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A family of small molecular weight proteins with thiol-disulfide exchange activity have been discovered, widely distributed from E. coli to mammalian systems, called thioltransferases or glutaredoxins. There are no substantiated reports of thioltransferases-glutaredoxins in plants; however, partially purified dehydroascorbate reductase from peas had thiol-disulfide exchange catalytic activity using glutathione as reductant and S-sulfocysteine as thiosulfate cosubstrate (unpublished data). Thus, this class of proteins is universally distributed. Based on mutagenesis studies, a sequence of Cys-Pro-Tyr(Phe)-Cys- followed by Arg-Lys- or Lys alone is critical for both the thiol-disulfide exchange reaction and the dehydroascorbate reductase activity. The dithiol-disulfide loop represented by this structure is unique since the cystine closer to the N-terminus has a highly acidic thiol pKa (3.8 as determined for the pig liver enzyme) that contributes to the protein's high S- nucleophilicity. Compared with the microbial enzyme, the mammalian thioltransferases (glutaredoxins) are extended at both N and C termini by 10-12 amino acid residues, including a second pair of cysteines toward the C-terminus with no known special function. Yeast thioltransferase is more like mammalian enzymes in length (106 amino acids) but more like E. coli glutaredoxin in being unblocked at the N-terminus and having only one set of cysteines; that is, at the active center. The three mammalian enzymes, for which sequences are available, are blocked at the N-terminus by an acetyl group linked to alanine with no known special function other than possibly to impart greater cellular turnover stability. A report of carbohydrate (8.6%) content in rat liver thioltransferase has not been verified by more sensitive methods of carbohydrate analysis, nor has carbohydrate been identified in samples of purified glutaredoxin from any source. Thiol transferase and glutaredoxin are two names for the same protein based on similarity of amino acid sequence, immunochemical cross-reactivity, and other enzyme properties. The inability of thioltransferase from some mammalian sources to act as an electron carrier in ribonucleotide reductase systems, whether homologous or heterologous in origin, remains to be explained in future studies.

Under anaerobic conditions, recombinant pig liver thioltransferase (glutaredoxin)(TT, GRX) (EC 1.8.4.1) was strongly inhibited by cis and carbo-platin and somewhat less sensitive to trans-platin, in vitro. By extrapolation to total inhibition, the ratio of platinum drug/thioltransferase was approximately 1.0 for cis and carbo-platin, but greater than 1.0 for trans-platin. When thioltransferase was not reduced, inhibition by preincubation with the platinum complexes required molar excesses of 1,300 and 675 to one for cis-platin and trans-platin, respectively or 400-500 microM for 50% inhibition. The inhibition of thioltransferase at high drug concentrations in the presence of oxygen was associated with cross-linking of monomers into dimers within 5 min and, in the case of cis-platin treatment, to trimers in 20 min incubation.

Chelation of Fe from the Fe-protein component (Av2) of Azotobacter vinelandii nitrogenase has been investigated. The chelation, which requires MgATP binding by Av2, is best described as a two-exponential process. The rates for the two phases differed by approximately 10-fold and increased as the concentration of MgATP was increased. The rates for both phases were 50% of maximum at approximately 1.5 mM MgATP. At MgATP concentrations greater than 100 microM, the more rapid phase represented approximately 25% of the total Fe chelated from Av2. However, below 100 microM MgATP, the proportion of the faster phase decreased until at 20 microM MgATP, only a single phase could be detected. The properties of Av2 were studied at various stages of Fe chelation. The partially chelated protein was isolated from the reaction by gel filtration and was subjected to a second MgATP-dependent Fe chelation. Material isolated after the completion of the first phase regained biphasic kinetics in subsequent chelation reactions. However, if MgATP was present during the isolation of Av2, then only a single phase was observed in the subsequent chelation studies. In addition, the enzymatic activity of Av2 decreased concomitantly with total Fe chelation. To account for these observations, a model is presented in which Av2 exists in two conformers. Fe chelation is proposed to occur from either conformer but only when two MgATP are bound. Both conformers bind MgATP with the same affinity but are distinguished by a 10-fold difference in chelation rate. The two conformers are in equilibrium and can interconvert only in the absence of MgATP. That is, MgATP binding prevents the conversion of the two conformational states.