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Near diffusion-controlled reaction of a Zn(Cys)4 zinc finger with hypochlorous acid.

ABSTRACT: Hypochlorous acid (HOCl) is one of the strongest oxidants produced in mammals to kill invading microorganisms. The bacterial response to HOCl involves proteins that are able to sense HOCl using methionine, free cysteines or zinc-bound cysteines of zinc finger sites. Although the reactivity of methionine or free cysteine with HOCl is well documented at the molecular level, this is not the case for zinc-bound cysteines. We present here a study that aims at filling this gap. Using a model peptide of the Zn(Cys)4 zinc finger site of the chaperone Hsp33, a protein involved in the defence against HOCl in bacteria, we show that HOCl oxidation of this model leads to the formation of two disulfides. A detailed mechanistic and kinetic study of this reaction, relying on stopped-flow measurements and competitive oxidation with methionine, reveals very high rate constants: the absolute second-order rate constants for the reaction of the model zinc finger with HOCl and its conjugated base ClO- are (9.3 ± 0.8) × 108 M-1 s-1 and (1.2 ± 0.2) × 104 M-1 s-1, the former approaching the diffusion limit. Revised values of the second-order rate constants for the reaction of methionine with HOCl and ClO- were also determined to be (5.5 ± 0.8) × 108 M-1 s-1 and (7 ± 5) × 102 M-1 s-1, respectively. At physiological pH, the zinc finger site reacts faster with HOCl than methionine and glutathione or cysteine. This study demonstrates that zinc fingers are potent targets for HOCl and confirms that they may serve as HOCl sensors as proposed for Hsp33.

SUBMITTER: Lebrun V

PROVIDER: S-EPMC6021785 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Project description:Zinc finger proteins that bind Zn(II) using a Cys2His2 coordination motif within a ??? protein fold are the most abundant DNA binding transcription factor domains in eukaryotic systems. These classic zinc fingers are typically unfolded in the apo state and spontaneously fold into their functional ??? folds upon incorporation of Zn(II). These metal-induced protein folding events obscure the free energy cost of protein folding by coupling the protein folding and metal-ion binding thermodynamics. Herein, we determine the formation constant of a Cys2His2/??? zinc finger domain, the C-terminal finger of the Wilms' tumor suppressor protein (WT1-4), for the purposes of determining its free energy cost of protein folding. Measurements of individual conditional dissociation constants, Kd values, at pH values from 5 to 9 were determined using fluorescence spectroscopy by direct or competition titration. Potentiometric titrations of apo-WT1-4 followed by NMR spectroscopy provided the intrinsic pKa values of the Cys2His2 residues, and corresponding potentiometric titrations of Zn(II)-WT1-4 followed by fluorescence spectroscopy yielded the effective pKa(eff) values of the Cys2His2 ligands bound to Zn(II). The Kd, pKa, and pKa(eff) values were combined in a minimal, complete equilibrium model to yield the pH-independent formation constant value for Zn(II)-WT1-4, Kf(ML) value of 7.5 × 10(12) M(-1), with a limiting Kd value of 133 fM. This shows that Zn(II) binding to the Cys2His2 site in WT1-4 provides at least -17.6 kcal/mol in driving force to fold the protein scaffold. A comparison of the conditional dissociation constants of Zn(II)-WT1-4 to those from the model peptide Zn(II)-GGG-Cys2His2 over the pH range 5.0 to 9.0 and a comparison of their pH-independent Kf(ML) values demonstrates that the free energy cost of protein folding in WT1-4 is less than +2.1 kcal/mol. These results validate our GGG model system for determining the cost of protein folding in natural zinc finger proteins and support the conclusion that the cost of protein folding in most zinc finger proteins is ?+4.2 kcal/mol, a value that pales in comparison to the free energy contribution of Zn(II) binding, -17.6 kcal/mol.

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Near diffusion-controlled reaction of a Zn(Cys)4 zinc finger with hypochlorous acid.

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