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

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.