THE EVOLUTIONARY SIGNIFICANCE OF THE
SPECIES VARIATION IN
CYTOCHROME c STRUCTURE
GORDON C. MILLS*
Professor of Biochemistry
University of Texas Medical Branch,
From: JASA 20 (June
Information relating to the differences in amino acid sequence of cytochrome c of various species is reviewed briefly. The author notes that previous investigators have explained the similarities in amino acid sequence on the basis that all of these types of cytochrome c were derived initially from a single precursor segment of DNA, presumably as a result of mutations. A major limitation in this hypothesis is noted and an alternative explanation for these similarities is proposed: that similarities in structure of cytochrome c of various species is a consequence of a single Divine knowledge behind the creation of all these species.
In recent years, application of newer analytical tech niques have permitted the determination of the amino acid sequence of a number of proteins with biological activity. Noteworthy among these are the sequences of amino acids in the polypeptide chains of hemoglobin and in cytochrome c. In the latter case, the amino acid sequence has been determined in cytochrome c from at least 35 different species. Since there are many similarities in structure in these different cytochrome c molecules, previous investigators have concluded that the genes responsible for the production of the protein structure of cytochrome c are all derived from a common archetypal piece of DNA1. The following quotation indicates the evolutionary significance that has been ascribed to these findings: "Presently under investigation, among others, are the structures of cytochrome c of wheat germ and from an insect. If, as already suspected, these proteins will fall into line with the others, we shall have accumulated overwhelming evidence for the common origin of a single gene and hence for the common ancestry of all present day forms of living aerobic organisms encompassing invertebrates and vertebrates, as well as primitive and higher plants"2. The evidence upon which this is based may be summarized as follows:
1. The polypeptide chains of different vertebrate cytochrome c molecules are all of about the same . length (104 amino acid residues) and arrange themselves into similar secondary and tertiary structures.
2. There are certain similarities in all vertebrate cytochrome c structures, e.g., in having cysteine residues at positions 14 and 17 and in having an eleven amino acid invariant chain from position 70 through position 80. In vertebrate cytochrome c molecules, there are 77 positions (out of 104) which possess identical amino acid residues. When yeast cytochrome c is compared with vertebrate cytochrome c, there are 58 positions with identical amino acid residues. For additional similarities, reference may be made to reviews by Smith and Margoliash2 and Margoliash and Schejter3.
3. Comparison of all the various cytochrome c molecules indicates a strict homology in structure; the closer the taxonomic relationship between two species, the greater the similarity in structure.*
4. Statistical considerations indicate that no matter how many species of cytochrome c were examined, 27 to 29 invariant residues would remain. These represent the amino acids that cannot be replaced without a major modification of the enzymic properties of the cytochrome c.**
As a consequence of the above evidence, Dr. Margoliash presents the following hypothesis: every amino acid similarity above 27-29 can therefore be ascribed to evolutionary homology (i.e., derivation from a common precursor). Consequently, since there are 8 to 12 variations in amino acid residues of the cytochrome c protein when comparing the cytochrome 1c of mammals with the cytochrome c of birds, this presumably represents approximately 8-12 mutations of the DNA which codes for the cytochrome c structure. In a similar fashion, a difference of approximately 18 amino acid residues when mammalian cytochrome c is compared with fish cytochrome c suggests that approximately 18-20 DNA mutations separate these two classes. Some of the other differences in cytochrome c molecules are summarized in Table 1, which is adapted from Margoliash and Schejter.3.
The major objection to this hypothesis of Margoliash and Smith (i.e., that differences in amino acid sequence are due to mutations of the DNA) comes from the finding that there is only one predominant molecular type of cytochrome c in each species. Currently, there is no theory to explain why a perfectly functional cytochrome c molecule should disappear from a population. Stated in another way, each mutation of the DNA for cytochrome c should increase by one the types of cytochrome c present in a given species. Only if the species population were markedly reduced in numbers (e.g., by a major catastrophe wiping out an entire population with only one or two survivors) would the laws of chance permit a mutant cytochrome c to become the predominant form.'***
*Dr. Margoliash has noted, however, that rattlesnake cytochrome c (Table 1) does not fit well with this generalization. It resembles too closely the cytoebrome c of man and monkey. "Note should be made, however, that the amino acid sequence of cytochrome c from bacteria and from photosynthetic organisms has no resemblance to the amino acid sequence in cytochrome c of yeast and vertebrates2.
The following illustration may present this view more clearly. Suppose there was a mutation in the germinal DNA of one individual causing the production of a cytochrome c variant in the offspring. If this cytochrome c variant is perfectly functional, there would appear to be no pressures (survival value, etc.), once the variant cytochrome c was established, to cause its disappearance. The relative abundance of this cytochrome c in the entire population would depend upon inheritance factors. At the same time, there would appear to be no pressures to cause the disappearance of the normal cytochrome c. Consequently, each mutation would therefore increase by one the number of different types of molecules of cytochrome C present in a given species. Dr. Margoliash has noted that all the different types of cytochrome c which have been isolated, appear to be perfectly functional; none appear to have superiority of function that would provide survival value.
Consequently, if the variations in cytochrome c structure which have been noted, do actually represent mutations, there appears to be no reason why there should not be many different types of functional cytochrome c molecules in a given species. Since the evidence to date indicates that there is only one (or at least very few) types of cytochrome c in each species, certainly there is justification for questioning the basic assumption of Margoliash and Smith (i.e., that the 8-12 variant residues between mammals and birds represent 8-12 mutations).""
In place of the assumption noted above, an alternative assumption may be proposed: that similarities in structures of cytochrome c of various species is a consequence of a single Divine knowledge behind the creation of all of these species. The similarities of amino acid sequence then, are a reflection of the knowledge of a common Creator. The differences in amino acid sequence may represent, in some cases ' mutations; but far more often would reflect a choice of the Creator in providing different species with immunologically different proteins for carrying out a specific enzymic task. The author is aware, of course, that invoking the intervention of Divine knowledge is not a new hypothesis; it was in vogue long before the current evolutionary hypothesis became popular.
The proposed hypothesis endeavors to provide a reasonable explanation for the experimental findings of
***The author had occasion to question Dr. Margoliash following his lecture entitled "Cytochrome c Structural Variability" (Fourth Annual Pathobiology Conference, Aspen, Colorado, August, 1967) in regard to this limitation to his hypothesis. Dr. Margoliash noted that finding of only one (or possibly two) predominant types of cytochrome c in each species was a major problem, but he surmised that population geneticists would have to find an answer. He agreed that it was difficult to suggest survival value as an answer since all of the cytochrome c molecules appear to be perfectly functional.
****In criticizing the hypotheses of Drs. Smith and Margoliasb, the author does not mean to be critical of their experimental work. The elucidation of the amino acid sequences of the different cytochromes represents a tremendous amount of careful and painstaking experimentation. Certainly, the work of these men and their collaborators represents a very noteworthy achievement in Biochemistry.
variation in protein structure that is consistent with both scientific evidence and the revealed Word of God. It must be emphasized that one of the basic assumptions of Smith and Margoliash (although not so stated), is that God did not enter into the processes involved in the formation and modification of life; or stated in -mother way, these authors assume that all modifications in living organisms are a consequence of chance happenings. The proposed hypothesis does not attempt to describe how the various cytochrome c molecules were formed in the various species. It is possible that God, in his sovereignty, brought about a change in the DNA structure to produce a slightly different cytochrome c. These modifications in protein structure might be ascribed to a purposeful "God-induced mutation" in the DNA causing the replacement of one or more amino acids of the original cytocbrome c to form a new, but equally functional, variant cytochrome c. In this case, the modifications of cytochrome c (and other proteins as well) would presumably be sufficiently extensivetolimit cross-fertility and hence ensure the establishment of a new species. On the other hand, it is equally consistent with the view presented, to propose that God might choose to produce an entirely new DNA for each species; in this case, however, retaining most of the basic "information" in the DNA for determining the structure of each protein. The resultant DNA would code for new proteins differing from the original proteins in only a relatively small portion of their amino acid sequences. It is certainly possible that other means could be proposed for the production of modified protein molecules, that are consistent with the view that God, in some manner, was involved in directing these modifications to achieve His desired purpose.
In view of the significance of the hypotheses mentioned herein to the basic structure of all proteins, a brief reference should be made to variation in the structure of hemoglobin. Presently, there are a large number of known variants of both the alpha and beta chains of human hemoglobin. In all cases, where the structure of the variant hemoglobin has been determined, these variants differ from normal hemoglobin by the replacement of a single amino acid, corresponding to a single base change in a DNA codon4. It appears likely that these hemoglobin variants are all due to mutations in portions of the DNA coding for the polypeptide chains of hemoglobin. With the possible exception of S hemoglobin (sickle-cell hemoglobin) all of these variants are rare, and in many cases (including S hemoglobin), the mutant hemoglobin is only pood], functional. Consequently, there is no reason to believe that any of these mutant hemoglobins will become the predominant form. In other respects, the arguments presented in relation to the structure of cytochrome c are equally valid for the hemoglobins. The hypothesis that the alpha and beta chains of human hemoglobin (which are similar in 61 amino acid positions) are derived from a single archetypal gene, has recently been challenged on mathematical grounds by Dr. Murray Eden5.
Amino acid differences in cytochrome c
Man - monkey 1
Man - horse 12
Man - rattlesnake 14
Man - dog 10
Pig - cow - sheep 0
Horse - cow 3
Man - chicken 13
Man - tuna 21
Kangaroo - tuna 20
Man - moth 31
Tuna - moth 33
Man - yeast 44
Adapted from Table III of the review by Scheiter3.