EPR of iron complexes
From NMR Wiki
Heme and hemo-proteins
Moura et. al [1] have applied EPR spectroscopy at 10K to a study of red-ox equilibria in Cytrochrome c3 from Desulfovibrio baculatus sulfate-reducing bacterium. Cytochrome c3 is a 13 kDa protein that has four heme prosthetic groups. Each heme center can be either in Fe(II) (reduced) or Fe(III) (oxidized) state. Thus the whole molecule can have five different red-ox states. Authors titrated Cyt c3 samples (830 μM, in 0.1 M TRIS-HCl pH 8.1 buffer in the presence of red-ox mediators: phenosaphranine, benzylviologen, methylviologen and 2-hydroxy-(1,4)-naphtoquinone at 10 μM each) with reducing (dithionite) or oxidizing (ferricyanide) reagents. Samples were transferred to EPR tubes and frozen at 77K. Spectra (recorded at 10K set by the flow cryostat) of the four EPR-active components of the equilibrium were deduced by substracting EPR spectra of samples at different red-ox potentials. Obtained difference spectra were used for the subsequent substractions. Result of the study was deduction of EPR parameters of the four heme groups and corresponding mid-point red-ox potentials. Oxidized forms of all four hemes were in the low-spin state. The authors have also attempted to assign heme moieties to the positions in the crystal structures based on the correlation of high (>3) gmax values and non-coplanar arrangement of axial hystidinyl ligands. Typical gmax max values were ~3, gmid ~2.25, gmin ~ 1.5.
