Cytochrome b^ 573 



Many of you may recall that Haas, more than twenty years ago, described a red 

 complex of old yellow enzyme (OYE) produced by reducing the enzyme in the presence 

 of excess triphosphopyridine nucleotide (TPN). Haas attributed the red complex 

 which displayed a shift in the absorption band from 465 m/t to 475 m/<, to free radical 

 formation, and this has frequently been cited as the best evidence for the formation 

 of a semiquinone intermediate in a biological oxidation reaction. Recently Beinert 

 ascribed to free radical formation a broad absorption band (550-660 m//.) with a 

 peak at about 565 m//, that occurred upon reduction and subsequent oxidation of a 

 number of flavin enzymes, chiefly acyl dehydrogenases. This absorption band was 

 accompanied by a change in colour from yellow to green-brown. With old yellow 

 enzyme, his results were equivocal, yet in no instance did Beinert find a red complex 

 or the spectral changes described by Haas. 



While preparing OYE, one of us obtained a red compound with spectral properties 

 similar to those described by Haas, when the protein was precipitated with alcohol at 

 — 5°C. Therefore we attempted to repeat Haas' experiment to determine by means of 

 paramagnetic resonance absorption whether the red complex was a free radical. Under 

 a variety of conditions we were able to get a red complex with shift of the two absorp- 

 tion peaks at 383 and 465 m/t to 392 and 475 mn. This was produced at room tempera- 

 ture by addition of a 10-15-fold excess of TPNH to a solution of OYE in neutral 

 phosphate buffer. The presence of the pyridine nucleotide in the reduced form appeared 

 to be essential but exclusion of oxygen or addition of dithionite was not necessary. 

 Despite careful search we could find no distinct peak at 565 m/<, but only a slight 

 increase in absorbancy of the order that Beinert found with OYE. This appeared to be 

 due to the shift of the large peak to 475 m/<. By ultracentrifugation of the red complex 

 in a separation cell and enzymic assay of the TPN remaining uncombined in the 

 supernatant, it was shown that the amount of TPN bound by the enzyme was equiva- 

 lent to its FMN content — that is, 2 moles/mole of protein. Free radical formation 

 was then demonstrated by paramagnetic resonance absorption, by using higher 

 concentrations of OYE (240 //m to 1 //m), and TPNH. A very large ESR signal was 

 obtained, which had a ^-value of 2002 indicating an organic free radical. The 

 derivative curves were indistinguishable in shape, width and ^-value from those 

 obtained by reduction of FMN with zinc in acid solution. Our results indicated that 

 the red complex contains TPN in addition to FMN, as Haas assumed. However, the 

 red complex is not identical with the free radical, since independent measurements 

 showed that free radicals comprised only about 15 % of the total enzyme concentration 

 (FMN equivalents). Free radicals appeared to accumulate until a steady-state was 

 reached and disappeared when the solution was exposed to air. 



Further studies suggested that the red complex contains TPN and FMN, both in the 

 oxidized form. Thus, it appears to be, in eff"ect, an oxidized ES complex, or an un- 

 dissociated enzyme-product (EP). 



One is reminded that complexing of FMN to the apoprotein to form OYE, shifts the 

 two absorption peaks of the former (372, 450 m/^) to 383 and 465 m/t, respectively. We 

 now have evidence that attachment of the pyridine nucleotide produces a further shift 

 toward long wave lengths to 391 and 475 m//, respectively. The shift of the peaks of 

 cytochrome b^ from 527 and 557 to 533 and 567, during an intermediate stage of 

 oxidation, that Morton and co-workers have described, might have an analogous 

 interpretation. The titration data of Boeri and colleagues suggest that a free radical 

 is likely, and the paramagnetic resonance absorption studies which he has planned 

 should afford a final answer to this. 



Autoxidation of Cytochrome h^ 



Margoliash : In connexion with the findings on the effect of ionic strength on the rate of 

 autoxidation of reduced cytochrome b.^ may I point out that a rather similar situation 

 occurs with cytochrome c. Although native mammalian heart cytochrome c has a very 

 slow rate of autoxidation this rate can be increased quite considerably by increasing 

 the ionic strength. Denatured cytochrome c shows a varying rate of autoxidation 



