The 695-µ band of ferricytochrome c at neutral pH can be abolished by increasing the temperature and by adding denaturing agents or iron-binding ligands. These spectroscopic modifications are fast and reversible. Between 0.07 and 1.7 mM concentrations Beer's law is obeyed by the 695-µ band of ferricytochrome c, intimating that changes in aggregation are not involved in the transformations that cause the disappearance of the band. Addition of each of several nitrogenous bases to heme and hemoprotein solutions devoid of a 695-µ band did not cause the appearance of this band, suggesting that the 695-µ band is determined by structural features other than the chemical nature of the iron ligands. This result and the effect of denaturing agents are interpreted as indications that the 695-µ band is intimately related to the overall conformation of the protein moiety of ferricytochrome c. The effect of temperature on the band can be considered as a conformational isomerism between a "cold" and a "hot" form of the hemoprotein, stable at low and high temperatures, respectively. At 25°this equilibrium is characterized by the following thermodynamic parameters: AF = +1.7 kcal/mole; AH = + 14.6 kcal/mole; AS = +43 eu. These values are of an order of magnitude compatible with changes in protein conformation. At 38°about 15% of the molecules are in the "hot" conformation, implying a possible role of this isomerism in biochemical processes in which cytochrome c participates.In its oxidized state, mammalian cytochrome c has an absorption band in the red region of the spectrum, characterized by a maximum at 695 µ and an inflection at 655 µ. This band was first described by Theorell and Akesson (1939), who also showed that it disappears at pH lower than 2.5 and higher than 9.35 (Theorell and Akesson, 1941).
The tyrosine-67 to phenylalanine mutated rat cytochrome c is similar to the unmutated protein in its spectral, reduction potential, and enzymic electron-transfer properties. However, the loss of the 695-nm band, characteristic of the ferric form of the normal low-spin physiologically active configuration, occurs 1.2 pH units higher on the alkaline side and 0.7 pH unit lower on the acid side. Similarly, the heme ironmethionine-80 sulfur bond is more stable to temperature, with the midpoint ofthe transition being 300C higher, corresponding to an increase in AH of 5 kcal/mol (1 cal = 4.184 J), partially mitigated by an increase of 11 entropy units in AS. Urea has only slightly different effects on the two proteins. These phenomena are best explained by considering that the loss of one of the three hydrogen-bonding side chains, tyrosine-67, asparagine-52, and threonine-78, which hold an internal water molecule on the "left, lower front" side of the protein [Takano, Biol. 153,, is sufficient to prevent its inclusion in the mutant protein, leading to a more stable structure, and, as indicated by preliminary proton NMR two-dimensional phase-sensitive nuclear Overhauser effect spectroscopy analyses, a reorganization of this area. This hypothesis predicts that elimination ofthe hydrogenbonding ability of residue 52 or 78 would also result in cytochromes c having similar properties. It is not obvious why the space-filling structure involving the internalized water molecule that leads to a destabilization energy of about 3 kcal/mol should be subject to extreme evolutionary conservation, when a more stable and apparently fully functional structure is readily available.
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