The simultaneous measurement of the decrease of available FeII ions and the increase of available FeIII ions allowed the analysis of the ferroxidase activity of two distinct apoferritins. Although recombinant human apoferritin (HuFtH) rapidly oxidizes FeII to FeIII, this iron is not properly stored in the ferritin cavity, as otherwise occurs in horse-spleen H/L-apoferritin (HsFt; H=heavy subunit, L= light subunit). Iron storage in these apoferritins was also studied in the presence of two copper-loaded mammalian metallothioneins (MT2 and MT3), a scenario that occurs in different brain-cell types. For HuFtH, unstored FeIII ions trigger the oxidation of Cu–MT2 with concomitant CuI release. In contrast, there is no reaction with Cu–MT2 in the case of HsFt. Similarly, Cu–MT3 does not react during either HuFtH or HsFt iron reconstitution. Significantly, the combination of ferritin and metallothionein isoforms reported in glia and neuronal cells are precisely those combinations that avoid a harmful release of FeII and CuI ions.

1. Departamento de Química Inorgánica
Ferritina-Metalotioneína: ¿Un tándem
regulador de iones metálicos en el
cerebro?
Departamento de Química Inorgánica
Instituto de Biotecnología
Universidad de Granada, SPAIN
Fernando Carmona Rodríguez-Acosta

2. Departamento de Química Inorgánica
Fe2+
apoferritin ferritin
Fe2+ + H2O2 ! OH· + OH- + Fe3+

3. 6 Hydrophobic Channels C48 Hydrophilic channels C3
Fe2+
Fe3+
Departamento de Química Inorgánica

4. WR Hagen
Fe2+ Fe3+
EC Theil
Departamento de Química Inorgánica

5. L21H3L12H12L0H24 L24H0
178 aa
21 KDa
Heavy Chain H
Fe(II) oxidation
171 aa
19 KDa
Light Chain L
Fe(III) nucleation
24 subunits
Departamento de Química Inorgánica

6. Apoferritin (H/L/HL)
1/1.000
+ Fe2+
+ [FeII(fz)3]2+
λmax= 562 nm
A test for measuring the ferroxidase activity:
Monitoring free Fe(II)
450 500 550 600 650
A562nm
batch
Sample 1
Sample 2
Sample 3
A562nm
1 2 3 4 5 6 7 8 9
time
Departamento de Química Inorgánica
ferrozine
fz

7. 0 50 100 150 200 250 300
A562nm
[FeII(fz)3]2+
Time (min)
Fe2+
Horse spleen
H3L21
Recombinant L
H0L24
Recombinant H
H24L0
5%Fe(II)
40%Fe(II)
44%Fe(II)
Fe(II)
oxidation
H
Departamento de Química Inorgánica
Fe(III)
nucleation
L
Control
ferrozine
fz

8. H HL L
> >
Recombinant H apoferritin
oxidizes the Fe(II)
in a few minutes…
> Fe(III)
nucleation
L
Fe(II)
oxidation
H
Ferroxidase activity
Departamento de Química Inorgánica

9. 0 50 100 150 200 250 300
0% [FeIII(Lf)]
[FeIII(Lf)]
λmax=464 nm
+
Apolactoferrin Lf
0% [FeIII(Lf)]
0% [FeIII(Lf)]
60% [FeIII(Lf)]
A562nm
Time (min)
5%Fe(II)/95%Fe(III)
40%Fe(II)/60%Fe(III)
44%Fe(II)/56%Fe(III)
Departamento de Química Inorgánica
Control
Carmona, Dominguez-Vera
Metallomics 2013, 5, 193

10. CONCLUSION:
H-rich apoferritin has strong ferroxidase
but low iron-storage activity.
Fe(II)
oxidation
H
Departamento de Química Inorgánica
Carmona, Theil, Dominguez-
Vera
Submitted

11. QUESTIONS:
• What happens if a HL-ferritin becomes more
and more rich in H subunits?
• Some influence on other (not only Fe) metal
metabolisms?
Departamento de Química Inorgánica

12. • Large [Fe] (as in liver!) found in Substantia Nigra
(Parkinson Disease, PD) and Hippocampus (Alzheimer
Disease, AD).
• Oxidative damage causes neuron death.
• Free Fe and Cu produce oxidative damage (Fenton
Reaction).
• Free metals induce aggregation of some proteins: β-
amyloid (AD) or α-synuclein (PD).
• “AD patients COULD have a dysfunction in brain as a
consequence of a disrupted ferritin H- and L-chain
synthesis” (J. Dobson 2010).
Departamento de Química Inorgánica

13. Substantia Nigra
(200 ppm Fe)
Nervous Cell Death in PD
Hippocampus
(50 ppm Fe)
Nervous Cell Death in AD
Fe(III)
nucleation
L
Fe(II)
oxidation
H
Carmona, Capdevila, Dominguez-Vera
Coord. Chem. Rev.
DOI: 10.1016/j.ccr.2013.03.034
Departamento de Química Inorgánica

14. + Fe2+
Cu2+
Zn2+
+ Fe2+
+ Fe2+
Cu2+ promotes
ferritin reconstitution
Zn2+ inhibits
ferritin reconstitution
Departamento de Química Inorgánica

15. Cu2+
Zn2+
Fe2+ Fe2+
Departamento de Química Inorgánica
Neuronal death

16. IN COLLABORATION WITH
MERCE CAPDEVILA
Departamento de Química Inorgánica

17. In NEURONS…
Departamento de Química Inorgánica

18. + Fe2+ + Cu-MT
H
fz
BTA
+ Fe3+
[FeII(fz)3]2+
λmax= 562 nm
[CuI(BTA)2]+
λmax= 480 nm
?
Departamento de Química Inorgánica

19. Departamento de Química Inorgánica
Fe3+ + Cu-MT Fe2+ + Cu+ + MTox

20. + Fe2+
H
Fe3+
+ Cu-MT
Fe2+
Cu+ Cu2+
Departamento de Química Inorgánica
Neuronal death
Carmona, Capdevila, Dominguez-Vera
Submitted

21. CONCLUSION:
In NEURONS, if apoferritin becomes H,
free Fe(II) leads to free Cu(I).
Fe and Cu are interrelated by apoferritin.
Departamento de Química Inorgánica
Fe2+ Fe2+
Fe2+Fe2+
Cu+
Cu+
Cu+
Cu-MTH-apoferritin
+

22. In MICROGLIA…
Departamento de Química Inorgánica

23. + Cu-MT
HL
+ Fe3+
+ Zn-MT
HL
+ Fe3+
?
?
Departamento de Química Inorgánica

24. 0
0,2
0,4
450 550 650
Absorbance
Wavelenght
Blank: Fe3++HLApo+fz
≈ free Fe2+
Blank: Fe3++Zn-MT+fz
Monitoring free Fe(II)
[FeII(fz)3]2+, λmax= 562 nm
0
0,2
0,4
450 550 650
Absorbance
Wavelenght
Zn-MT+
HLApo
Blank: Fe3++HLApo+fz
" free Fe2+
Cu-MT+
HLApo
Blank: Fe3++Cu-MT+fz
Departamento de Química Inorgánica

25. + Zn-MT
Fe2+
HL
+ Fe3+
Zn2+
+ Cu-MT
Fe2+
Cu+
HL
+ Fe3+
Cu2+
Cu2+ promotes
ferritin reconstitution
Zn2+ inhibits
ferritin reconstitution
Carmona, Capdevila, Dominguez-
Vera
submitted
Departamento de Química Inorgánica

26. CONCLUSIONS:
In MICROGLIA, Cu-MT promotes the IRON
STORAGE in ferritin.
Zn-MT inhibits the ferritin iron loading and leads
to free Fe(II) and Zn(II).
Departamento de Química Inorgánica

27. In progress: HARDER STILL.
More complexes and MORE realistic
scenarios: α-synuclein or β-amyloid !
+ Cu-MT Cu++ Fe2+
Cu2+
IN COLLABORATION WITH
CLAUDIO FERNANDEZ
(Rosario, Argentina)
H
Departamento de Química Inorgánica

28. Daniela
http://www.ugr.es/~josema
fcar@ugr.es
Rafa Josema
Nati
Elsa
Migue
Puri
Paulina

29. MERCE CAPDEVILA GROUP
UNIVERSITAT AUTONOMA DE BARCELONA
SPAIN
SILVIA ATRIAN GROUP
UNIVERSITAT DE BARCELONA
SPAIN

30. Project CTQ2009-09344
CTQ2012-32236
…and the financial support of:

31. La Alhambra desde el “Carmen de la Victoria”
(Universidad de Granada)
Gracias!