Bioavailability and Chemistry of IronIII Porphyrin Complexes

1
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)