The nature of the thermal equilibrium affecting the iron coordination of ferric cytochrome c.

In cytochrome c, ligation of the heme iron by the methionine-80 sulfur plays a major role in determining the structure and the thermodynamic stability of the protein. In the ferric state, this bond is reversibly broken by moderately acid or alkaline pH's (pK's 2.5 and 9.4, respectively) and by exogenous ligands. NMR studies of horse ferricytochrome c in which the Met-65 and Met-80 methyl groups were chemically enriched with 13C demonstrate that, at 59 degrees C, a temperature at which the protein is still folded, the sulfur-iron bond is already partially broken. This structural change corresponds to the reversible disappearance upon moderate heating of the 695 nm band, characteristic of the sulfur-iron coordination of this protein. The thermal effect results from a shift in the alkaline pK from 9.4 at 25 degrees C to 8.2 at 59 degrees C. The exchange rate from iron-bound to free methionine-80 at 59 degrees C is 1.8 s-1, as measured by saturation transfer experiments. The free and bound methionine-80 epsilon-methyl groups in the 1H spectrum are assigned as (1.87, 2.25) and -21.43, respectively; in the 13C spectrum they are assigned as 15.6 and 12.8, respectively (all these values are in ppm from 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt).

Effects of mutating Asn-52 to isoleucine on the haem-linked properties of cytochrome c.

Asn-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was changed to isoleucine by site-directed mutagenesis and the mutated proteins expressed in and purified from cultures of transformed yeast. This mutation affected the affinity of the haem iron for the Met-80 sulphur in the ferric state and the reduction potential of the molecule. The yeast protein, in which the sulphur-iron bond is distinctly weaker than in vertebrate cytochromes c, became very similar to the latter: the pKa of the alkaline ionization rose from 8.3 to 9.4 and that of the acidic ionization decreased from 3.4 to 2.8. The rates of binding and dissociation of cyanide became markedly lower, and the affinity was lowered by half an order of magnitude. In the ferrous state the dissociation of cyanide from the variant yeast cytochrome c was three times slower than in the wild-type. The same mutation had analogous but less pronounced effects on rat cytochrome c: it did not alter the alkaline ionization pKa nor its affinity for cyanide, but it lowered its acidic ionization pKa from 2.8 to 2.2. These results indicate that the mutation of Asn-52 to isoleucine increases the stability of the cytochrome c closed-haem crevice as observed earlier for the mutation of Tyr-67 to phenylalanine [Luntz, Schejter, Garber and Margoliash (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3524-3528], because of either its effects on the hydrogen-bonding of an interior water molecule or a general increase in the hydrophobicity of the protein in the domain occupied by the mutated residues. The reduction potentials were affected in different ways; the Eo of rat cytochrome c rose by 14 mV whereas that of the yeast iso-1 cychrome c was 30 mV lower as a result of the change of Asn-52 to isoleucine.

The reactivity of cytochrome c with soft ligands.

The spectral changes caused by binding soft ligands to the cytochrome c iron and their correlation to ligand affinities support the hypothesis that the iron-methionine sulfur bond of this heme protein is enhanced by delocalization of the metal t2g electrons into the empty 3d orbitals of the ligand atom. These findings also explain the unique spectrum of cytochrome c in the far red.

Direct observation of the methionine residues of cytochrome c by 13C nuclear magnetic resonance spectroscopy.

The two Cepsilon-methyl methionine groups in cytochrome c have been chemically enriched (45%) with 13C. Their 13C NMR signals have been monitored in both the oxidized and reduced states and under various solution conditions. Methionine residue 80 showed characteristic chemical shift positions for the reduced Fe(II) and cyano-Fe(III) forms. No signal for methionine 80 was observed in the oxidized Fe(III) form due to the paramagnetic effect of the iron atom to which it is bonded, but the position of the methionine 65 signal was shifted, indicating that it is sensitive to the change of oxidation state. Two well resolved signals were observed at pH 11 for the Fe(III) form but only one was resolved at pH 2, indicating that while methionine 80 is definitely displaced from the iron atom at alkaline pH, it may not be in acid conditions.