Protein Unfolding: 1H‐NMR Studies of Paramagnetic Ferricytochrome c‐550 from Horse Heart

Electronic transfer protein cytochrome c-550 from horse heart is studied in the unfolded state by means of paramagnetic 1H NMR. The protein contains 104 aminoacid residues and a heme group with low spin FeIII ion in the oxidized form of protein. The global secondary structure is of the α-helix type as occurs in the case of very other cytochromes c investigated such as cyt c-550 from Thiobacillus versutus or cyt c-551 from Pseudomonas aeruginosa. We have studied the coordination characteristic and electronic properties of heme iron horse heart ferricytochrome c-550 at increasing denaturing conditions (up to 3.1 M GuHCl and 288-323 K). The 1H T1 values of the signals were measured and some resonance assignments made based on EXSY experiments. The electronic structure of the iron(III) is discussed on the basis of the temperature dependence of the isotropic shifts and relaxation times. These results show that it is produced a change of spin, from low-spin iron(III) (2T2, S=1/2) in the folded state to high-spin iron(III) (6A1, S=5/2) in the unfolded state. It seems to be possible that in the opened structure the ferricyt c-550 loses one axial ligand (His/-) appearing the spin transition.