422 IRON-CONTAINING PROTEINS AND ENZYMES
thioether linkages holding the cytochrome c in place appear more strained in
the ferrocytochrome c in 30% ACN (PDB: 1LC1) than in any of the other
structures. Sivakolundu and Mabrouk discuss the various proposed mecha-
nisms for ferro - and ferricytochrome folding, electron transfer within cyto-
chrome c and its redox partners, and the role of cytochrome c in cell apoptosis
in relation to their studies of these cytochromes in nonaqueous solvents. These
topics will be discussed in Section 7.7.4.
7.7.4 Cytochrome c Folding, Electron Transfer, and Cell Apoptosis
7.7.4.1 Cytochrome c Folding. Harry B. Gray and Jay R. Winkler have
studied the cytochrome c folding landscape extensively. In a 2002 publication,
the researchers used energy landscape theory to interpret experimental inves-
tigation of protein folding.^124 Using horse, tuna, and yeast cytochrome c, the
workers substituted cobalt(III) ions for iron, fi nding that this did not change
the secondary structure (comparison of tuna cytochrome X - ray crystallo-
graphic structure, PDB: 1LFM^124 with PDB: 1CYT^115 ) or the absorption spectra.
The replacement of the iron ion with cobalt(III) slows down the fi nal step of
the cobalt(III) – cytochrome c (Co – cyt c) folding process so that early folding
intermediates can be examined by many different physical methods. The
Co(III) substitution does not change the situation around the heme, and the
X - ray crystallographic structures overlap almost perfectly (see bond distances
in Table 7.8 ). The absorption spectra of horse, tuna and yeast Co – cyt c are
virtually identical indicating that their heme environments are the same. For
Co – cyt c, the so - called Soret maximum occurs at 427 nm and the Q bands occur
at 534 and 568 nm. After addition of the denaturing reagent guanidine hydro-
chloride (GuHCl), unfolded protein is indicated by blue shifts to 422, 530,
and 564 nm. Far - UV circular dichroism (CD) minima for Co – cyt c at 208 and
222 nm are characteristic of α - helical structures in the folded protein. After
addition of the denaturing reagent GuHCl, the far - UV spectra indicate
random - coil conformation of the Co – cyt c protein. Trp59 fl uorescence, fully
quenched by energy transfer to heme in the folded protein, shows considerable
fl uorescence intensity in the unfolded Co – cyt c protein.
Kinetic experiments by these researchers have shown that horse and tuna
Co – cyto c folds several orders of magnitude more slowly than the native pro-
teins.^125 The refolding kinetics are biphasic because misfolding occurs during
protein unfolding and folding events. In horse cytochrome c, his26 and his33
are likely “ misfolding ” ligands, thereby replacing met80, whereas in tuna cyto-
chrome c, which lacks a his33 residue, one of 16 possible lysine residues prob-
ably becomes a “ misfolding ” ligand. To investigate the amino acid lysine ’ s
contribution to misfolding events, the reference 125 researchers replaced all
lysines (19 in horse cytochrome c, 16 in tuna) with homoarginine (Har) (see
Figure 7.35 ) and reinvestigated the refolding kinetics.
Homoarginine is not expected to coordinate to the cobalt(III) metal centers.
Results show that the tuna Har – Co – cyt c refolded in a single kinetic event