Bimolecular reactions of pyridinyl radicals in water and the mechanism of NAD+-NADH dehydrogenase reactions

Abstract

I-Methyl-3-carbamido- (3.) and 4-carbamidopyridinyl (4.) radicals, generated through pulse radiolysis via l e addition to the pyridinium ions, disappear by rather different pathways in water. 3. dimerizes in a pH-independent reaction, whereas 4. reacts by electron transfer with protonated 4. (4H+.) and 4H+- reaFts with other 4H+., leading to a pH-dependent reaction yielding pyridinium ion and a dihydropyridine. The latter reaction was defined with respect to both kinetics and products by a study o f the disappearnce of 1-ethyl- and I-tert-butyl-4-carbomethoxypyridinyinl aqueous solutions between pH 8 and 9. At higher pH, ester hydrolysis produces the highly reactive carboxylate-substituted radical. A log k-pH plot suggests pK, differences between ester and amide radicals. Dimerization and electron-transfer reactions of 2-, 3., and 4. pyridinyl radicals generated by radiolytic techniques can be distinguished by careful measurement of pH changes of unbuffered solutions. Both the 3. dimer and the dihydropyridine from 4. react with water in pH-dependent reactions to yield derivatives absorbing at much shorter wave lengths. The pK, for 3- i s 1.4, but the structurally similar radical from nicotinamide adenine dinucleotide (NAD.) is unprotonated at pH 0.3. The NAD. dimer reacts with water at about 0.03 of the rate found for 3. dimer. Some properties of the product of combination of the hydroxy-tert-butyl radical and 3. are compared to those of the 3- dimer. The suggested le, H+, pathway for NAD+-NADH enzyme-catalyzed reactions is made more attractive by our results.