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

ABSTRACT: Liquid chromatography was used with an online 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, called Mn1100, had a mass of ?1100 Da and a concentration of ?2 ?M. Mammalian mitochondria contained a second Mn species with a mass of ?2000 Da at a comparable concentration. The major Fe complex in mitochondria isolated from exponentially growing yeast cells had a mass of ?580 Da; the concentration of Fe580 in mitochondria was ?100 ?M. When mitochondria were isolated from fermenting cells in postexponential phase, the mass of the dominant LMM Fe complex was ?1100 Da. Upon incubation, the intensity of Fe1100 declined and that of Fe580 increased, suggesting that the two are interrelated. Mammalian mitochondria contained Fe580 and two other Fe species (Fe2000 and Fe1100) at concentrations of ?50 ?M each. The dominant LMM Zn species in mitochondria had a mass of ?1200 Da and a concentration of ?110 ?M. Mammalian mitochondria contained a second major LMM Zn species at 1500 Da. The dominant LMM Cu species in yeast mitochondria had a mass of ?5000 Da and a concentration in yeast mitochondria of ?16 ?M; 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 a concentration of ?1 ?M. 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 glutathione or glutathione disulfide.

SUBMITTER: McCormick SP

PROVIDER: S-EPMC4627607 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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

McCormick Sean P SP Moore Michael J MJ Lindahl Paul A PA

Biochemistry 20150527 22

Liquid chromatography was used with an online 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, called Mn1100, had a mass of ∼1100 Da and a concentration of ∼2 μM. Mammalian mitochondria contained a second Mn sp ...[more]

PMID: 26018429

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Project description:Fluorescence-based chelators are commonly used to probe labile low-molecular-mass (LMM) metal pools in the cytosol of eukaryotic cells, but such chelators destroy the complexes of interest during detection. The objective of this study was to use chromatography to directly detect such complexes. Towards this end, 47 batches of cytosol were isolated from fermenting S. cerevisiae yeast cells and passed through a 10 kDa cut-off membrane. The metal contents of the cytosol and resulting flow-through solution (FTS) were determined. FTSs were applied to a size-exclusion LC column located in an anaerobic refrigerated glove box. The LC system was coupled to an online inductively-coupled-plasma mass spectrometer (ICP-MS) for detection of individual metals. Iron-detected chromatograms of cytosolic FTSs from WT cells exhibited 2-4 major species with apparent masses between 500-1300 Da. Increasing the iron concentration in the growth medium 40-fold increased the overall intensity of these peaks. Approximately 3 LMM cytosolic copper complexes with apparent masses between 300-1300 Da were also detected; their LC intensities were weak, but these increased with increasing concentrations of copper in the growth medium. Observed higher-mass copper-detected peaks were tentatively assigned to copper-bound metallothioneins Cup1 and Crs5. FTSs from strains in which Cup1 or the Cox17 copper chaperone were deleted altered the distribution of LMM copper complexes. LMM zinc- and manganese-detected species were also present in cytosol, albeit at low concentrations. Supplementing the growth medium with zinc increased the intensity of the zinc peak assigned to Crs5 but the intensities of LMM zinc complexes were unaffected. Phosphorus-detected chromatograms were dominated by peaks at apparent masses 400-800 Da, with minor peaks at 1000-1500 Da in some batches. Sulfur chromatograms contained a low-intensity peak that comigrated with a glutathione standard; quantification suggested a GSH concentration in the cytosol of ca. 13 mM. A second LMM sulfur peak that migrated at an apparent mass of 100 Da was also evident.

Project description:The solubility of molecular transition metal complexes can vary widely across different redox states, leaving these compounds vulnerable to electron transfer-initiated heterogenization processes in which oxidation or reduction of the soluble form of the redox couple generates insoluble molecular deposits. These insoluble species can precipitate as suspended nanoparticles in solution or, under electrochemical conditions, as an electrode-adsorbed material. While this electrochemically-driven solubility cycling is technically reversible, the reverse electron transfer to regenerate the soluble redox couple state is a practical challenge if sluggish electron transfer kinetics result in a loss of electronic communication between the molecular deposits and the electrode. In this work, we present an example of this electrochemically-driven solubility cycling, report a novel strategy for catalytically enhancing the oxidation of the insoluble material using homogeneous redox mediators, and develop the theoretical framework for analysing and digitally simulating the action of a homogeneous catalyst on a heterogeneous substrate via cyclic voltammetry. First, a mix of electrochemical and spectroscopic methods are used to characterize an example of this electrochemically-driven solubility cycling which is based on the two-electron reduction of homogeneous [Ni(PPh 2NPh 2)2(CH3CN)]2+ (PPh 2NPh 2 = 1,3,5,7-tetraphenyl-1,5-diaza-3,7-diphosphacyclooctane). The limited solubility of the doubly-reduced product in acetonitrile leads to precipitation and deposition of molecular [Ni(PPh 2NPh 2)2]. While direct oxidation of this heterogeneous [Ni(PPh 2NPh 2)2] at the electrode surface is possible, this electron transfer is kinetically limited. We demonstrate how a freely diffusing redox mediator (ferrocene) - which shuttles electrons between the electrode and the molecular material - can be used to overcome these slow electron transfer kinetics, enabling catalytic regeneration of soluble [Ni(PPh 2NPh 2)2]2+. Finally, mathematical models are developed that describe the current-potential response for a generic EC' mechanism involving a homogeneous catalyst and surface-adsorbed substrate. This novel strategy has the potential to enable reversible redox chemistry for heterogeneous, molecular deposits that are adsorbed on the electrode or suspended as nanoparticles in solution.

Dataset Information

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

Publications

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

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