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

Abstract

The complexes formed between Zn2+ or Cd2+ and half-reduced flavins (isoalloxazines) in neutral aqueous as well as non-aqueous media were studied by electron spin resonance. Formation of 1 : 1 radical chelates between anionic radicals and Zn2+ as well as Cd2+ were found to occur. The stability constants at 20" for the chelates were estimated. A number of flavin derivatives were investigated. The spin density distribution was mapped by means of isotopic substitutions (ZH, 1 5 N , 6*Zn, 67Zn, 1Wd, 113Cd, and 114Cd). The spin distribution within the ligand is similar to that found earlier for the anionic flavin radical. Spin delocalization to the metals was observed and h y p e r h e coupling constants for magnetic isotopes determined by direct measurements and spectra simulations. The difference in spin delocalization between Zn and Cd is discussed.Evidence has accumulated indicating that flavin radicals (flavosemiquinones) occur as intermediates in the reactions of certain metal containing flavoenzyme [3-51. From microwave saturation experiments with electron spin resonance it was suggested [6] that metal-flavin interaction exists within the metalloflavoproteins. The nature of this interaction Among these five species, l?lR-, obtained by one equivalent reduction of Fl,,R, is the only one to show appreciable affinity for heavy metal ions [7,10,111 and therefore is capable of forming flavosemiquinone, or flavinradical, chelates (Mel?lR)+ (Fig.l).At neutral pH the free disproportionated system will "comproportionate') upon addition of d-metal Fig. 1. The structures of the oxidized, anionic radical and radical chelate form of flavins (isoalloxazines). Ring numbering system according to IUPAC-IUB nomenclature tentative rules, J . Biol. Chem. 241 (1966) 2987 is not yet understood and flavin chelates can therefore a t present not be excluded. The essential constituents of the half-reduced flavocoenzyme system a t equilibrium in dilute aqueous solution of pH 5 to 9 are the following molecular species : The quinonc Fl,,R (Fig.l), the hydroquinone Fl,,dRH, and its anion Fl,,RH-, and finally the semiquinonesl?lRHand PlR- (Fig

Novel free radicals in synthetic and natural pheomelanins: distinction between dopa melanins and cysteinyldopa melanins by ESR spectroscopy.

“…The splitting from "3Cd is much larger than that found in eumelanin systems (ca. 3.5 G), but is similar to that (17.5 G) measured in complexes with small flavin semiquinones (23).…”

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

“…Plrtvosemiquinones of type VIII, I X are sufficiently stable to permit electron spin resonance spectra to be obtained. The spectra of these $lR2-type radicals [9] are practically identical with those of the analogous anions &R- [lo]. This is understandable since in VIII, IX the additional R-substituent of @IR2 is located in the pyrimidine part of the flavin nucleus (together with the negative charge of PIR-), where very little spin density is observed [S-lo].…”

“…Thus, different apoproteins might favor the formation of different semiquinones, while the free chemical system under neutral conditions exhibits the blue (N[5]-protonated) tautomer only. I n some flavoproteins the red radical anion could conceivably be stabilized by chelate formation with a redox-inactive metal ion [9,31].…”

Proton Magnetic Resonance Studies of Flavoquinone-Metal Complexes

“…Furthermore, it indicates the presence at most ring atoms of a Ti-electron spin polarized antiparallel to the metal ion electron spin. The sign, the magnitude and the distribution of the unpaired spin (very different from those of the unpaired spin in flavosemiquinone radicals) 7 cannot correspond to direct d,?r-electron delocalisation between the ligand and the metal Orbitals, but must be due to 0,^-polarisation at the coordination site. Thus flavoquinone-metal binding is mainly of o-type regardless of the nature of the divalent transition ion.…”