Spectroscopic studies of arsenic(III) binding to Escherichia coli RI methyltransferase and to two mutants, C223S and W183F

Abstract

The interactions of an arsenic (III) reagent, (CH3)2AsSCH2CONH2, with two Escherichia coli RI methyltransferase mutants, W183F and C223S, have been studied by phosphorescence, optically detected magnetic resonance, and fluorescence spectroscopy. The phosphorescence spectrum of the W183F mutant containing only one tryptophan at position 225 reveals a single 0,0-band that is red-shifted by 9.8 nm upon binding of As(III). Fluorescence titration of W183F with (CH3)2AsSCH2CONH2 produces a large tryptophan fluorescence quenching. Analysis of the quenching data points to a single high-affinity As(III) binding site that is associated with the fluorescence quenching. Triplet-state kinetic measurements performed on the perturbed tryptophan show large reductions in the lifetimes of the triplet sublevels, especially that of the T chi sublevel. As(III) binding to the enzyme at a site very close to the Trp225 residue induces an external heavy-atom effect, showing that the perturber atom is in van der Waals contact with the indole chromophore. In the case of the C223S mutant, a single tryptophan 0,0-band also is observed in the phosphorescence spectrum, but no change occurs upon addition of the As(III) reagent. Fluorescence titration of C223S with As(III) shows essentially no quenching of tryptophan fluorescence, in contrast with W183F. These results, along with previous triplet-state and biochemical studies on the wild-type enzyme [Tsao, D. H.H., & Maki, A. H. (1991) Biochemistry 30, 4565-4572], show that As(III) binds with high affinity to the Cys223 residue and that the Trp225 side chain is located close enough to that of Cys223 to produce a heavy-atom perturbation when As(III) is bound.

Binding of the General Anesthetics Chloroform and 2,2,2-Trichloroethanol to the Hydrophobic Core of a Four–α-Helix Bundle Protein¶

“…The quenching of fluorescence and the shortening of the phosphorescence lifetime are characteristics of the heavy atom effect (39). Both mechanisms require close contact (orbital overlap) between the quencher and the indole ring (40)(41)(42), although there is some evidence that quenching can occur at distances of 3-5 Å (43,44). The ability of chloroform and halothane to enhance electron spin intersystem crossing may influence electron transfer rates as proposed for xenon (45) and perhaps underlies some of the biological effects of these anesthetics.…”

Identification of arsenic-binding proteins in human breast cancer cells

“…Direct binding to arsenic is believed to be important in at least two aspects: 1) binding and resulting inhibition of enzymes and other proteins may cause the chemotherapeutic effects; 2) binding to certain proteins may be a detoxification process (14). It is known that trivalent arsenic is chemically more reactive than pentavalent species, and binds many proteins, for example, tubulin and actin (15), IKKα/β (16), E. coli arsR protein (17), E. coli RI methyltransferase (18), hemoglobin (19,20), nicotinic receptor (21), metallothionein (22), galectin 1 and thioredoxin peroxidase II (23,24), thioredoxin and protein disulfide isomerase (25), glucocorticoid receptor (26), and estrogen receptor α (27). Some other proteins were also shown to bind arsenic, yet their identities are still unknown (25,28).…”

Transition metal ions and selenite modulate the methylation of arsenite by the recombinant human arsenic (+3 oxidation state) methyltransferase (hAS3MT)

“…and Cys residues are in close proximity to each other [55]. Another peak appeared at about 408 nm when Co 2+ or Mn 2+ but not Zn 2+ was added.…”