Anders Ehrenberg

Ionic base radicals in γ-irradiated DNA

Abstract The free radicals induced by γ-irradiation at 77°K in moist oriented DNA have been studied by electron spin resonance. The hyperfine structure of the ESR spectra with the magnetic field of the spectrometer parallel and perpendicular to the DNA helix axis has been analyzed. Two radical components produced in comparable amounts with different line-widths and hyperfine coupling schemes have been observed. Possible radical models for the two components are given. Spectra simulations according to the proposed hyperfine coupling schemes give a reasonable fit to the experimental spectra. Due to the symmetry character, the spectra are assigned to DNA base radicals with the unpaired electron in a delocalized π-orbital. The interpretation of the hyperfine couplings is in agreement with the preliminary results of a study on the very slow exchange of carbon bound hydrogen for deuterium on the DNA bases. Spin densities are estimated from the observed coupling constants. From comparison with theoretical spin densities in the literature we have further assigned one component to an anionic radical on thymine (or cytosine), and the other one to a cationic radical on guanine (or cytosine).

Heterogeneity of cytochrome P-450 in rat liver microsomes: selective interaction of metyrapone and SKF 525-A with different fractions of microsomal cytochrome P-450.

It is well established that different substrates affect the light absorption of microsomal cytochrome P-450 differently. The substrates have been classified into two types [ 1 ] : type I substrates which cause a shift of the Soret band absorption to shorter wavelength, indicating an increase in high spin character, and type II substrates which cause a shift of the Soret band to longer wavelength, indicating an increase in low spin character. The different spectral changes resulting from the interaction of various substrates with microsomal cytochrome P-450 poses the question whether the substrates interact with different cytochrome P-450 species or with different sites on a single species of cytochrome P-450. Many studies concerned with purification of liver microsomal cytochrome P-450 show evidence for different forms of the hemoprotein [2-71. We will report here observations by light absorption and EPR suggesting that SKF 525-A (diethylaminoethyl-2,2-diphenylvalerate) and metyrapone (MP), inhibitors of cytochrome P-450~linked reactions [8], react selectively with different fractions of microsomal cytochrome P-450. The reactions are indepen-

The effects of some cations and anions on spin labeled cytoplasmic membranes of bacillus subtilis

Abstract The effects of CaCl2, MgCl2, LaCl3 and some alkali halides on Bacillus subtilis cytoplasmic membranes were studied using stearic acid spin labels. The results indicate that two mechanisms are operating when these ions interact with B. subtilis membranes. At low ionic concentrations (0 to 0.1 M) there is direct cation binding to the anionic membranes which in the case of Ca2+, Mg2+ and La3+, confers rigidity on the membranes and reaches saturation when the number of cations present equals the number of anionic sites on the lipids. At high concentrations there is a further effect on the membranes that parallels the known organizing/disorganizing effects that the ions studied have on water structure.

Endor studies on the covalently bound flavin at the active center of succinate dehydrogenase

Hemmerich et al. [l] recently reported that the probable site of attachment of the covalently bound FAD [2] to the peptide chain at the active center of succinate dehydrogenase (SD) is through the 8a group of the isoalloxazine ring system. This conclusion was based on a systematic comparison of acid hydrolyzed SD flavin with &substituted and other model compounds in terms of EPR and optical spectroscopy, pH-fluorescence, and chemical reactivity and stability. The strongest evidence that the peptide is attached at this position comes from the nearly identical EPR hyperfine structure of the cation radical and hyposochromic optical shifts shown by SD flavin and &-substituted model compounds. It was, nervertheless, desirable to confirm this conclusion by an independent method. This was done with a flavin peptide preparation [2] . In our previous studies [l] acid hydrolyzed peptides were used, which lack the amino acids but still contain a terminal group attached to the 8cr carbon (cf fig. 5A for structure). It has been pointed out [3] that ENDOR (electron nuclear double resonance) is particularly suited to investigate methyl groups

Flavin-radical-metal chelates.

Publisher Summary This chapter presents an overview of flavin–radical–metal chelates. The existence of flavin-radical-metal chelates and of flavoquinonemetal charge-transfer chelates has been well established in the model systems and their properties have been determined. So far, however, there is no single case where any such chelate has been demonstrated in a flavin-metal containing protein. With metalloflavoproteins containing Mo and/or Fe, it has been observed in the native enzyme that only part of the flavin and the metal can under the conditions explored be recovered as species that give ESR signals. The possibility has been discussed that the missing portions of both species could be present as radical-metal chelates, which would become indetectable by ESR. This could possibly be demonstrated by the characteristic paramagnetic susceptibility of such a chelate. Such measurements would require great sensitivity and accuracy because several centers in the metalloflavoprotein must change their paramagnetism and each individual change depends critically on the redox state of the protein. In this chapter, an overview of chelate stability, electron spin resonance, and electron nuclear double resonance is presented. Light absorption is analyzed, and concepts of paramagnetic susceptibility are explained.

Magnetic susceptibility of succinate dehydrogenase: the 4-iron preparation.

Abstract Measurements were made at 20°C of the magnetic susceptibility of soluble succinate dehydrogenase (succinate: (acceptor) oxidoreductase, EC 1.3.99.1) (4 Fe/flavin) from beef heart. The resting enzyme and enzyme in the presence of fumarate or oxaloacetate appeared to be nearly diamagnetic. The addition of succinate, malonate, or malonate plus succinate, and other treatments resulted in an increase in paramagnetism: ΔχM,Fe = 1200×10−6c.g.s units (average) per penzyme iron atom. An excess of dithionite caused a larger increase in paramagnetism: ΔχM,Fe = 2500×10−6c.g.s. units (average). The change due to dithionite was partially reversible upon autoxidation. We cannot account quantitatively for these changes in terms of the paramagnetic species observable by electron spin resonance.