ON THE MONOHEME NATURE OF CYTOCHROME c ′ ( Rhodopseudomonas palustris )

Various purple photosynthetic bacteria contain two predominant classes of soluble c-type cytochromes,l namely, cytochromes c2 which resemble mammalian cytochromes c with regard to size and many other molecular properties, and cytochromes cc' which bear two covalently bound heme groups on a single polypeptide chain2 roughly twice the size of cytochromes c2 and which exhibit absorption spectra and chemical properties different from those of the mammalian c-type cytochromes.3 Cytochromes cc' have previously been referred to as"RHP"type cytochromes (cf. ref. 1). We now report the first example of a member of this class of heme proteins which contains only one heme group per molecule. It was noted earlier4 that the heme protein, designated as Rps. palustris (van Niel strain #2.1.37) cytochrome cc', differed from other cytochromes of this class in that it exhibited a more positive oxidation-reduction potential (Em,7 = +0.105 v) than previously encountered in the cytochromes cc', a relatively low molecular weight (<20,000), a basic isoelectric point (pH >9.0), and a blocked NHrterminal group. A recent improvement5 in the purification procedure for this protein has made available highly purified samples suitable for a careful study of the amino acid composition of this protein. Table 1 shows its amino acid composition,6 as compiled from experiments using acid hydrolyses with 6 N HCl at 105°C for 24 and 48 hours, as well as from complete enzymic hydrolyses of the protein with pronase followed by leucine aminopeptidase. The contents of cysteine + cystine and of methionine were determined as cysteic acid and methionine sulfone, respectively, from hydrolysates of performic acid-oxidized samples.7 The absence of tryptophan in the enzymic hydrolysate was confirmed by spectrophotometric titration of protein samples with N-bromosuccinimide.8 The amino acid composition given in Table 1 is in fairly good agreement with data published previously from this laboratory1 as well as with the data of Henderson and Nankiville,9 the main improvement being a more accurate characterization and determination of the acid-labile amino acid residues of the protein. We find a higher lysine content (about four residues) than that reported by Henderson and Nankiville,9 who comment on the difficulty of explaining the markedly basic isoelectric point of the protein on the basis of their amino acid composition. In consideration of the presence of at least eight amide groups, a comparison of the total basic (19 lysine and 2 arginine) and acidic (18 aspartic acid plus glutamic acid) amino acid residues indicates an excess of three basic residues, which is in good agreement with the strongly basic isoelectric point observed for this protein.9 Similarly, our present analysis indicates a higher content in alanine than was observed by Henderson and Nankiville.9 Unusually high alanine contents are a characteristic property of all cytochromes cc' so far investigated. 1 The residue numbers in Table 1 are based on the occurrence of a single residue