Classical Cys₂His₂Zinc Finger Peptides Are Rapidly Oxidized by Either H₂O₂or O₂Irrespective of Metal Coordination

Abstract

ZIF268, a member of the classical zinc finger protein family, contains three Cys(2)His(2) zinc binding domains that together recognize the DNA sequence 5'-AGCGTGGGCGT-3'. These domains can be fused to an endonuclease to make a chimeric protein to target and cleave specific DNA sequences. A peptide corresponding to these domains, named ZIF268-3D, has been prepared to determine if the zinc finger domain itself can promote DNA cleavage when a redox active metal ion, Fe(II), is coordinated. The UV-vis absorption spectrum of Fe(II)-ZIF268-3D is indicative of Fe(II) coordination. Using fluorescence anisotropy, we demonstrate that Fe(II)-ZIF268-3D binds selectively to its target DNA in the same manner as Zn(II)-ZIF268-3D. In the presence of added oxidant, H(2)O(2) or O(2), DNA cleavage is not observed by Fe(II)-ZIF268-3D. Instead, the peptide itself is rapidly oxidized. Similarly, Zn(II)-ZIF268-3D and apo-ZIF268-3D are rapidly oxidized by H(2)O(2) or O(2), and we propose that ZIF268-3D is highly susceptible to oxidation.

Oxidation of Zn(Cys)4 Zinc Finger Peptides by O2 and H2O2: Products, Mechanism and Kinetics

“…[6,7] Recently, it was reported that zinc protects the cysteines of tris-tetraproline, a ZnA C H T U N G T R E N N U N G (Cys) 3 A C H T U N G T R E N N U N G (His) zinc finger, from oxidation, [8] but this was not evidenced for a classical ZnA C H T U N G T R E N N U N G (Cys) 2 A C H T U N G T R E N N U N G (His) 2 zinc finger. [9] However, proteins containing a reactive zinc finger site were discovered about 20 years ago. The first one was the protein Ada, which repairs mutagenic DNA methyl phosphotriester lesions by transfer of the methyl group to a highly nucleophilic cysteine residue of its ZnA C H T U N G T R E N N U N G (Cys) 4 site.…”

“…Comparison between the Zn 2+ ‐loaded and the apo form of the Sp1 transcription factor pointed to the conclusion that Zn 2+ protects the cysteines of the neutral Zn(Cys) 2 (His) 2 site from oxidation by O 2 and H 2 O 2 , thereby enforcing the idea that zinc‐bound cysteines are poorly reactive and that zinc fingers are unreactive in comparison with free cysteines 6. 7 Recently, it was reported that zinc protects the cysteines of tris‐tetraproline, a Zn(Cys) 3 (His) zinc finger, from oxidation,8 but this was not evidenced for a classical Zn(Cys) 2 (His) 2 zinc finger 9…”

Switching Metal Ion Coordination and DNA Recognition in a Tandem CCHHC-type Zinc Finger Peptide

“…28,52,63–65 The native protein, NZF-1-F2F3 was expected to bind to this DNA with nanomolar affinity, based upon previous studies. 28 Similarly, the CCFHC mutant, which maintains the metal coordinating histidine, was also expected to bind to this DNA target with nM affinity.…”

Regulation of the Central Dogma through Bioinorganic Events with Metal Coordination for Specific Interactions

“…Studies on the coordination of ZFs have limitations due to the spectroscopically silent zinc ions, which have a fully occupied d‐orbital despite seeming to be free from oxidation in coordination chemistry. Fortunately, Co 2+ coordinates with Cys and His ligands with distorted T d geometries and provides basic biophysical evidences of Co 2+ coordination 19,24,30,32–40 . These spectroscopic approaches provide valuable information for biophysical assays, including association constants that can be further applied for the quantification of oxidized states 5,39,40 .…”

“…Fortunately, Co 2+ coordinates with Cys and His ligands with distorted T d geometries and provides basic biophysical evidences of Co 2+ coordination 19,24,30,32–40 . These spectroscopic approaches provide valuable information for biophysical assays, including association constants that can be further applied for the quantification of oxidized states 5,39,40 . ZF proteins show significantly low K d values to Zn 2+ (10 −12 to −15 M), indicating that Zn 2+ is tightly regulated in physiological systems 10,41–46 .…”