Enzymatic reduction of labile iron by organelles of the rat liver. Superior role of an NADH-dependent activity in the outer mitochondrial membrane.

The enzymatic system mainly responsible for the reduction of labile iron ions in mammalian cells is still unknown. Using isolated organelles of the rat liver, i.e. mitochondria, microsomes, nuclei and the cytosol, we here demonstrate that Fe(III), added as Fe(III)-ATP complex, is predominantly reduced by an NADH-dependent enzyme system associated with mitochondria (65% of the overall enzymatic Fe(III) reduction capacity within liver cells). Microsomes showed a significantly smaller Fe(III) reduction capacity, whereas the cytosol and nuclei hardly reduced Fe(III). Studying the mitochondrial iron reduction, this NADH-dependent process was not mediated by superoxide, ascorbic acid, or NADH itself, excluding low-molecular-weight reductants. No evidence was found for the involvement of complex I and III of the respiratory chain. Submitochondrial preparations revealed the highest specific activity reducing Fe(III) in the outer membrane fraction. In conclusion, an NADH-dependent mitochondrial enzyme system, most likely the NADH-cytochrome c reductase system, located at the outer membrane, should decisively contribute to the enzymatic reduction of labile iron within liver cells, especially under pathological conditions.

NAD(H) enhances the Cu(II)-mediated inactivation of lactate dehydrogenase by increasing the accessibility of sulfhydryl groups.

Copper ions are known to inactivate a variety of enzymes, and lactate dehydrogenase (LDH) is exceptionally sensitive to the presence of this metal. We now found that NADH strongly enhances the Cu(II)-mediated loss of LDH activity. Surprisingly, NADH was not oxidized in this process and also NAD+ promoted the Cu(II)-dependent inactivation of LDH. Catalase only partly protected the enzyme, whereas hypoxia even enhanced LDH inactivation. NAD(H) accelerated sulfhydryl (SH) group oxidation of LDH by 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB), and, vice versa, LDH-mediated Cu(II) reduction. LDH activity was preserved by thiol donators and pyruvate and partially preserved by lactate and oxamate. Our results suggest that reactive oxygen species (ROS) are of minor importance for the inactivation of LDH induced by Cu(II)/NADH. We propose that conformational changes of the enzymes' active sites induced by NAD(H)-binding increase the accessibility of active sites' cysteine residues to Cu(II) thereby accelerating their oxidation and, consequently, loss of catalytic activity.