Comparative Solvent Perturbation of Horse Heart Cytochrome c and Rhodospirillum rubrum Cytochrome CA-*

cytochrome

The extent of exposure of heme to solvent in horse heart cytochrome c and Rhodospirillum rubrum cz was investigated to determine whether a correlation exists between the properties of these oxidation-reduction proteins and their heme environments.
Solvent perturbation absorption difference spectra were measured using ethylene glycol, glycerol, and sucrose at concentrations between 0 and 30%. Cytochrome c appears to exhibit a somewhat greater extent of heme exposure than cytochrome cg for both the oxidized and reduced states. These results suggest that the lower oxidationreduction potential of cytochrome c may in part be due to a greater extent of exposure of the heme. The oxidized state of both proteins appears to exhibit a greater exposure than that of the reduced state which is consistent with a more favorable environment for the charge on the ferric heme coordination center.
X-ray crystallographic studies have shown that mitochondrial cytochrome c and the bacterial cytochrome c2 of Rhodosgirillum rubrum exhibit extensive structural similarities (l-3). The heme moiety in both cytochromes is situated in a hydrophobic crevice with exposure of heme occurring at only one edge which is surrounded by a ring of positively charged lysine side chains on the surface of the protein. The heme iron in both proteins is coordinated to the imidazole group of a histidyl residue and the sulfur of a methionyl residue in positions 5 and 6, respectively.
However, despite such structural similarities, qualitative differences have been observed in the near-ultraviolet circular dichroism spectra of cytochromes c and cz which have been interpreted in terms of subtle differences in the heme environments of these cytochromes (4). Significant differences in other chemical and physical properties of these proteins have also been reported. Different rates of oxidation and reduction have been observed for cytochromes c and cz in their reactions with iron hexacyanides (5-7). The reduction potential of cytochrome c2 is 60 mV higher than that of cytochrome c. Furthermore, both the rates of oxidation and reduction (7) and the oxidation-reduction potential of cytochrome c2 (8) have been found to be dependent on pH in the range between 5 and 8, whereas with cytochrome c these properties are independent of pH in this range (9,10).
It has been suggested by Kassner (11,12) that the hydrophobic character of the heme environments in these proteins may account for the differences between the oxidation-reduc- The exposure of the heme to solvent has also been considered as a factor in the rates of electron transfer reactions. Marcus theory (13) has been found useful in predicting reaction rates of cytochrome c with a variety of inorganic reactants (14-16) and in accounting for the observed self-exchange rate constant (17) of cytochrome c. The self-exchange rate constant for cytochrome c has been rationalized as being the product of the self-exchange rate constant for the heme moiety and a steric factor which accounts for the fact that the effective area of the heme group occupies only a fraction of the total surface area of the protein (18). Therefore, differences between the self-exchange rate constants for cytochromes c and c2 may be related to differences in exposure of the heme to solvent in these proteins.
The purpose of this study is to explore the differences in the exposure of the heme moiety in cytochrome c and cytochrome c2 and to use them as a basis for understanding the difference in oxidation-reduction properties of these cytochromes.
The relative exposure of the hemes in cytochromes c and cz is determined through the application of solvent perturbation difference spectroscopy which has had wide application in determining the relative exposure of chromophores in heme proteins (19)(20)(21)

RESULTS
In order to determine the relative exposure of heme to solvent in ferrous and ferric cytochromes c and ~2, solvent perturbation absorption difference spectra were measured using ethylene glycol, glycerol, and sucrose at concentrations between 0 and 30%. The principal effect of each of the perturbants on cytochromes c and cz is an enhancement of the Soret absorbance.
A linear relationship between AE/E and perturbant concentration has been used as an indication that solvent perturbation occurs without changes in the native protein conformation (23) and has been shown to be adequate in detecting solvent denaturation in cytochrome c (19) and other heme proteins (20,21). In the present study, the relationship between A/e values and perturbant concentration was found to be linear in all cases over the range investigated as shown for   Tables I and II. The heme exposure in ferrous and ferric cytochromes c and cz was found not to be a function of the size of perturbants used. These results are in agreement with previous results for ferrous cytochrome c (19) and similar to those observed for other heme proteins (20, 21).
As seen in Table I, the extent of enhancement, AJE, and the resultant exposure of the heme relative to the model chromophore are somewhat greater for ferric cytochrome c than for ferric cytochrome c2. The same effect is observed for the ferrous cytochromes as shown in Table II. A further  comparison  of Tables I and II indicates that for both cytochromes c and c2 the relative exposure in the ferric state appears greater than that in the ferrous state. DISCUSSION The solvent perturbation results summarized in Tables I  and II indicate that the heme moiety in both cytochromes c and cz is partially exposed to solvent and that for cytochrome c2 the folding of the polypeptide chain about the heme restricts its contact with solvent to a greater extent than that in cytochrome c. It has been proposed that a more hydrophilic heme environment stabilizes the ferric state relative to a hydrophobic environment and thus results in a lowering of the oxidation-reduction potential (11). On this basis, one would predict that cytochrome c2 should have a higher oxidation-reduction potential than cytochrome c since a more hydrophobic environment would in general be expected for a smaller degree of exposure to solvent. The observation that the oxidation-reduction potential of cytochrome c2 is about 60 mV higher than that of cytochrome c suggests that a corre-lation exists between solvent exposure and oxidation-reduction potential. This observation is in contrast to the lack of correlation between the oxidation-reduction potentials of equine heart cytochrome c and Prosthecochloris aestuarii cytochrome cs5 and the per cent exposure of the heme group in these cytochromes (21). Although cytochrome c555 has also been shown to be characterized by the same axial ligands as cytochrome c and c2, spectroscopic and sequence differences have suggested that it be classified with other cytochromes (33).
The observation that the heme exposure for the ferricytochromes c is greater than that for the ferrocytochromes c suggests that a conformational change toward a tighter heme crevice occurs upon reduction in both cytochromes c and c2. This result is consistent with the expectation that the oxidized state would be stabilized by a more polar heme environment (11,12,34). These results are then also consistent with the concept that differences between the oxidation-reduction potentials of these cytochromes may be due to differences in the change in conformational energy between the oxidized and reduced states.
It has recently been shown that a correlation exists between the oxidation-reduction potential and methionine methyl resonances of various ferrocytochromes which indicates that there is a variation in the iron-sulfur bond length with variation in oxidation-reduction potential (35). This concept suggests that the iron-sulfur bond length in these proteins is determined by the minimal conformational energy of the polypeptide and, therefore, exhibits a value intermediate between the bond lengths of unconstrained ferrous and ferric complexes. However, the change in conformation indicated by the difference in per cent exposure of oxidized and reduced states suggests that the iron-sulfur bond length may not be rigidly constrained.
It should be acknowledged that an evaluation of the solvent perturbation of these cytochromes in terms of a per cent exposure of the heme to solvent requires a comparison of the data for the proteins to that of a model chromophore completely exposed to the solvent. In this study, the heme octapeptide in the appropriate oxidation state plus imidazole was used as the model chromophore.
Although this complex should suffice as a model chromophore, a more appropriate model would be the methionine. histidine s heme complex. However, added methionine appears to coordinate incompletely to the ferric heme octapeptide at accessible concentrations (30). The solvent perturbation results show that a decrease in the extent of exposure is not observed with an increase in the size of perturbant.
This indicates that the heme crevice in cytochromes c and c2 does not selectively restrict the access of the perturbants to the heme according to size and suggests that a portion of the heme protrudes out of the hydrophobic crevice into the solvent for both the oxidized and reduced states of cytochromes c and c2. These observations and the aforementioned differences between the heme exposures of cytochromes c and c2 are also pertinent to proposed mechanisms of electron transfer. As previously mentioned, Marcus theory has been used in the analysis of the reactions of cytochrome c with various inorganic reactants involving an outer sphere mechanism and in the determination of the selfexchange rate constant of cytochrome c. This self-exchange rate constant has been rationalized as being the product of the self-exchange rate constant for the heme moiety and a steric factor which is essentially an expression of the exposure of the heme moiety. It, therefore, should follow that the difference in the self-exchange rate constants for these two cytochromes should be dependent on differences in the exposure of the heme moiety. Since the heme in cytochrome cz is less exposed than the heme in cytochrome c, one would predict a smaller steric factor for cytochrome c2 which in turn would result in a smaller self-exchange rate constant for cytochrome cp than for cytochrome c. However, calculated self-exchange rates based on cross-reaction rates with ferricyanide or ferrocyanide indicate that cytochrome c2 has a greater self-exchange rate than cytochrome c (36). Thus, the present results suggest that electron transfer may occur through a mechanism where exposure of the heme is not a limiting factor.