Spectroscopic studies of zinc(II)- and cobalt(II)-associated Escherichia coli formamidopyrimidine-DNA glycosylase: Extended X-ray absorption fine structure evidence for a metal-binding domain
Gw. Buchko et al., Spectroscopic studies of zinc(II)- and cobalt(II)-associated Escherichia coli formamidopyrimidine-DNA glycosylase: Extended X-ray absorption fine structure evidence for a metal-binding domain, BIOCHEM, 39(40), 2000, pp. 12441-12449
Formamidopyrimidine-DNA glycosylase (Fpg) is a 30.2 kDa protein that plays
an important role in the base excision repair of oxidatively damaged DNA in
Escherichia coli. Sequence analysis and genetic evidence suggest that zinc
is associated with a C4-type motif, C-244-X-2-C-247-X-16-C-264-X-2-C-267,
located at the C-terminus of the protein. The zinc-associated motif has bee
n shown to be essential for damaged DNA recognition. Extended X-ray absorpt
ion fine structure (EXAFS) spectra collected on the zinc-associated protein
(ZnFpg) in the lyophilized state and in 10% frozen aqueous glycerol soluti
on show directly that the metal is coordinated to the sulfur atom of four c
ysteine residues. The average Zn-S bond length is 2.33 +/- 0.01 and 2.34 +/
- 0.01 Angstrom, respectively, in the lyophilized state and in 10% frozen a
queous glycerol solution. Fpg was also expressed in minimal medium suppleme
nted with cobalt nitrate to yield a blue-colored protein that was primarily
cobalt-associated (CoFpg). The profiles of the circular dichroism spectra
for CoFpg and ZnFpg are identical, suggesting that the substitution of Co2 for Zn2+ does not alter the structure of Fpg. A similar conclusion is reac
hed upon the analysis of two-dimensional N-15/H-1 HSQC spectra of uniformly
N-15-labeled samples of ZnFpg and CoFpg; the spectra are similar and displ
ay features characteristic of a structured protein. Biochemical assays with
a 54 nt DNA oligomer containing 7,8-dihydro-8-oxoguanine at a specific loc
ation show that CoFpg and ZnFpg are equally active at cleaving the DNA at t
he site of the oxidized guanine. EXAFS spectra of CoFpg indicate that the c
obalt is coordinated to the sulfur atom of four cysteine residues with an a
verage Co-S bond length of 2.28 +/- 0.01 and 2.29 +/- 0.01 Angstrom, respec
tively, in the lyophilized state and in 10% frozen aqueous glycerol solutio
n. The structural similarity between CoFpg and ZnFpg suggests that it is bi
ologically relevant to use the paramagnetic properties of Co2+ as a structu
ral probe.