A basic cytochrome was isolated from the phytomastigophorean protozoan Euglena gracilis and a similar protein from the zoomastigophorean protozoan Crithidia oncopelti. In both cases chromatography on CM-cellulose in first the reduced and then the oxidized form proved to be an efficient means of purification. The two cytochromes can be classed in the cytochrome c family but they have certain atypical features. The alpha peak of the absorption spectrum is shifted towards the red and is asymmetrical. The pyridine ferrohaemochrome has an alpha-peak maximum intermediate between that of c-type cytochromes and proteins containing protohaem IX. The test for free vinyl groups was positive. The amino acid sequences of the two cytochromes were determined. Attention is drawn in the text to those parts of the evidence that are less satisfactory. Both sequences are homologous with the family of cytochrome c, but are unusual in having only one cysteine residue so that the haem is attached through only one thioether bond. Detailed evidence for the amino acid dequences of the two proteins has been deposited as Supplementary Publication SUP 50042 (70 pages) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Wetherby, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975) 145, 5.

An EXAFS study on the oxidized and reduced forms of cytochromes c' from Rhodospirillum rubrum and Rhodospirillum molischianum was performed at pH 7. The cytochromes c' have an apparent coordination number of 5 in both oxidation states. Average Fe-ligand bond lengths of 2.02 +/- 0.025 and 2.06 +/- 0.025 A are obtained in their oxidized and reduced forms, respectively. By use of suitable values for the Fe-NHis bond length and Fe out-of-plane displacement, as determined by small molecule crystallographic techniques, the Fe-Npyrrole bond lengths and the porphyrin center-to-Npyrrole distance have been estimated for cytochrome c' in both of its oxidation states. With this model, estimates of the Fe-Npyrrole bond lengths are 2.01 +/- 0.03 and 2.05 +/- 0.03 A, for the oxidized and reduced cytochromes c', respectively. The center-to-Npyrrole distance is estimated to be 1.99 +/- 0.03 A for oxidized cytochrome c' and 2.03 +/- 0.03 A for reduced cytochrome c'.

The cytochromes c' are electron transport proteins widely distributed in photosynthetic and aerobic bacteria. We report the amino acid sequences of the proteins from 12 different bacterial species, and we show by sequences that the cytochromes c-556 from 2 different bacteria are structurally related to the cytochromes c'. Unlike the mitochondrial cytochromes c, the heme binding site in the cytochromes c' and c-556 is near the COOH terminus. The cytochromes c-556 probably have a methionine sixth heme ligand located near the NH2 terminus, whereas the cytochromes c' may be pentacoordinate. Quantitative comparison ofcytochrome c' and c-556 sequences indicates a relatively low 28% average identity.

It has been proposed that phylogenetic trees, intended to show divergence of eukaryotic protein and nucleic acid sequences, be extended to include those from bacteria. However, we have compared the amino acid sequences of 18 of the most divergent mitochondrial cytochromes c with those of 18 bacterial cytochromes c2 and have found that the average percentage difference between these mitochondrial cytochromes c and cytochromes c2 was not significantly greater than that among the cytochromes c2 alone. The large discontinuities in physical-chemical properties recognized between the prokaryote and eukaryote cytochromes render it highly improbable that members of the two classes should be no more different from one another than members of either class alone, assuming that sequence differences can accurately reveal evolutionary divergence. Instead, we propose that divergent amino acid sequences approach a limit of change considerably less than for comparison of random sequences. This limit of change presumably is determined by the structure/function relationship. When two homologous protein sequences have reached such a limit, convergence or back-mutations and parallel mutations become as frequent as divergent mutations. As two diverging proteins approach this steady-state condition, sequence differences no longer reflect the numbers ofmutations resulting in amino acid substitution and therefore species cannot be positioned on a phylogenetic tree. Insertions and deletions are less reversible than are amino acid substitutions and, provided they are well-documented, might be more reliable indicators of bacterial relationships. Nevertheless, we suggest that data available on bacterial protein sequences do not permit construction of all-inclusive phylogenetic trees. Comparisons of protein and rRNA trees suggest that similar restrictions apply to use of rRNA sequence data.Phylogenetic trees constructed from protein sequences of eukaryotes agree reasonably well with those deduced from the fossil record, provided one is allowed to exclude a small number of unexplained anomalies (e.g., rattlesnake cytochrome c) (1-3). It has been natural to hope that success would attend the use of sequence determination to derive such trees for prokaryotes and thus extend phylogeny beyond the fossil record. Basically, a phylogenetic tree is intended to show the relative order of divergence proceeding on the assumption that time intervals are proportional to the number of accepted mutations and can be estimated from protein or nucleic acid sequence differences. For using the "matrix method," large sequence differences are related to mutations by mathematical treatments, either deduced from empirical observations of proteins that have diverged to only a small degree (4) or on theoretical grounds (3). That the numbers of mutations resulting in amino acid substitutions could be accurately estimated from protein sequence differences was not possible to test experimentally in the past because very different, yet homologous, sequences were n...

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