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Reports in the literature indicate that the flavin prosthetic group of Chromatium cytochrome c552 is not acid-extractable but is dissociated from the protein by various treatments, including prolonged incubation with urea solution, which are not expected to break covalent bonds. In the present paper it is established by isolation of peptic and of tryptic-chymotryptic flavin peptides that the flavin component is covalently linked to the protein. The peptide chain is attached to the 8α group of the FAD, as in other enzymes containing covalently bound flavin, as evidenced by a hypochromic shift of the near ultraviolet absorption maximum, the characteristics of the hyperfine EPR spectrum, and the release of 8-carboxy-FMN from the cytochrome on oxidation with cold performic acid with concomitant hydrolysis of the pyrophosphate linkage. The fluorescence of the flavin peptides, at the FAD, FMN and riboflavin level, is extensively quenched, with less than 1% of the quantum yield of fluorescence, compared with riboflavin, in the peptic peptide. On oxidation of the tryptic-chymotryptic peptide with performic acid the fluorescence increases to 50% of that given by an equimolar concentration of riboflavin. This increase is accompanied by a further hypochromic shift of the near ultraviolet absorption maximum. This behavior, the tendency of the flavin peptide to undergo autooxidation, and the positive chloroplatinic acid test resemble the properties of cysteinylflavin thioether and its peptides and suggest that the flavin is bonded to a cysteine residue, as in monoamine oxidase. The presence of cysteine in both flavin peptides from cytochrome c552 has been verified by the liberation of cysteine on acid hydrolysis. Despite these similarities to peptides containing a cysteinyl flavin thioether. the peptic and tryptic-chymotryptic peptides from cytochrome c552 show several properties which preclude a thioether linkage. Evidence is summarized to indicate that the flavin is linked via a thiohemiacetal bond to a cysteinyl residue in the polypeptide chain. Thus, the flavin released from the peptides by acid hydrolysis is in every respect identical to 8-formylriboflavin. Further, two flavin components were detected on high voltage electrophoresis after aminopeptidase M digestion of the peptic peptide, which had mobilities expected for the aminoacylflavin and a thiazolidine derivative. A thiohemiacetal structure also ac-counts for the much greater lability of the flavin peptide linkage than that found for monoamine oxidase. Thus from the evidence presented, it is concluded that the structure of the covalently bound flavin in Chromatium cytochrome c552 is 8α-S-cysteinyl-8α-hydroxy-FAD. [ABSTRACT FROM AUTHOR]

A model is proposed to explain the biphasic reduction of ferricytochrome c at alkaline pH assuming an equilibrium distribution between a reducible and non-reducible conformer. An analysis of this model is given in an appendix to this paper. Protein denaturants, increasing pH and temperature are found to disturb the conformer equilibrium in favour of the irreducible form. The thermodynamic parameters governing this equilibrium have been determined. Kinetic parameters defined in the model were measured under a variety of conditions. The kinetic and equilibrium data strongly suggests that only the reducible conformer possesses the 695 nm band. Possible origins of the 695 nm band are discussed. [ABSTRACT FROM AUTHOR]

Amino acid analyses of L-lactate dehydrogenase from baker's show that the minimum molecular weight (53 000 daltons) of the protein is much lower than found in the literature (80 000). This result, combined with those reported in the following papers, leads to a revision of the dimeric model generally accepted for cytochrome b2 [ABSTRACT FROM AUTHOR]