Spectroscopic studies of arsenic(III) binding to Escherichia coli RI methyltransferase and to two mutants, C223S and W183F.
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.
Lam
W C
WC
Department of Chemistry, University of California, Davis 95616.
Tsao
D H
DH
Maki
A H
AH
Maegley
K A
KA
Reich
N O
NO
D001151:Arsenic / Q000378:metabolism*; D001665:Binding Sites; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008163:Luminescent Measurements; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D014364:Tryptophan; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D003545:Cysteine; D001151:Arsenic