Wolfram H. Walker

Structure of the covalently bound flavin of monoamine oxidase

Abstract Flavin peptides derived from monoamine oxidase, free from succinate dehydrogenase flavin, were obtained by digestion of outer membranes of beef liver mitochondria with trypsin and chymotrypsin and purification by various chromatographic methods. The flavin peptides show the same hypsochromic shift of the optical absorption spectrum as flavin peptides from succinate dehydrogenase: the 370 mμ band of the neutral oxidized flavin is shifted to 340 mμ, whereas the cation shows a peak at 370 mμ. The ESR spectrum of the monoamine oxidase flavin cation radical also resembles that of succinate dehydrogenase flavin in that the total width is reduced from 49 G (in riboflavin) to at least 45 G, and the line width from 3.8 G (in riboflavin) to 2.3 G. The covalently bound flavins of monoamine oxidase and succinate dehydrogenase differ, however, in that the former shows the same fluorescence intensity between pH 3.4 and 8, while the latter is quenched with a pK of 4.5 ± 0.1. These observations indicate that the FAD of monoamine oxidase is covalently linked to the peptide chain through the 8α-CH 3 group of riboflavin but histidine is not the immediate substituent, as in succinate dehydrogenase. Hydrolysis of flavin peptides from monoamine oxidase in 6 N HCl at 95° yields a derivative chromatographically distinct from free flavins which is ninhydrin-positive and thus contains an amino acid bound to the 8α-position.

Sequence and structure of a cysteinyl flavin peptide from monoamine oxidase

Abstract Previous studies in this laboratory have shown that the active center of hepatic monoamine oxidase contains flavin dinucleotide covalently linked to the peptide chain via the 8α position of the flavin but that, unlike in succinate dehydrogenase, the linkage is not to histidine but to another amino acid. A pure flavin pentapeptide has now been isolated from monoamine oxidase which yields on acid hydrolysis or digestion with aminopeptidase M 1 mole each of serine and tyrosine and 2 of glycine and gives a positive test for sulfur. Oxidation of the peptide with performic acid yields, in addition to the amino acids mentioned, cysteic acid. The physical and chemical properties of the peptide are in accord with the conclusion that the amino acid substituted on the 8α group of the flavin is cysteine in thioether linkage. Edman degradation followed by dansylation revealed the sequence:

Amino acid sequence at the active center of succinate dehydrogenase

Abstract The amino acid sequence of the coenzyme binding site of beef heart succinate dehydrogenase is serine-histidine-threonine-valine-alanine. Flavin adenine dinucleotide is covalently bound to a ring nitrogen of histidine through the 8α position of the isoalloxazine ring system.

Synthesis and properties of 8α-substituted riboflavins of biological importance

* Abbreviations: t-BOC = t-butyloxycarbonyl; Rfl = riboflavin; TA = tetraacetyl; FAD = flavin-adeninedinucleotide; ESR = electron spin resonance. linked to the imidazole N-3 nitrogen of histidine as determined by degradative studies and by chemical synthesis [l-4]. The amino acid sequence around the histidyl-8cu-flavinhas also been determined [S]. The prosthetic group of mitochondrial monoamine oxidase has been identified as flavin in thioether linkage to a cysteinyl peptide [6-9].8cuCysteinylriboflavin was chemically synthesized [lo]. Recently, a third novel covalently bound flavin prosthetic group was reported which was isolated from Chromatium flavocytochrome c-552 enzyme [ 1 l]. Hendriks and Cronin [ 121 showed that this flavin is probably FAD linked through the 8a-position of the flavin. This assignment was definitely established from the ESR hyperfine structure of the cation radical and by oxidation ,of the flavin with performic acid to 8-carboxy-riboflavin and evidence was obtained for the probable presence of sulfur in the immediate vicinity of the 8a-position [ 13, 141. In view of the varieties of covalently bound flavins so far discovered, other types of flavins linked to functional groups other than in histidine and in cysteine may exist in nature. Their characteristics and properties are of considerable importance. In this paper, the chemical syntheses and properties of 8a-(e-N-lysyl)-riboflavin and 8a-(O-tyrosyl)riboflavin (compounds IVb and Vb, respectively, scheme 1) are described and a comparison with previously characterized 8a-substituted flavins is made.

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

Summary 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.