Extreme Zinc-Binding Thermodynamics of the Metal Sensor/Regulator Protein, ZntR Yutaka Hitomi, Caryn E. Outten, and Thomas V. O’Halloran* Department of Chemistry, Northwestern UniVersity EVanston, Illinois 60208-3113 ReceiVed May 5, 2001 ReVised Manuscript ReceiVed July 15, 2001 Cells accumulate Zn(II) and employ this metal ion in a wide range of enzyme active sites. Working models for zinc homeo- stasis, the cumulative processes that maintain the cellular zinc quota within an optimal range, have benefited from recent discoveries of zinc uptake, export, sequestration, and sensing proteins. 1 A key feature of most zinc homeostasis models is the idea that unbound ion accumulates in a so-called “free zinc pool” in the cytosol; however, the magnitude of this pool has proved difficult to evaluate. Here we address this issue by developing a method to directly determine the zinc-binding thermodynamics of ZntR, an intracellular zinc metalloregulatory protein from Escherichia coli. While coupled activity assays indicate that ZntR has a femtomolar sensitivity to Zn(II), 2 the direct binding constant is not known. We now show that ZntR exhibits the highest equilibrium Zn(II) binding constant measured for a native zinc- protein to date, although some zinc-finger peptides approach this affinity. The unusual thermodynamic affinity of zinc for this sensor requires a reevaluation of some tenets of cellular zinc homeostasis mechanisms. ZntR, a homologue of MerR, is a dimeric metalloregulatory protein that functions as a Zn(II)-responsive genetic switch. 3 As zinc concentrations outside the cell rise, ZntR turns on production of proteins that remove any excess zinc ions from the cell. Specifically, the Zn(II) bound form of ZntR, but not the apo- form, stimulates transcription of zntA, a gene encoding a zinc efflux pump. 3 ZntR regulates transcription by a metal-induced DNA-distortion mechanism 3c in which zinc-induced structural changes in the ZntR protein lead to restructuring of the bound DNA. Metal occupancy of ZntR was monitored by changes in tyrosine fluorescence. ZntR has no tryptophan but five tyrosine residues, one of which is located near the putative zinc-binding site while others are near the helix-turn-helix DNA-binding domain. Excita- tion of apo-ZntR at 278 nm yields a maximum fluorescence emission at 303 nm, characteristic of tyrosine and not tyrosinate. 4 In the presence of excess zinc, the emission intensity increases by 2.6-fold with no change in the spectral shape, indicating a Zn(II)-induced change in protein conformation that alters the local environment of one or more tyrosines (Figure 1a). The activity of ZntR is very sensitive to free zinc, therefore a strong zinc chelator is required to see the lowest levels of transcript in the absence of zinc ion. 3c To directly measure the extremely high zinc affinity under such conditions, we employed a metal-buffered solution method developed for characterization of metal-chelator interactions. 5 N,N,N′,N′-Tetrakis(2-pyridyl- methyl)ethylenediamine (TPEN) is a zinc chelator with a zinc dissociation constant of 9.2 × 10 -14 M at pH 7.0, 6 and can be used to buffer the free zinc concentrations between 10 -16 and 10 -14 M. The concentrations of free zinc were calculated using the program SPE based on the published pK a and logK Zn-TPEN values of TPEN. 6b The results of a typical fluorescence experiment at pH 7.0 are shown in Figure 1a. Several models for the apparent zinc dissociation constant were fit to the observed binding isotherm. The best fit was obtained for noncooperative 1:1 binding of Zn(II) to the ZntR dimer according to eq 1, with a dissociation constant of log K d )-14.8 ( 0.03 (Figure 1b): where F and Z represent the fluorescence intensity and the free zinc concentration, respectively. Although previous results indicate that the analogous MerR protein binds one Hg(II) per dimer, the ZntR dimer can bind two zinc ions per dimer. 3c Therefore two other zinc-binding models can be considered which are consistent with the noncooperative binding isotherm (Figure 1b) where the value for a refined Hill coefficient is 1.00: (1) zinc binding to two equivalent sites in the ZntR dimer equally enhances tyrosine fluorescence intensity; or (2) the ZntR dimer has two inequivalent (1) (a) Williams, R. J. P.; Frausto da Silva, J. J. R. Coord. Chem. ReV. 2000, 200-202, 247-348. (b) Cousins, R. J.; McMahon, R. J. J. Nutr. 2000, 130, 1384S-7S. (c) Eide, D. J. Annu. ReV. Nutr. 1998, 18, 441-69. (d) Patzer, S. I.; Hantke Mol. Microbiol. 1998, 31, 893. (e) Palmiter, R.; Findley EMBO J. 1995, 14, 639. (2) Outten, C. E.; O’Halloran, T. V. Science 2001, 292, 2488-2492. (3) (a) Binet, M. R.; Poole, R. K. FEBS Lett. 2000, 473, 67-70. (b) Brocklehurst, K. R.; Hobman, J. L.; Lawley, B.; Blank, L.; Marshall, S. J.; Brown, N. L.; Morby, A. P. Mol. Microbiol. 1999, 31, 893-902. (c) Outten, C. E.; Outten, F. W.; O’Halloran, T. V. J. Biol. Chem. 1999, 274, 37517-24. (4) Lakowicz, J. R. Principles of Fluorescence Spectorscopy; 2nd ed.; Kluwer Academic/Plenum: New York, 1999. (5) (a) Fahrni, C. J.; O’Halloran, T. V. J. Am. Chem. Soc. 1999, 121, 11448-11458. (b) Tsien, R.; Pozzan, T. Methods Enzymol. 1989, 172, 230- 62. (6) (a) Martell, A. E.; Smith, R. M. NIST Critical Stability Constants of Metal Complexes. NIST Standard Reference Database 46, 1998; Vol. 5.0.(b) Martell, A. E.; Motekaitis, R. J. The Determination and Use of Stability Constants; VCH: New York, 1988. Figure 1. Fluorescence emission spectra (a) and normalized fluorescence intensity (b) in the presence of 10.0 μM ZntR dimer and a range of free zinc ion concentrations, monitered with excitation wavelengths of 278 nm. Conditions: 1.0 mM TPEN, 20 mM Tris-Bis Tris buffer (pH 7.0, I ) 0.1 M (NaCl)) at 25 °C. The solid line shows the fitted curve for eq 1 with log Kd )-14.8 (r 2 ) 0.999). F ) (F min K d + F max Z)/(K d + Z) (1) 8614 J. Am. Chem. Soc. 2001, 123, 8614-8615 10.1021/ja016146v CCC: $20.00 © 2001 American Chemical Society Published on Web 08/09/2001