A complete amino acid sequence is proposed for the cytochrome c-550 isolated from the gram-negative chemoorganotrophic bacterium Aquaspirillum itersonii. The sequence, a single polypeptide chain of 11 1 residues, was deduced from the sequences of peptides obtained by tryptic, thermolytic or chymotryptic digestion.The cytochrome shows a high degree of sequence homology with the cytochrome c2 from the photosynthetic bacterium Rhodospirillum rubrum, and the evolutionary implications of this are considered.Aquaspirillum itersonii is a non-photosynthetic freshwater bacterium. Although previously classified as Spirillum itersonii [l] the Spirilla were extensively studied and reclassified by Hylemon et al. [2] and Carney et al. [3], who proposed that S. itersonii should be reclassified in the (newly described) genus Aquaspirillum. However, on the basis of 16SRNA oligonucleotide catalogue data [4, 51 it has been proposed that this genus lacks phylogenetic coherence, and a close relationship between A. itersonii and a subset of the purple photosynthetic bacteria that includes Rhodospirillum rubrum has been proposed.Comparative analysis of c-type cytochrome amino acid sequences has been used as a guide to possible phylogenetic relationships between prokaryotes [6]. The amino acid sequence of the cytochrome c2 from Rs. rubrum was one of the first prokaryotic proteins to be determined [7, 81. A. itersonii has also been reported to posses a c-type cytochrome which has a cytochrome-c2-like spectrum [9] and a molecular mass similar to that of Rs. rubrum cytochrome c2 [lo].In this paper the complete amino acid sequence of the cytochrome c-550 from A. itersonii is reported and the structure is discussed in relation to the function of the protein and the phylogeny of the bacterium.
EXPERIMENTAL PROCEDUREPreparation of cytochrome c-550. A. itersonii was grown, and the cytochrome c-550 isolated, as described in the accompanying paper [lo]. The purity of the cytochrome was assessed by SDS/polyacrylamide gel electrophoresis and isoelectric focusing gels. The amino acid composition is shown in Table 1.Amino acid sequence determination. The amino acid sequence was determined by the general methods described in [ll, 121 for other bacterial cytochromes c, and to similar standards. Protein (1.5 -2.0 pmol) was treated with HgC12 in 8 M urea/O.l M HC1 at 37°C for 16 h to remove the haem moiety, and after gel filtration and freeze-drying the sample was digested with protease. The peptides were fractionated by gel filtration followed by high-voltage paper electrophoresis then analysed quantitatively, using a Rank-Hilger ChromoCorrespondence fo K. Woolley, Department of Botany, University of Glasgow, Glasgow, Scotland G12 8QQ spek amino acid analyser, for amino acid composition and purity. Peptide sequences were mainly investigated by the 4-N,N-dimethylaminoazobenzene-4-isothiocyanate (Dabitc) method [13 ~ 161, but the dansyl/phenylisothiocyanate method was sometimes used. In a few cases digestion with carboxypeptidase A was used to investig...
