Evidence That a Respiratory Shield in \u3cem\u3eEscherichia coli\u3c/em\u3e Protects a Low-Molecular-Mass Fe-\u3csup\u3eII\u3c/sup\u3e Pool from O\u3csub\u3e2\u3c/sub\u3e-Dependent Oxidation

Iron is critical for virtually all organisms, yet major questions remain regarding the systems\u27 level understanding of iron in whole cells. Here, we obtained Mössbauer and EPR spectra of Escherichia coli cells prepared under different nutrient iron concentrations, carbon sources, growth phases, and O2 concentrations to better understand their global iron content. We investigated wild-type cells and those lacking Fur, FtnA, Bfr, and Dps proteins. The coarse-grain iron content of exponentially growing cells consisted of iron-sulfur clusters, variable amounts of nonheme high-spin FeII species, and an unassigned residual quadrupole doublet. The iron in stationary-phase cells was dominated by magnetically-ordered FeIII ions due to oxyhydroxide nanoparticles. Analysis of cytosolic extracts by size-exclusion chromatography detected by an online inductively-coupled plasma mass spectrometer revealed a low-molecular-mass (4LMM) FeII pool consisting of two iron complexes with masses of ~ 500 (major) and ~1300 (minor) Da. They appeared to be high-spin FeII species with mostly O donor ligands, perhaps a few N donors, and probably no S donor. Surprisingly, the iron content of E. coli and its reactivity with O2 were remarkably similar to those of mitochondria. In both cases, a respiratory shield composed of membrane-bound iron-rich respiratory complexes may protect the LMM FeII pool from reacting with O2. When exponentially growing cells transition to stationary phase, the shield deactivates as metabolic activity declines. Given the universality of oxidative phosphorylation in aerobic biology, the iron content and respiratory shield in other aerobic prokaryotes might be similar to those of E. coli and mitochondria

Characterization and Speciation of the Low Molecular Mass Iron Pool in Blood

The foci of this work were in characterizing and speciating the low molecular mass (LMM) iron pool in blood. We operationally define this pool as non-proteinaceous iron species with masses ＜10 kDa. In iron overload disorders (e.g., hemochromatosis) some portion of this pool is involved in organ damage and is termed non-transferrin bound iron (NTBI). NTBI has been poorly characterized and is thought to be ferric citrate. The blood’s major iron transport protein, transferrin (Tf), is typically 30% saturated; when saturation levels are elevated, NTBI concentrations typically increase due to the unregulated trafficking of iron into the blood. The first part of this work involves work done on characterizing the LMM pool of iron in blood. Characterization (i.e., mass, concentration, number of species, chemical identity) of the LMM pool of iron was performed using an anaerobic and chilled liquid chromatography system, coupled to an inductively coupled plasma mass spectrometer (LC-ICP-MS). Flow-through solutions (FTS) from filtered plasma were injected onto a size-exclusion column (SEC). In the second part of this work, the intact circulatory and digestive system of an iron-deficient pig was used to detect endogenously formed NTBI by tracing labeled nutrient iron (57FeII-ascorbate). Blood was sampled from before and after the liver to observe NTBI formation and the effect of the liver via LC-ICP-MS. Two to six conserved LMM species with apparent masses ranging from 400- 2500 Da and concentrations ranging from 10-100 nM were detected in blood from various healthy mammals and hemochromatosis (HC) humans. Of these, the species in the 400 -500 Da range were the most reproducible. HC human FTS did not show any difference relative to that of a healthy human, possibly due to the treatment of their disease. Comparison of the observed species in FTSs with ferric citrate standards showed similar, though not identical, elution profiles. Ferric citrate was not the predominate species in healthy blood plasma, though it could be a constituent. 57Fe traced nutrient iron was predominately bound to Tf with no detectable intermediates, suggesting that nutrient iron is channeled to Tf with forming stable-intermediate species. Endogenous (56Fe) LMM iron species were observed with masses ranging from 400- 1600 Da. The liver both absorbed and released LMM iron species to a modest degree, indicative of its role in iron metabolism. The endogenous species detected are thought to be generated from red blood cell recycling

Characterization and Speciation of the Low Molecular Mass Iron Pool in Blood

The foci of this work were in characterizing and speciating the low molecular mass (LMM) iron pool in blood. We operationally define this pool as non-proteinaceous iron species with masses ＜10 kDa. In iron overload disorders (e.g., hemochromatosis) some portion of this pool is involved in organ damage and is termed non-transferrin bound iron (NTBI). NTBI has been poorly characterized and is thought to be ferric citrate. The blood’s major iron transport protein, transferrin (Tf), is typically 30% saturated; when saturation levels are elevated, NTBI concentrations typically increase due to the unregulated trafficking of iron into the blood. The first part of this work involves work done on characterizing the LMM pool of iron in blood. Characterization (i.e., mass, concentration, number of species, chemical identity) of the LMM pool of iron was performed using an anaerobic and chilled liquid chromatography system, coupled to an inductively coupled plasma mass spectrometer (LC-ICP-MS). Flow-through solutions (FTS) from filtered plasma were injected onto a size-exclusion column (SEC). In the second part of this work, the intact circulatory and digestive system of an iron-deficient pig was used to detect endogenously formed NTBI by tracing labeled nutrient iron (57FeII-ascorbate). Blood was sampled from before and after the liver to observe NTBI formation and the effect of the liver via LC-ICP-MS. Two to six conserved LMM species with apparent masses ranging from 400- 2500 Da and concentrations ranging from 10-100 nM were detected in blood from various healthy mammals and hemochromatosis (HC) humans. Of these, the species in the 400 -500 Da range were the most reproducible. HC human FTS did not show any difference relative to that of a healthy human, possibly due to the treatment of their disease. Comparison of the observed species in FTSs with ferric citrate standards showed similar, though not identical, elution profiles. Ferric citrate was not the predominate species in healthy blood plasma, though it could be a constituent. 57Fe traced nutrient iron was predominately bound to Tf with no detectable intermediates, suggesting that nutrient iron is channeled to Tf with forming stable-intermediate species. Endogenous (56Fe) LMM iron species were observed with masses ranging from 400- 1600 Da. The liver both absorbed and released LMM iron species to a modest degree, indicative of its role in iron metabolism. The endogenous species detected are thought to be generated from red blood cell recycling

COA6 is structurally tuned to function as a thiol-disulfide oxidoreductase in copper delivery to mitochondrial cytochrome c oxidase

In eukaryotes, cellular respiration is driven by mitochondrial cytochrome c oxidase (CcO), an enzyme complex that requires copper cofactors for its catalytic activity. Insertion of copper into its catalytically active subunits, including COX2, is a complex process that requires metallochaperones and redox proteins including SCO1, SCO2, and COA6, a recently discovered protein whose molecular function is unknown. To uncover the molecular mechanism by which COA6 and SCO proteins mediate copper delivery to COX2, we have solved the solution structure of COA6, which reveals a coiled-coil-helix-coiled-coilhelix domain typical of redox-active proteins found in the mitochondrial inter-membrane space. Accordingly, we demonstrate that COA6 can reduce the copper-coordinating disulfides of its client proteins, SCO1 and COX2, allowing for copper binding. Finally, our determination of the interaction surfaces and reduction potentials of COA6 and its client proteins provides a mechanism of how metallochaperone and disulfide reductase activities are coordinated to deliver copper to CcO.Fil: Soma, Shivatheja. Texas A&M University. Department of Biochemistry and Biophysics; United States.Fil: Morgada, Marcos N. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Morgada, Marcos N. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Biológica. Área Biofísica; Argentina.Fil: Naik, Mandar T. Texas A&M University. Department of Biochemistry and Biophysics; United States.Fil: Naik, Mandar T. Brown University. Department of Molecular Pharmacology, Physiology, and Biotechnology; United States.Fil: Boulet, Aren. University of Saskatchewan. Department of Biochemistry, Microbiology and Immunology; Canada.Fil: Roesler, Anna A. University of Saskatchewan. Department of Biochemistry, Microbiology and Immunology; Canada.Fil: Dziuba, Nathaniel. Texas A&M University. Department of Biochemistry and Biophysics; United States.Fil: Ghosh, Alok. Texas A&M University. Department of Biochemistry and Biophysics; United States.Fil: Ghosh, Alok. University of Calcutta. Department of Biochemistry; India.Fil: Yu, Qinhong. University of California. Department of Chemistry; United States.Fil: Lindahl, Paul A. Texas A&M University. Department of Biochemistry and Biophysics; United States.Fil: Lindahl, Paul A. Texas A&M University. Department of Chemistry; United States.Fil: Ames, James B. University of California. Department of Chemistry; United States.Fil: Leary, Scot C. University of Saskatchewan. Department of Biochemistry, Microbiology and Immunology; Canada.Fil: Vila, Alejandro J. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Vila, Alejandro J. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Biológica. Área Biofísica; Argentina.Fil: Gohil, Vishal M. Texas A&M University. Department of Biochemistry and Biophysics; United States