On the irreversible destruction of reduced nicotinamide nucleotides by hypohalous acids

Degradation of the reduced pyridine nucleotides NMNH and NADH by HOCl involves two distinct stages: a fast reaction, k = 4.2 x 10(5) M(-1) s(-1), leads to generation of stable pyridine products (Py/Cl) with a strong absorption band at 275 nm (epsilon = 12.4 x 10(3) M(-1) cm(-1) in the case of NMNH); secondarily, a subsequent reaction of HOCl, k = 3.9 x 10(3) M(-1) s(-1), leads to a complete loss of the aromatic absorption band of the pyridine ring. HOBr and HOI(I(2)) react similarly. Apparent rate constants of the primary reactions of HOX species with NMNH at pH 7.2 increase in the order HOCl (3 x 10(5) M(-1) s(-1)) < HOBr( approximately 4 x 10(6) M(-1) s(-1)) < HOI(I(2))( approximately 6.5 x 10(7) M(-1) s(-1)). HOBr reacts fast also with the primary product Py/Br, k approximately 9 x 10(5) M(-1) s(-1), while the reactions of HOI and I(2) with Py/I are slower, approximately 1.4 x 10(3) M(-1) s(-1) and >6 x 10(3) M(-1) s(-1), respectively. Halogenation of the amide group of NMN(+) by HOX species is many orders of magnitude slower than oxidation of NMNH. Taurine inhibits HOCl-induced oxidation of NADH, but HOBr-induced oxidation is not inhibited because the taurine monobromamine rapidly oxidizes NADH, and oxidation by HOI(I(2)) is not inhibited because taurine is inert toward HOI(I(2)). Also sulfur compounds (GSH, GSSG, and methionine) are less efficient in protecting NADH against oxidation by HOBr and HOI(I(2)) than against oxidation by HOCl. The results suggest that reactions of HOBr and HOI(I(2)) in a cellular environment are much more selectively directed toward irreversible oxidation of NADH than reactions of HOCl. It is noteworthy that the rather inert N-chloramines react with iodide to generate HOI(I(2)), i.e., the most reactive and selective oxidant of reduced pyridine nucleotides. NMR investigations show that the primary stable products of the reaction between NMNH and HOCl are various isomeric chlorohydrins originating from a nonstereospecific electrophilic addition of HOCl to the C5&dbond;C6 double bond of the pyridine ring. The primary products (Py/X) of NMNH all exhibit similar absorption bands around 275 nm and are hence likely to result from analogous addition of HOX to the C5&dbond;C6 bond of the pyridine ring. Since the Py/X species are stable and inert toward endogeneous reductants like ascorbate and GSH, they may generally be useful markers for assessing the contribution of hypohalous acids to inflammatory injury.