Chromatium Cytochrome Research Articles

Binding of cyanide to cytochrome c′ from Chromatium vinosum

Spectroscopic evidence is presented which demonstrates the binding of cyanide to the ferric cytochrome c′ from Chromatium vinosum. The cytochrome was shown to bind one equivalent of cyanide with an equilibrium constant of 2.1·10 4 at pH 7.0 and 25°C. This finding represents the first observation of the binding of an anionic ligand to the heme iron in a ferric cytochrome c′. These results suggest that the binding site of the ferric Chromatium cytochrome c′ may be significantly more accessible than previously indicated.

Binding of ethyl isocyanide to cytochrome c′ from Chromatium vinosum

Alkyl isocyanides were investigated as potential ligands for the cytochromes c′. Spectroscopic evidence is presented which demonstrates the binding of ethyl isocyanide to cytochrome c′ from Chromatium vinosum. Chromatium cytochrome c′ was shown to bind one molecule of ethyl isocyanide with an equilibrium constant of 3.29·10 3 at pH 7.0 and 25°C. This new finding indicates that the binding site of Chromatium cytochrome c′ may be significantly more accessible than that of Rhodospirillum rubrum cytochrome c′ and further provides an opportunity to examine the structure of the heme environments of the cytochromes c′ through a study of the binding of alkyl isocyanides to these proteins.

Preparation of subunits of flavocytochromes c derived from Chlorobium limicola f. thiosulfatophilum and Chromatium vinosum

The subunits of Chlorobium limicala f. thiosulfatophilum cytochrome c-553 and of Chromatium vinosum cytochrome c-552 have been obtained. Chlorobium cytochrome c-553 is split into the cytochrome and flavoprotein subunits by treatment with trichloroacetic acid; after the cytochrome is precipitated by 1–2% trichloroacetic acid, the cytochrome subunit is extractable with buffer, while the flavoprotein subunit is not dissolved. The subunits of Chromatium cytochrome c-552 can not be obtained by the trichloroacetic acid-treatment. The flavoprotein subunit of the Chromatium cytochrome is obtained by isoelectric focusing in the presence of 6 M urea and 1% mercaptoethanol, while the cytochrome subunit is prepared by gel filtration in the presence of 6 M urea with Sephacryl S-200. Molecular weights of the cytochrome and flavoprotein subunits from the Chlorobium cytochrome are 11,000 and 47,000, respectively, while those of the two subunits from the Chromatium cytochrome are 21,000 and 46,000, respectively. The molecule of each flavocytochrome c is composed of one molecule of each of the cytochrome and flavoprotein subunits.

Evidence for a thioether linkage between the flavin and polypeptide chain of Chromatium cytochrome c 552.

Evidence for a thioether linkage between the flavin and polypeptide chain of Chromatium cytochrome c 552.

The covalently bound flavin of Chromatium cytochrome c552. 1. Evidence for cysteine thiohemiacetal at the 8 alpha position.

The covalently bound flavin of Chromatium cytochrome c552. 1. Evidence for cysteine thiohemiacetal at the 8 alpha position.

The covalently bound flavin of Chromatium cytochrome c552. 2. Sequence of flavin peptides and flavin-tyrosine interaction.

The previous paper in this series described the isolation of tryptic-chymotryptic and peptic flavin peptides from Chromatium cytochrome c552 in homogeneous form and presented evidence that the FAD component is covalently linked to a cysteinyl residue via a hemiacetal linkage to the 8α position of the isoalloxazine ring. The amino acid sequence of the peptic and tryptic-chymotryptic flavin peptides were found to be Tyr-Thr-Cys(FAD)-Tyr and Thr-Cys(FAD)-Tyr, respectively. However, the presence of the NH2-tyrosyl residue in the former, imparts markedly greater stability to the cysteinyl flavin linkage, and even results in essentially quenched fluorescence of the flavin after performic acid oxidation of the sulfur moiety. Oxidation at 40 °C, which destroys tyrosine, results in the same level of fluorescence as found when the tryptic-chymotryptic peptide is oxidized at 0 °C. Removal of this tyrosyl residue from the peptic peptide, oxidized at 0 °C, by aminopeptidase M also leads to an increase in fluorescence. Evidence for a tyrosyl-flavin interaction has been further obtained from CD spectra. The peptic peptide has a broad, positive Cotton effect with a maximum at 484 to 490 nm, which is enhanced after treatment with pyrophosphatase and phosphatase and negative bands at 305 and 375 nm. The tryptic-chymotryptic peptide, on the other hand, has a positive band at 340 nm, as in FAD, but only a small negative band at 380 nm. Thus the tyrosyl-flavin interaction appears to be responsible for the stabilization of the thiohemiacetal bond in the peptide and perhaps also in the protein.

Structure of the flavin site of [formula omitted] flavocytochrome [formula omitted

Two flavin peptides have been isolated from Chromatium cytochrome c 552 by digestion with pepsin and with trypsin-chymotrypsin, respectively. Acid hydrolysis and aminopeptidase digestion of the peptic peptide shows the presence of 1 mole each of threonine and cysteine and 2 of tyrosine, per mole of FAD. Edman degradation gives the sequence: tyr-thr-cys (flavin)-tyr. Tryptic-chymotryptic digestion yields a flavin tripeptide of the structure: thr-cys (flavin)-tyr. The N-terminal tyrosine in the peptic tetrapeptide shows a strong interaction with the flavin, as judged by CD spectra, and this may account for the resistance of the bridge sulfur to oxidation by cold performic acid. Both peptides show abnormally low fluorescence in the pure state (1 to 5% relative to riboflavin) and positive iodoplatinic test; the peptic peptide yields a positive, the trypticchymotryptic peptide negative ninhydrin reaction. The electrophoretic mobility of the major product of aminopeptidase digestion (presumably the thiazolidine form of cysteinyl-8α-FAD thiohemiacetal) and other properties of the two peptides are in accord with previous suggestions that the linkage of the flavin to the peptide is a thiohemiacetal.

Recent advances in the chemistry of covalently bound flavin coenzymes.

Following elucidation of the structures of the flavin components of succinate dehydrogenase (SD) as N (3) -histidyl-8α-FAD and of monoamine oxidase (MAO) as cysteinyl-8α-FAD and determination of the peptide sequences around the flavin sites of these enzymes, attention has been focused on the covalently bound FAD of Chromatium cytochrome c-552. As documented in preliminary communications, the FAD moiety of this enzyme is also substituted at the 8α-position, as judged from ESR hyderfine structure of the free radical cation and the characteristic hypsochromic shift of the second absorption band of the neutral flavoquinone in purified preparations of the flavin. Definite proof has come from the liberation of 8-carbxyriboflavin on performic acid treatment of the enzyme. In regard to ESR and optical spectra and the tendency of the purified flavin (liberated by proteolysis) to undergo autooxidation with a further hypsochromic shift of the second absorption band and increased fluorescence, the flavin resembles the MAO flavin. The fact that fluorescence is >90% quenched at all pH values even at the FMN level and doees not vary with pH between 3.2 and 8 also suggests a thioether linkage as in cysteinyl riboflavin. In many respects, however, the Chromatium flavin differs from cysteinyl riboflavin. Highly purified preparations from tryptic-chymotryptic digests give a positive chloroplatinic test. Electrophoresis clearly shows the presence of carboxyl and amino groups but the peptide gives no characteristic ninhydrin reaction and amino acid analysis of performic acid oxidized samples yields cysteic acid and threonine in amounts less than equimolar to the flavin. The amino acid environment around the flavin may account for these results although a linkage other than a thioether remains a possibility.

Properties of the covalently bound flavin of chromatium cytochrome c-552 and its conversion to 8-carboxy-riboflavin

Previous work has shown that cytochrome c-552 from Chromatium contains a covalently bound flavin, which is not released by denaturation but is liberated by proteolysis or various chemical treatments and that the protein is probably attached at the 8α position of the FAD. In the present study unambiguous evidence was obtained for 8α substitution from (a) ESR hyperfine spectra of the flavin cation radical and (b) from identification of the flavin released by performic acid oxidation from cytochrome c-552 as riboflavin-8α-carboxylic acid. Various lines of evidence suggest that in the native enzyme a reduced sulfur may be linked to the 8α group of the flavin, similarly to monoamine oxidase, which contains cysteinyl 8α-FAD, although the conditions required for cleavage of the flavin from the two enzymes appear to be quite different.

The flavin of [formula omitted] cytochrome [formula omitted]-552

A flavin obtained from Chromatium cytochrome c -552 has been purified and characterized with respect to its neutral and acid absorption spectra, fluorescence-pH relationship, and behavior on partition and molecular exclusion chromatography. The chromatographic results are consistent with a flavin adenine dinucleotide type structure. Neutral and acid absorption spectra taken after degradation of the flavin to the riboflavin level differ in the near UV from those of riboflavin and are similar to those of flavins modified in the dimethylisoalloxazine ring system, such as the histidyl-riboflavin obtained from mammalian succinate dehydrogenase. However, cytochrome c -552 flavin when at the riboflavin level and histidyl-riboflavin are quite dissimilar when compared fluorometrically and by molecular exclusion chromatography.

Molecular weights of some cytochromes cc'

The molecular weights of seven different cytochromes cc' have been determined by sedimentation equilibrium centrifugation. Studies were conducted in a variety of solvents and it was found that 6 M guanidine promoted the dissociation of three of the cytochromes cc' into subunits. The apparent size of Chromatium cytochrome cc' was unaffected by 6 M guanidine. However, the Chromatium cytochrome was completely dissociated into subunits of approx. 12 000 molecular weight in the presence of 100 mM NaOH. This last result suggests that the existence of a diheme peptide as previously reported should be re-evaluated.

Optical Rotatory Dispersion of Chromatium Cytochrome c552

Abstract Chromatium cytochrome c552 contains two covalently linked hemes and one tightly bound flavin. The optical rotatory dispersion of the oxidized, reduced, and carbon monoxide-reduced forms of the cytochrome was studied between 185 and 600 nm. It showed a rather featureless optical rotatory dispersion in the visible and aromatic regions but large anomalous rotations with a prominent peak flanked by two small troughs in the Soret region. Reduction or reaction with carbon monoxide increased the magnitude of rotation at the positive extremum, accompanied by a general sharpening of the optical rotatory dispersion profile. Analysis of these dispersion curves gave a set of Cotton effects of opposite signs. In the oxidized form, four Cotton effects were resolved from the dispersion curve and were located at 426.4, 416.1, 406.1, and 360.6 nm with rotational strengths of -0.25, -0.24, +0.55, and -0.07 Debye magneton, respectively. The net rotational strength of these Cotton effects approximated zero. Similar results were obtained in the reduced form which had transitions at 420.6(-), 413.4(+), and 347.2 nm(-), and in the carbon monoxide-reacted form with transitions at 416.6(-), 412.0(+), 410.2(+), and 342.1 nm(-). The Kronig-Kramer transform computed for each form of the cytochrome gave a split circular dichroic spectrum comparable to that experimentally observed. These results could possibly mean that (a) coupled heme interaction might exist in the molecule, (b) the immediate environment of the prosthetic groups might be affected by the oxidation state of the hemes or their reaction with carbon monoxide, and (c) the sharing of the carbon monoxide by the two hemes might not be exactly equal.