Sites of electron transfer to tetrazolium salts in the succinoxidase system.

With the increased use of tetrazolium salts in biological systems (l-20), and with the increased availability of tetrazolium salts with widely different oxidation-reduction potentials (3-5, 11, 14, 32), differences in the effect of anaerobiosis on the reaction rate (3, 6, 9, 10, 16, lQ), in the effect of disrupting influences such as freeze-thawing or section thinness (6, 10, 16, lQ), and in the inhibitory action of cyanide (6, 15, 18,21-24), have become more evident. It has occurred to us that these differences could be due to the fact that the various tetrazolium salts accept electrons from different sites along the electron transport chain. Although recent publications have dealt with the sites of electron transfer to a variety of acceptors, little attention has been given to tetrazolium salts (25-27), other than to point out both similarities and dissimilarities with other oxidation-reduction dyes, such as methylene blue and 2,6-dichlorophenolindophenol (9, 28-31). In order to test the above hypothesis, three pairs of tetrazolium salts were selected for study. These were two monotetrazolium salts, without and with nitro-substitution (2,3,5-triphenyl ktrazolium chloride and 2-p-nitrophenyl-3-p-iodophenyl-5phenyltetrazolium chloride); two ditctrazolium salts without and with nitro-substitution (2,2’, 5,5’-tetraphenyl-3,3’-(3-3’dimethoxy-4,4’-biphenylene)ditetrazolium chloride and 2,2’-di-p-nitrophenyl-5,5’diphenyl-3,3’(3,3’-dimethoxy-4,4’-biphenylene) ditetrazolium chloride; and two ditetrazolium salts without methoxyl substitution, and without and with nitro-substitution (2,2’5,5’-tetraphenyl-3,3’-(p-biphenylene)ditetrazolium chloride and 2,2’-diphenyl5,5’-di-m-nitrophenyl-3,3’-(p-biphenylene)dit&razolium chloride). Not only do these nitro groups increase the oxidation-reduction potential (32), but they apparently make the tetrazolium salt a more active acceptor of electrons in dehydrogenase systems, whereas the methoxyl group increases the possibility of steric hindrance in reactions with components of dehydrogenase systems. Although the final organization of electron transport in the succinoxidase system has not been determined, a simplified scheme which incorporates current opinion (33-39) is shown in Scheme 1, together with the special conditions or inhibitors that were used in this study. By notation of the effects of these in-