substances. The long-wavelength band ol this enzyme had previously 

 been attributed to the presence of a heavy metal, but recent work 

 (17) has made it likely that it is due to the interaction of a com- 

 ponent of the enzyme protein itself with its own prosthetic flavin. 

 No analogies are available from interactions of simple flavin com- 

 pounds which woidd assist in clarifying the kind of reaction involved. 



The characteristics of the appearance and disappearance of the 

 bands at about 560 ny and 900 ni/x indicate that they belong to in- 

 termediates in the oxidation-reduction of flavin compounds (1) . Be- 

 cause of its concentration and temperature dependence the band 

 at 900 m^Li has been attributed to a dimer of the semiquinone form 

 of flavins. This band has never been seen wdth flavoproteins. It is 

 of considerable interest that such quinhydrone-type dimers show in 

 the relatively unexplored near-infrared region. 



The band at 560 m^x is thought to indicate the monomeric semi- 

 quinone at neutral pH. Recent work by paramagnetic resonance 

 (EPR) spectroscopy has confirmed this view (4) . A broad band 

 ^\•ith maximum absorption at wavelengths from 530 to 570 m^ also 

 appears temporarily during oxidation-reduction of several flavopro- 

 teins by dithionite or by substrate. In the cases which were studied, 

 EPR has again provided confirmatory evidence that semiquinones 

 are formed under these conditions. There are, however, several flavo- 

 proteins which show a persistent, and not a transient, absorption band 

 of very similar shape and intensity when substrate is added. 



Although one is inclined to infer from spectral analogy that this 

 also indicates semiquinone formation, no semiquinone was detectable 

 by EPR in the only case which has so far been thoroughly investigated 

 (4). 



Semiquinone Formation of Flavins and Flavoproteins 

 The evidence for semiquinone formation of flavins and flavopro- 

 teins obtained by optical methods and by EPR methods, and the 

 correlation of the results obtained by both methods, was discussed at 

 a recent symposium by Beinert and Sands (4) . It may therefore 

 suffice to state here the conclusions they reached. The appearance of 

 an absorption band with a maximum from 530-570 ni/^ in many cases 

 coincides with the appearance of EPR signals indicative of free-radical 

 formation, particularly when the absorption band makes a transient 

 appearance. The appearance of a persistent absorption band in this 

 region, however, does not necessarily coincide with formation of 



