Stereochemistry of hydrogen-transfer in the energy-linked pyridine nucleotide transhydrogenase and related reactions

Abstract

The stereochemistry of hydrogen-transfer in the energy-linked and non-energy-linked pyridine nucleotide transhydrogenase, the respiratory chain-linked NADH dehydrogenase, and the DT diaphorase-catalyzed reactions have been investigated with tritiated pyridine nucleotides. Both the energy-linked and the non-energy-linked pyridine nucleotide transhydrogenase reactions, catalyzed by stibmitochondrial particles from beef heart, involve the 4A hydrogen atom of NADH, and the 4B hydrogen atom of NADPH. The reactions involve no intrinsic exchange of hydrogen atoms between the reduced pyridine nucleotides and water. The aerobic oxidation of NADH, and the energy-linked reduction of NAD+ by succinate, catalyzed by the same particles, involve the 4B hydrogen atom of NADH, and are connected with a rapid exchange of hydrogen atoms between the latter and water. The reaction catalyzed by purified rat-liver DT diaphorase involves the 4A hydrogen atom of either NADH or NADPH, and involves no exchange of hydrogen atoms between the reduced pyridine nucleotides and water. Studies with 14C-labelled NADH indicate that the energy-linked pyridine nucleotide transhydrogenase reaction proceeds without an exchange of the nicotinamide moieties of the two pyridine nucleotides. These results are discussed in relation to the mechanism of the energy-linked pyridine nucleotide transhydrogenase reaction.