Bioavailability and Chemistry of Iron(III) Porphyrin Complexes

1. Results Heterotrophic Bacteria ~4 nM of the 5 nM Fe(III) Coproporphyrin added and 2nM of the 5 nM Fe(III) DFO added was internalized after 40 hours by the heterotrophic bacteria. Killed control uptake was low. Coproporphyrin Thalassiosiraweissflogii Copro CuCopro After 38 hours T.weissflogii took up ~0.6 nM of the Fe(III) Coproporphyrin but none of the Fe(III) DFO. However the killed control uptake was high as becomes more apparent when uptakes are normalized to cell numbers as shown below. Bioavailability and Chemistry of Iron(III) Porphyrin Complexes Brian Hopkinson1, Joshawna Nunnery2, Kathy Barbeau1 1Scripps Institution of Oceanography/ UCSD 2Southampton College – Long Island University Binding Studies Uptake Experiments • Abstract • As an essential but scarce micronutrient, iron is a valuable commodity to life in the ocean. The bioavailability of iron is dependent on its chemical form and uptake mechanisms of the resident microbes. In seawater dissolved iron is chelated by organic ligands and although the identity of these ligands is speculative, porphyrins have been suggested to constitute a fraction. In addition, iron porphyrins make up a significant portion of the intracellular iron pool so that their bioavailability may be particularly important in understanding pathways of iron recycling. • We studied the potential for porphyrins to bind iron in seawater, and the bioavailability of a well-characterized Iron(III) Coproporphyrin complex. • A method for synthesizing radiolabelled 55Fe(III) Coproporphyrin was developed. • The 55Fe(III) Coproporphyrin was bioavailable to an assemblage of heterotrophic marine bacteria. The coastal diatom T.weissflogii took up a small amount of 55Fe(III) Coproporphyrin. • Preliminary binding studies indicated that iron(III) did not bind to porphyrins at high nM concentrations. Copper did bind to the porphyrin tested. Binding studies at high nanomolar porphyrin and metal concentrations were done both to assess the tetrapyrroles’ potential to be natural iron ligands and as a route to radiotracer synthesis. 50 - 100nM Coproporphyrin was added to seawater followed by 10 - 100nM Fe(III), Fe(II), Cu(II), or Ni(II). The mixture was allowed to react in the dark for 24 hours using UV/Vis scans to monitor progress of any reaction. After 24 hours the porphyrins were concentrated onto a hydrophobic resin (HP20s) and eluted with an organic solvent. Extracted porphyrins were analyzed by HPLC and mass spectrometry. Only Cu(II) was found to bind to the porphyrin tested under these conditions. Our studies suggest the kinetics of iron insertion into tetrapyrroles is too slow to be of environmental importance. However, the experiments should be done at lower concentrations (~1 nM) more representative of natural conditions before making any firm conclusions. The bioavailability of 55Fe(III) Coproporphyrin was tested on heterotrophic marine bacteria and the coastal diatom Thalassiosira weissflogii. Heterotrophic bacteria internalized nearly all the added 55Fe(III) Coproporphyrin clearly demonstrating its bioavailability to these bacteria. The diatoms took up the radiotracer and there was little degradation of the Fe(III) Coproporphyrin. However the killed control activity was relatively high, making the extent of active uptake somewhat ambiguous. Methods Uptake experiments were conducted in acid cleaned polycarbonate bottles under fluorescent lights. Control treatments were killed with glutaraldhyde (bacteria) or KCN (diatoms). After allowing 1 hour to kill the controls, 55Fe(III) Coproporphyrin was added to one set of replicate live and killed control bottles while 55Fe(III) DFO was added to another. Final concentration of the radiotracer was 5nM in all treatments. In a parallel experiment 25nM Fe(III) Coproporphyrin was added to sterile media and its concentration was monitored by UV/Vis throughout the experiment to check for degradation of the porphyrin. Samples from the experimental and killed control treatments were taken every 12-24 hours for 1-2 days and washed with Ti(III)-EDTA-Citrate to remove surface bound iron. Activity was measured by scintillation counting. Heterotrophic marine bacteria were grown up by enriching GF/F filtered seawater from the Scripps Pier with glucose, NO3, and PO4. Axenic T.weissflogii (CCMP 1051) were cultured in Aquil and rinsed and resuspended in artificial seawater with nutrients prior to starting the uptake experiment. Summary of Metal Binding Radiotracer Synthesis Iron Complexation Neither Iron(III) nor Iron(II) bound to Coproporphyrin in seawater as shown by HPLC of the extracted porphyrins and UV/Vis spectra of the solution (not shown). Blue Chromatogram: 10nM Fe(III): 50nM Copro extract Red Chromatogram: same + Fe(III)Copro spike In order to do bioavailability studies a method was developed to synthesize a radiolabelled iron porphyrin. Iron(III) Coproporphyrin was chosen for its solubility in seawater and structural similarity to biological porphyrins. Complete iron chelation was achieved with a modified Acetic Acid/Acetate method in which Coproporphyrin, 1M sodium acetate, and 55FeCl3 5 times in excess of the porphyrin were dissolved in glacial acetic acid and refluxed for 16 hours. After distilling off most of the glacial acetic acid, the 55Fe(III)Coproporphyrin was resuspended in 5mL Milli-Q water. This step was necessary in order to purify the compound by extraction onto a hydrophobic resin (HP20s). Once the 55Fe(III) Coproporphyrin was stuck onto the resin it was washed with Milli-Q water and Ti(III)-EDTA-citrate wash to remove the excess 55Fe. To ensure that all the excess 55Fe was removed we did a “mock” synthesis in which the standard synthesis and purification steps were followed except no Coproporphyrin was added to the reaction mixture. Our purification protocol was adequate as very little 55Fe was found in the methanol eluent from the mock synthesis. Copper Complexation Copper(II) bound to Coproporphyrin when incubated overnight in seawater. Identity of the Cu(II) Coproporphyrin was established using UV/Vis, HPLC, and mass spectrometry. Summary of Synthesis and Purification 1. Started with 4µM Coproporphyrin and 1M Sodium Acetate in Glacial Acetic Acid. 2. Added 55FeCl3 for a final concentration of 20µM 55Fe(III). 3. Refluxed 16 hours. 4. Distilled off Glacial Acetic acid until only ~0.5mL is left, then added 5mL water. 5. Extracted 55Fe(III) Coproporphyrin onto a column of HP20s resin. 6. Washed column with 10mL of: water (2x), Ti wash (2x), water(4-6x). 7. Eluted 55Fe(III) Coproporphyrin with 5-10mL methanol. HPLC of Cu(II):Coproporphyrin experiment Blue Chromatogram: 50nM Cu(II), 50nM Copro extract Black Chromatogram: 0nM Cu(II), 50nM Copro extract Note: retention times have shifted slightly between runs UV/Vis of Copro and Cu(II)Copro Coproporphyrin • Conclusions • A synthesis for 55Fe(III) Coproporphyrin was developed since mixing 55Fe(III) and Coproporphyrin in seawater did not result in binding. • Heterotrophic bacteria were able to take up 55Fe(III) Coproporphyrin and 55Fe(III) DFO quite rapidly indicating their iron uptake mechanisms are capable of accessing diverse forms of iron. The coastal diatom, T.weissflogii, took up a small amount of 55Fe(III) Coproporphyrin but no 55Fe(III) DFO. Future goals include repeating the diatom experiment with Fe-stressed cells and a better killed control. Cu(II)Copro, synthesized Cu(II)Copro, SW incubation Above: MS and MS/MS of Cu(II) Coproporphyrin Below: Identification of mass fragments Acknowledgements: We would like to thank Ralf Goericke for help with work on porphyrins. Funding from: American Chemical Society/ Petroleum Research Fund, Grant 380662 (to KB), and NDSEG Graduate Fellowship (to BH)