Evidence That apo-Reduced Nicotinamide Adenine Dinucleotide Dehydrogenase and apo-Electron-transferring Flavoprotein from Peptostreptococcus elsdenii Are Identical

Electron-transferring flavoprotein (ETF) and NADH dehydrogenase, containing a novel orange flavin, have been highly purified from the strict anaerobe Peptostreptococcus elsdenii. Both enzymes couple the oxidation of NADH to the reduction of dyes but only ETF couples the oxidation of NADH to the reduction of butyryl coenzyme A dehydrogenase (ETF activity). Disc gel electrophoresis in sodium dodecyl sulfate and urea, immunological studies, and amino acid analyses have provided strong evidence that the apoproteins of ETF and NADH dehydrogenase are very similar or identical. The two enzyme preparations differ structurally in the proportions of their flavin chromophores, FAD and two novel modified flavins, 6-hydroxy-7,8-dimethyl-10-(ribityl-5'-ADP)-isoalloxazine and 7-methyl-8-hydroxy-10-(ribityl-5'-ADP)-isoalloxazine (8-OH-FAD). Preparations with ETF activity contain FAD as the predominant prosthetic group and low amounts of the two modified flavins. NADH dehydrogenase preparations contain all three flavins, about 50% FAD and 50% modified flavins; there is a high proportion of 8-OH-FAD which has an intense absorption band at 475 nm and imparts an orange color to the protein. Differences in catalytic activity, absorption spectrum, electrophoretic mobility, and isoelectric point of ETF and NADH dehydrogenase are most likely due to different proportions of the three flavins.