STRUCTURAL ELUCIDATION AND PROPERTIES OF 8ALPHA-(N1-HISTIDYL)RIBOFLAVIN - FLAVIN COMPONENT OF THIAMINE DEHYDROGENASE AND BETA-CYCLOPIAZONATE OXIDOCYCLASE

In addition to 8.alpha.-(N3-histidyl)riboflavin, 8.alpha.-(N1-histidyl)riboflavin is also formed during the reaction of N.alpha.-blocked histidine with 8.alpha.-bromotetraacetylriboflavin in a yield of 20-25% of the total histidylflavin fraction. The properties of 8.alpha.-(N1-histidyl]riboflavin are identical with those of the histidylflavin isolated from thiamine dehydrogenase and .beta.-cyclopiazonate oxidocylase but differ from those of 8.alpha.-(N3-histidyl)riboflavin. These properties include pKa of fluorescence quenching, electrophoretic mobility at pH 5.0, stability to storage, and reduction by NaBH4. Proof for 8.alpha. substitution is shown by the EPR and electron-nuclear double resonance spectra of the cationic semiquinone form, as well as by the PMR spectrum of the oxidized form. The site of histidine substitution by the 8.alpha.-methylene of the flavin moiety was shown by methylation of the imidazole ring with methyl iodide, cleavage of the methylhistidine-flavin bond by acid hydrolysis at 150.degree. C, and identification of the methylhistidine isomer by electrophoresis. 3-Methylhistidine is the product from the N1-histidylfavin isomer, while 1-methylhistidine is produced from the N3 isomer. The flavin product from reductive Zn cleavage of either isomer was identified as riboflavin. The compound obtained on acid treatment of 8.alpha.-(N3-histidyl)riboflavin (previously thought to be the N1 isomer) differs from the parent compound only in the ribityl side chain, since chemical degradation studies show 1-methylhistidine as a product and a flavin product which differs from riboflavin only in mobility in TLC, but not in absorption, fluorescence, and EPR spectral properties. Proof that acid modification involves only the ribityl chain has come from the observations that alkaline irradiation of this flavin yields lumiflavin, that the PMR spectrum of the compound differs from that of riboflavin in the region of the ribityl proton resonance, and that its periodate titer is lower than that of authentic riboflavin. The identity of 8.alpha.-(N1-histidyl)riboflavin with the histidylflavin from thiamine dehydrogenase and .beta.-cyclopiazonate oxidocyclase shows that both isomeric forms of 8.alpha.-histidylflavin occur in nature.