Amino acid sequence of a heme peptide with two heme groups.

We have isolated an unusual heme peptide containing 27 amino acid residues and two heme groups from the variant heme protein, RHP, of the obligate photoanaerobe, Chromatium, strain D. This protein has been obtained in pure form and characterized previously (1) as a compound with molecular weight of 36,000 containing two functional heme groups. The heme peptide mixture, obtained by the peptic digestion method of Tuppy, Bodo, and Paleus (2, 3) accounts quantitatively for all the heme present in the original protein. Two major components in approximately equal amounts and one minor component are present (see Table I), as shown by Celite column chromatography with the solvent system, n-butanol-acetic acidwater (2). The major components differ only with respect to the NHz-terminal amino acid; one terminates in alanine, whereas the other contains the additional amino acid, phenylalanine, as the NHz-terminal group. The sequence proposed for the peptic heme peptide is shown as Sequence (a) in Table I. A partial list of peptide fragments which provide the basis for the sequence proposed is given in Table II. Established sequences are indicated by all peptides lacking braces and commas. Quantitative determination of heme content as pyridine hemochromogen establishes the presence of two heme groups in the peptide. The spectrum of the hemochromogen is identical with that derived from cytochrome c. One heme group is attached in the usual manner through thioether linkages between the vinyl side chains and the cysteine residues Nos. 5 and 8 (Table I). The placement of the other covalently linked heme is still uncertain, but may be assigned tentatively to cysteine No. 20 with which it may form a monothioether adduct. The possibility of the presence of a free sulfhydryl group is still under investigation. Quantitative end group analyses of the 2,4-dinitrophenylalanine of the one major heme peptide, together with assay of lysine residues as e-amino-DNPI derivatives, are in agreement with a molecular weight corresponding to the composition shown. No quantitative analysis of amino acids has been performed other than end-group determination by the Sanger dinitrofluorobenzene method. Visual judgment of the ninhydrin color on chromatograms is the basis for the analysis shown in Table I.