Zinc metallothionein imported into liver mitochondria modulates respiration

Abstract

Metallothionein (MT) localizes in the intermembrane space of liver mitochondria as well as in the cytosol and nucleus. Incubation of intact liver mitochondria with physiological, micromolar concentrations of MT leads to the import of MT into the mitochondria where it inhibits respiration. This activity is caused by the Nterminal ␤-domain of MT; in this system, the isolated C-terminal ␣-domain is inactive. Free zinc inhibits respiration at concentrations commensurate with the zinc content of either MT or the isolated ␤-domain, indicating that MT inhibition involves zinc delivery to mitochondria. Respiratory inhibition of uncoupled mitochondria identifies the electron transfer chain as the primary site of inhibition. The apoform of MT, thionein, is an endogenous chelating agent and activates zinc-inhibited respiration with a 1:1 stoichiometry ([zinc binding sites]͞[zinc]). Carbamoylation of the lysines of MT significantly attenuates the inhibitory effect, suggesting that these residues are critical for the passage of MT through the outer mitochondrial membrane. Such an import pathway has been proposed for other proteins that also lack a mitochondrial targeting sequence, e.g., apocytochrome c, and possibly Cox17, a mitochondrial copper chaperone that is the only protein known so far to exhibit significant primary sequence homology to MT. The presence and respiratory inhibition of MT in liver, but not heart, mitochondria suggest a hitherto unknown biological modulating activity of MT in cellular respiration and energy metabolism in a tissue-specific manner. Z inc has a multitude of known cellular functions (1), but knowledge regarding its functions in mitochondria is still scant. Mitochondrial zinc constitutes a significant fraction of total cellular zinc. It has been estimated that in B cell-rich islets 32% of the total zinc is in mitochondria (2). The concentration of zinc in rat liver mitochondria is lower than that in the cytosol (3), indicating the existence of mechanisms to maintain this gradient and to regulate the amount of mitochondrial zinc, which seems to be quite variable. Thus, orally administered zinc salts increase zinc in rat liver mitochondria (4), and mitochondria of epithelial prostate cells are particularly rich in zinc (5), but its concentration remains lower than in the cytosol. Molecules that transport zinc to mitochondria and distribute it within them, as well as the control of these processes, remain to be identified and defined. So far, metallothionein (MT) is the only protein that has been implicated in cellular zinc distribution (6). We became interested in the function of MT in relation to mitochondria because commercial preparations of cadmium-containing MT have previously been shown to inhibit oxygen consumption of intact mitochondria (7,8). Addition of zinc to isolated mitochondria inhibits respiration, an effect that has been traced to multiple sites in the electron transport chain (9). Simpkins et al. (7) compared the inhibitory effects of MT and zinc and concluded that the effect o...

Terminal oxidation and the effects of zinc in prostate versus liver mitochondria

“…Hunter and Ford (1955) first reported that micro-molar levels of free zinc ions dramatically inhibited coupled liver mitochondrial oxygen consumption stimulated by β-hydroxybutyrate. Similar inhibitory effects of zinc were obtained by Ye et al (2001) with uncoupled liver mitochondria. Skulachev et al (1967) found that μM free zinc ions inhibited electron transfer between cytochrome b and c 1 of complex III and had no effect on the cytochrome c oxidase; and our results are in general agreement with those observations.…”

“…We have reported that metallothionein is an effective chaperone for the delivery of zinc to the mitochondrial inner membrane for direct exchange with zinc uptake transporter, so it is likely that this relationship is applicable to zinc inhibition in liver cells. This was suggested by Ye et al (2001) who found that zinc-metallothionein entered the mitochondrial intermembrane space where it inhibited the respiration of liver mitochondria. They showed that, in the absence of a significant free Zn + + pool, zinc could be transferred from metallothionein to zinc-acceptor site; and that localized oxidation of zinc-metallothionein would enhance the effect by facilitating its release of zinc.…”

Mitochondrial function, zinc, and intermediary metabolism relationships in normal prostate and prostate cancer

“…Once delivered to the intermembranous space, the ZnLigand could be exposed to conditions (such as oxidative environment; pH change) that facilitate the release of zinc from the ligand; thereby providing a localized pool of free Zn + + as the reactive form of zinc. This possibility was proposed for the effects of zinc metallothionein (ZnMT) on terminal oxidation of liver mitochondria (Maret, 1994;Ye et al, 2001). Alternatively or additionally, the ZnLigand can donate its zinc to a recipient molecule by a direct transfer that does not involve free Zn + + ions as an intermediary (Halthout et al, 2001).…”

Catalytic selenols couple the redox cycles of metallothionein and glutathione

“…MT could translocate into another cellular compartment in which the environment is more oxidizing. Indeed, MT is now known to be a constituent of the intermembrane space of liver mitochondria [27]. Generating a transient reactive oxidizing species could also change the redox environment.…”