Gregory P. Holmes-Hampton

Biophysical Characterization of Iron in Mitochondria Isolated from Respiring and Fermenting Yeast

The distributions of Fe in mitochondria isolated from respiring, respiro-fermenting, and fermenting yeast cells were determined with an integrative biophysical approach involving Mossbauer and electronic absorption spectroscopies, electron paramagnetic resonance, and inductively coupled plasma emission mass spectrometry. Approximately 40% of the Fe in mitochondria from respiring cells was present in respiration-related proteins. The concentration and distribution of Fe in respiro-fermenting mitochondria, where both respiration and fermentation occur concurrently, were similar to those of respiring mitochondria. The concentration of Fe in fermenting mitochondria was also similar, but the distribution differed dramatically. Here, levels of respiration-related Fe-containing proteins were diminished approximately 3-fold, while non-heme HS Fe(II) species, non-heme mononuclear HS Fe(III), and Fe(III) nanoparticles dominated. These changes were rationalized by a model in which the pool of non-heme HS Fe(II) ions serves as feedstock for Fe-S cluster and heme biosynthesis. The integrative approach enabled us to estimate the concentration of respiration-related proteins.

Catalytic mechanism and three-dimensional structure of adenine deaminase.

Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k(cat) = 2.0 s(-1); k(cat)/K(m) = 2.5 × 10(3) M(-1) s(-1)). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn(2+) prior to induction, the purified enzyme was substantially more active for the deamination of adenine with k(cat) and k(cat)/K(m) values of 200 s(-1) and 5 × 10(5) M(-1) s(-1), respectively. The apoenzyme was prepared and reconstituted with Fe(2+), Zn(2+), or Mn(2+). In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe(II)/Fe(II)]-ADE was oxidized to [Fe(III)/Fe(III)]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe(III)/Fe(III)]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Mössbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 Å resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction mechanism and the identity of the rate-limiting steps.

Iron Content of Saccharomyces cerevisiae Cells Grown under Iron-Deficient and Iron-Overload Conditions

Fermenting cells were grown under Fe-deficient and Fe-overload conditions, and their Fe contents were examined using biophysical spectroscopies. The high-affinity Fe import pathway was active only in Fe-deficient cells. Such cells contained ~150 μM Fe, distributed primarily into nonheme high-spin (NHHS) Fe(II) species and mitochondrial Fe. Most NHHS Fe(II) was not located in mitochondria, and its function is unknown. Mitochondria isolated from Fe-deficient cells contained [Fe(4)S(4)](2+) clusters, low- and high-spin hemes, S = (1)/(2) [Fe(2)S(2)](+) clusters, NHHS Fe(II) species, and [Fe(2)S(2)](2+) clusters. The presence of [Fe(2)S(2)](2+) clusters was unprecedented; their presence in previous samples was obscured by the spectroscopic signature of Fe(III) nanoparticles, which are absent in Fe-deficient cells. Whether Fe-deficient cells were grown under fermenting or respirofermenting conditions had no effect on Fe content; such cells prioritized their use of Fe to essential forms devoid of nanoparticles and vacuolar Fe. The majority of Mn ions in wild-type yeast cells was electron paramagnetic resonance-active Mn(II) and not located in mitochondria or vacuoles. Fermenting cells grown on Fe-sufficient and Fe-overloaded medium contained 400-450 μM Fe. In these cells, the concentration of nonmitochondrial NHHS Fe(II) declined 3-fold, relative to that in Fe-deficient cells, whereas the concentration of vacuolar NHHS Fe(III) increased to a limiting cellular concentration of ~300 μM. Isolated mitochondria contained more NHHS Fe(II) ions and substantial amounts of Fe(III) nanoparticles. The Fe contents of cells grown with excessive Fe in the medium were similar over a 250-fold change in nutrient Fe levels. The ability to limit Fe import prevents cells from becoming overloaded with Fe.

A Nonheme High-Spin Ferrous Pool in Mitochondria Isolated from Fermenting: Saccharomyces cerevisiae †

Mössbauer spectroscopy was used to detect pools of Fe in mitochondria from fermenting yeast cells, including those consisting of nonheme high-spin (HS) Fe(II) species, Fe(III) nanoparticles, and mononuclear HS Fe(III) species. At issue was whether these species were located within mitochondria or on their exterior. None could be removed by washing mitochondria extensively with ethylene glycol tetraacetic acid or bathophenanthroline sulfonate (BPS), Fe(II) chelators that do not appear to penetrate mitochondrial membranes. However, when mitochondrial samples were sonicated, BPS coordinated the Fe(II) species, forming a low-spin Fe(II) complex. This treatment also diminished the levels of both Fe(III) species, suggesting that all of these Fe species are encapsulated by mitochondrial membranes and are protected from chelation until membranes are disrupted. 1,10-Phenanthroline is chemically similar to BPS but is membrane soluble; it coordinated nonheme HS Fe(II) in unsonicated mitochondria. Further, the HS Fe(III) species and nanoparticles were not reduced by dithionite until the detergent deoxycholate was added to disrupt membranes. There was no correlation between the percentage of nonheme HS Fe(II) species in mitochondrial samples and the level of contaminating proteins. These results collectively indicate that the observed Fe species are contained within mitochondria. Mossbauer spectra of whole cells were dominated by HS Fe(III) features; the remainder displayed spectral features typical of isolated mitochondria, suggesting that the Fe in fermenting yeast cells can be coarsely divided into two categories: mitochondrial Fe and (mostly) HS Fe(III) ions in one or more non-mitochondrial locations.

Mössbauer and EPR Study of Iron in Vacuoles from Fermenting Saccharomyces cerevisiae

Vacuoles were isolated from fermenting yeast cells grown on minimal medium supplemented with 40 μM (57)Fe. Absolute concentrations of Fe, Cu, Zn, Mn, Ca, and P in isolated vacuoles were determined by ICP-MS. Mössbauer spectra of isolated vacuoles were dominated by two spectral features: a mononuclear magnetically isolated high-spin (HS) Fe(III) species coordinated primarily by hard/ionic (mostly or exclusively oxygen) ligands and superparamagnetic Fe(III) oxyhydroxo nanoparticles. EPR spectra of isolated vacuoles exhibited a g(ave) ~ 4.3 signal typical of HS Fe(III) with E/D ~ 1/3. Chemical reduction of the HS Fe(III) species was possible, affording a Mössbauer quadrupole doublet with parameters consistent with O/N ligation. Vacuolar spectral features were present in whole fermenting yeast cells; however, quantitative comparisons indicated that Fe leaches out of vacuoles during isolation. The in vivo vacuolar Fe concentration was estimated to be ~1.2 mM while the Fe concentration of isolated vacuoles was ~220 μM. Mössbauer analysis of Fe(III) polyphosphate exhibited properties similar to those of vacuolar Fe. At the vacuolar pH of 5, Fe(III) polyphosphate was magnetically isolated, while at pH 7, it formed nanoparticles. This pH-dependent conversion was reversible. Fe(III) polyphosphate could also be reduced to the Fe(II) state, affording similar Mössbauer parameters to that of reduced vacuolar Fe. These results are insufficient to identify the exact coordination environment of the Fe(III) species in vacuoles, but they suggest a complex closely related to Fe(III) polyphosphate. A model for Fe trafficking into/out of yeast vacuoles is proposed.

Biophysical Investigation of the Iron in Aft1-1up and Gal-YAH1 Saccharomyces cerevisiae

Aft1p is a major iron regulator in budding yeast Saccharomyces cerevisiae. It indirectly senses cytosolic Fe status and responds by activating or repressing iron regulon genes. Aft1p within the Aft1-1(up) strain has a single amino acid mutation which causes it to constitutively activate iron regulon genes regardless of cellular Fe status. This leads to elevated Fe uptake under both low and high Fe growth conditions. Ferredoxin Yah1p is involved in Fe/S cluster assembly, and Aft1p-targeted iron regulon genes are also upregulated in Yah1p-depleted cells. In this study Mössbauer, EPR, and UV-vis spectroscopies were used to characterize the Fe distribution in Aft1-1(up) and Yah1p-depleted cells. Aft1-1(up) cells grown in low Fe medium contained more Fe than did WT cells. A basal level of Fe in both WT and Aft1-1(up) cells was located in mitochondria, primarily in the form of Fe/S clusters and heme centers. The additional Fe in Aft1-1(up) cells was present as mononuclear HS Fe(III) species. These species are in a nonmitochondrial location, assumed here to be vacuolar. Aft1-1(up) cells grown in high Fe medium contained far more Fe than found in WT cells. The extra Fe was present as HS Fe(III) ions, probably stored in vacuoles, and as Fe(III) phosphate nanoparticles, located in mitochondria. Yah1p-deficent cells also accumulated nanoparticles in their mitochondria, but they did not contain HS Fe(III) species. Results are interpreted by a proposed model involving three homeostatic regulatory systems, including the Aft1 system, a vacuolar iron regulatory system, and a mitochondrial Fe regulatory system.

Changing iron content of the mouse brain during development.

Iron is crucial to many processes in the brain yet the percentages of the major iron-containing species contained therein, and how these percentages change during development, have not been reliably determined. To do this, C57BL/6 mice were enriched in (57)Fe and their brains were examined by Mössbauer, EPR, and electronic absorption spectroscopy; Fe concentrations were evaluated using ICP-MS. Excluding the contribution of residual blood hemoglobin, the three major categories of brain Fe included ferritin (an iron storage protein), mitochondrial iron (consisting primarily of Fe/S clusters and hemes), and mononuclear nonheme high-spin (NHHS) Fe(II) and Fe(III) species. Brains from prenatal and one-week old mice were dominated by ferritin and were deficient in mitochondrial Fe. During the next few weeks of life, the brain grew and experienced a burst of mitochondriogenesis. Overall brain Fe concentration and the concentration of ferritin declined during this burst phase, suggesting that the rate of Fe incorporation was insufficient to accommodate these changes. The slow rate of Fe import and export to/from the brain, relative to other organs, was verified by an isotopic labeling study. Iron levels and ferritin stores replenished in young adult mice. NHHS Fe(II) species were observed in substantial levels in brains of several ages. A stable free-radical species that increased with age was observed by EPR spectroscopy. Brains from mice raised on an Fe-deficient diet showed depleted ferritin iron but normal mitochondrial iron levels.

The catalase activity of diiron adenine deaminase

Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn2+ before induction, the post‐translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo‐enzyme was prepared and reconstituted with two equivalents of FeSO4. Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high‐spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [FeII/FeII]‐ADE catalyzed the conversion of H2O2 to O2 and H2O. The values of kcat and kcat/Km for the catalase activity are 200 s−1 and 2.4 × 104 M−1 s−1, respectively. [FeII/FeII]‐ADE underwent more than 100 turnovers with H2O2 before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high‐spin ferric with gave = 4.3 EPR signal and no evidence of anti‐ferromagnetic spin‐coupling. A model is proposed for the disproportionation of H2O2 by [FeII/FeII]‐ADE that involves the cycling of the binuclear metal center between the di‐ferric and di‐ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.