Detection of Labile Low-Molecular-Mass Transition Metal Complexes in Mitochondria

Abstract

Liquid chromatography was used with an on-line inductively coupled plasma mass spectrometer to detect low-molecular-mass (LMM) transition metal complexes in mitochondria isolated from fermenting yeast cells, human Jurkat cells, and mouse brain and liver. These complexes constituted 20 – 40% of total mitochondrial Mn, Fe, Zn, and Cu ions. The major LMM Mn complex in yeast mitochondria had a mass of ca. 1100 Da and a concentration of ~ 2 μM. Mammalian mitochondria contained a second Mn species with a mass of ca. 2000 Da at a comparable concentration. The major Fe complex in mitochondria isolated from exponentially growing yeast cells had a mass of ca. 580 Da; the concentration of Fe580 in mitochondria was ca. 100 μM. When mitochondria were isolated from fermenting cells in post-exponential phase, the mass of the dominant LMM Fe complex was ca. 1100 Da. Upon incubation, the intensity of Fe1100 declined and Fe580 increased, suggesting that the two are interrelated. Mammalian mitochondria contained Fe580 and 2 other Fe species (Fe2000 and Fe1100) at concentrations of ca. 50 μM each. The dominant LMM Zn species in mitochondria had a mass of ca. 1200 Da and a concentration of ca. 110 μM. Mammalian mitochondria contained a second major LMM Zn species at 1500 Da. The dominant LMM Cu species in yeast mitochondria had a mass of ca. 5000 Da and a concentration in yeast mitochondria of ca. 16 μM; Cu5000 was not observed in mammalian mitochondria. The dominant Co species in mitochondria, Co1200, had a concentration of 20 nM and was probably a cobalamin. Mammalian but not yeast mitochondria contained a LMM Mo species, Mo730, at ca. 1 μM concentration. Increasing Mn, Fe, Cu, and Zn concentrations 10 fold in the medium increased the concentration of the same element in the corresponding isolated mitochondria. Treatment with metal chelators confirmed that these LMM species were labile. The dominant S species at 1100 Da was not free GSH or GSSG.

Labile Low-Molecular-Mass Metal Complexes in Mitochondria: Trials and Tribulations of a Burgeoning Field

“…43 Only one LMM Cu species, with a molecular mass of ~5000 Da (Cu 5000 ), was reproducibly observed in yeast mitochondria (Figure 2). We did not detect a major Cu-containing peak at 13 kDa, nor did we consistently observe any intense Cu features with masses of <5 kDa.…”

“…43 Zn 1200 was reproducibly present in LC traces of FTSs from fermenting yeast and mammalian mitochondrial extracts. FTSs from mammalian samples contained a second Zn species that migrated according to a mass of ~1500 Da.…”

“…In our most recent LC–ICP-MS study, we found that FTSs of mitochondrial extracts from fermenting yeast contain either of two LMM Fe complexes (Figure 2), depending on whether cells are harvested in the exponential or postexponential growth phase. 43 In the former case, a LMM species with an estimated mass of 580 Da (called Fe 580 ) is present, whereas in the latter, a complex corresponding to a mass of 1100 Da (Fe 1100 ) is observed. Curiously, Fe 1100 converts into Fe 580 when the FTS is allowed to incubate inside an anaerobic glovebox for a few days, suggesting that these two compounds are somehow related.…”

“…In our most recent LC–ICP-MS study, 43 we isolated mitochondria from fermenting yeast, human Jurkat cells, mouse brain, and mouse liver. Purified mitochondria were treated with detergent, and the soluble supernatant fraction was passed through a 10 kDa cutoff membrane in an Amicon (Merck Millipore Corp.) stirred cell.…”

“…Although sufficiently stable to remain intact down an LC column, the observed metal-containing species were sufficiently labile to be affected by treatment with 1,10-phenanthroline (Phen). 43 …”