Elemental analyses, Mössbauer, and EPR data are reported to show that endonuclease III of Escherichia coli is an iron-sulfur protein. Mössbauer spectra of protein freshly prepared from E. coli grown on 57Fe-enriched medium demonstrate that the native enzyme contains a single 4Fe-4S cluster in the 2+ oxidation state, with a net spin of zero. Upon treatment with ferricyanide, a fraction (less than 25%) of the clusters is oxidized into a state which yields an EPR spectrum near g = 2.01 typical of a 3Fe-4S cluster. The magnetic field dependence of the linear electric field effect verifies this assignment. Electron spin echo modulation on the g = 2.01 form of the protein in deuterated solvent indicates the presence of exchangeable protons in the vicinity of the 3Fe-4S cluster. The data obtained show that the [4Fe-4S]2+ cluster of the native enzyme is resistant to either oxidation or reduction, although photoreduction elicited a g = 1.94 type EPR signal characteristic of a [4Fe-4S]1+ cluster. These studies show that endonuclease III is unique in being both a DNA repair enzyme and an iron-sulfur protein. The function of the 4Fe-4S cluster remains to be established.

The gene which codes for endonuclease III of Escherichia coli has been sequenced. The nth gene was previously subcloned and defined as the gene which led to overproduction of endonuclease III when present on a multicopy plasmid and which created a deficiency in endonuclease III activity when mutated. The nth gene was sequenced and translated into a predicted polypeptide. The molecular weight (23,546), the amino-terminal amino acid sequence, and the amino acid composition of the polypeptide predicted from the nucleotide sequence are excellent agreement with those same properties determined for the purified protein. Thus, the nth gene is the structural gene for endonuclease III. Inspection of the nucleotide sequence reveals that there is an open reading frame immediately upstream of the nth gene, suggesting that it might be part of an operon. There is a region of dyad symmetry which could form a hairpin stem and loop structure if transcribed into RNA characteristic of a rho-dependent terminator downstream from the nth gene. The nth gene of Escherichia coli has been cloned onto a lambda PL expression vector which yields approximately 300-fold overproduction of endonuclease III. We have purified the enzyme to apparent homogeneity using two chromatographic steps. Our purification scheme allowed the preparation of 117 mg of protein from 190 g of E. coli with a 70% yield. The purified protein has both AP endonuclease activity and DNA N-glycosylase activity. The protein has a Stokes radius of 2.25 nm, a sedimentation coefficient of 2.65 S, a molecular weight of 26,300 in the native state and 27,300 in the denatured state, and a frictional ratio of 1.13.(ABSTRACT TRUNCATED AT 250 WORDS)

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The solid-state 113Cd nuclear magnetic resonance of cadmium adsorbed on montmorillonite was investigated. An analysis of three montmorillonite samples and a brief comparison with other clays are included. The spectra contain cadmium oxoanion components of differing line widths with differential sensitivities to preparation conditions. Echo train experiments at various temperatures and delays indicate that the line widths are governed by dynamic processes rather than chemical shift dispersion.These same experiments show the heterogeneity of the cadmium sites. Included in the study are the effects of pH variation, exchanged clay form, pretreatment with alginic and humic acids, and airand freeze-drying. These results are described in terms of ions in different environments and ion motion. For montmorillonite some of these sites are considered to be near iron in the octahedral layer. The interlayer ion diffusion is related to metal ion transport in clay layers and the potential consequences of migration.(1) SUNY at Albany.

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