The Chemical and Electronic Structure of the Neutral Flavin Radical as Revealed by Electron Spin Resonance Spectroscopy of Chemically and Isotopically Substituted Derivatives

Abstract

The neutral flavosemiquinone has been studied in detail by electron spin resonance spectroscopy. Isotopic (W, 2H) and chemical substitutions have been carried out. A hyperhe coupling scheme of the neutral flavosemiquinone is described where N(5), N(10), CH,(10), CH3(8) and H(6) are involved. The highest spin density is located a t N(5) as has also been found for the anionic and chelated form of the flavosemiquinone. The exchangeable proton in the neutral flavosemiquinone is attached to N(5) having a coupling constant of 7.6 G which is about the same magnitude as the coupling constant to N(5). The hyperfine couplings are interpreted in terms of spin densities and comparison is made with available quantum chemical calculations. Evidence is presented that two tautomeric forms of the neutral flavosemiquinone have to be considered which can be obtained in a pure form by suitable substitution of flavin derivatives. The two types of neutral flavosemiquinones are easily distinguishable by their electron spin resonance and light absorptions.Since the discovery of flavin radicals by Michaelis and co-workers [l] it has been tacitly assumed that the addition of a hydrogen atom to flavoquinone (I) would occur a t N(1) yielding a neutral flavin radical PlRH of structure 11. This was in analogy to the (in the meantime well established) protonation of flavoquinone, yielding the flavoquinone cation 111 [2]. I1 B~R H IIaThis idea was also supported by the fact that PlRH showed an acidity comparable to that of flavohydroquinone (IV) as established by Michaelis and Schwarzenbach potentiometrically [3]. These authors found pKglBE g 7 as compared to pKfireaRH, = 6.5. Clearly the most acidic proton in IV (R = H) must be assumed to dissociate from the cyclic imide group in either position 1 or 3, and not from the pyrrole" nitrogen in position 5 of the isoalloxazine ring. The validity of this assumption for l75-dihydroflavins was proved by the fact that 5,10-dihydro-l,3-dimethyllumichrome (V) does not exhibit a measurable acidity. Nomenclature. The term flavin means the 10-alkylated 6,7-benzo-pteridine-2,4-dione, i. e. the isoalloxazine nucleus, irrespective of the redox state. Its oxidized form is called "flavoquinone", its fully reduced form "flavohydroquinone", 1,5-dihydroflavin or, for convenience, leucoflavin, though the free leucoflavin is, in fact, not colorless. The term "flavosemiquinone" is assigned to the intermediate radical form. Lumiflavin means 7,8,10-trimethylflavin.' <

Flavin Radical Chelates as Studied by Electron Spin Resonance and Isotopic Substitution

“…Hence @" > @Me. The H (5) coupling constant is found to be comparable to that for N(5), i. e., 7-8 G [25]. From the relationship (10) we see that instead e H < @Me (Me = Zn2+ or Cd2+).…”

“…From these observations we conclude that the metal ion replaces the electron spin resonance-active proton of the PlRH when forming (MePlR)+. The dissociable proton is presumably attached to N(5) [1,25].…”

Flavin and 5‐deazaflavin: A chemical evaluation of ‘modified’ flavoproteins with respect to the mechanisms of redox biocatalysis

“…The blue radical species 5-Hill can be further stabilized chemically by alkylation, as shown with the derivatives 5-R61 [49] or biochemically via binding to a suitable apoprotein, as found in the wide-spread group of 'blue radical flavoproteins' [7,9] . In all cases where flavin radicals could be shown to act as catalytically essential intermediates, the radicals are of the blue variety 5-H61, and the stabilization is thought to be brought about by a regiospecific hydrogen bridge pointing from the apoprotein towards N(5) (see below).…”

“…Upon addition of a single electron to Fl,x, the red radical anion fil-is formed which, in the protein-free system, is protonated with a pK -8.5 [.50] to yield the blue neutral species 5-Hcl, whose structure was established by comparison with the corresponding alkyl derivatives S-Rfil [49] . This blue radical is of moderate thermodynamic stability in the protein-free system.…”

Essential Histidyl Residues in Glucose Oxidase. Chemical Modification of Histidyl Residues with Diethylpyrocarbonate†

“…We have studied amino acid residues responsible for the binding of apoprotein to the FAD coenzyme. 7 ,S) Muller et al 9 ) have classified the flavoproteins as those of which the blue or red semiquinone was stabilized by an H + -donating group (e.g., -COOH of Glu) or by an H+ -withdrawing group (e.g., -NH2 of Lys) in the vicinity of the active site, the flavin N(5) or 0(4) atom, and those in which such semiquinone species were not stabilized, rather, these semiquinones could be stabilized by way of flavin-N(5)-imidazole bonding, depending * Present address: Research Laboratory, Amana Pharmaceutical Co., Ltd., Nishiharu, Nishikasugai-gun, Aichi 481, Japan.…”