Phytochelatins as a Dynamic System for Cd(II) Buffering from the Micro- to Femtomolar Range

Abstract

Phytochelatins (PCs) are short Cys-rich peptides with repeating γ-Glu-Cys motifs found in plants, algae, certain fungi, and worms. Their biosynthesis has been found to be induced by heavy metals—both biogenic and toxic. Among all metal inducers, Cd(II) has been the most explored from a biological and chemical point of view. Although Cd(II)-induced PC biosynthesis has been widely examined, still little is known about the structure of Cd(II) complexes and their thermodynamic stability. Here, we systematically investigated glutathione (GSH) and PC2–PC6 systems, with regard to their complex stoichiometries and spectroscopic and thermodynamic properties. We paid special attention to the determination of stability constants using several complementary techniques. All peptides form CdL complexes, but CdL 2 was found for GSH, PC2, and partially for PC3. Moreover, binuclear species Cd x L y were identified for the series PC3–PC6 in an excess of Cd(II). Potentiometric and competition spectroscopic studies showed that the affinity of Cd(II) complexes increases from GSH to PC4 almost linearly from micromolar (log K 7.4 GSH = 5.93) to the femtomolar range (log K 7.4 PC4 = 13.39) and additional chain elongation does not increase the stability significantly. Data show that PCs form an efficient system which buffers free Cd(II) ions in the pico- to femtomolar range under cellular conditions, avoiding significant interference with Zn(II) complexes. Our study confirms that the favorable entropy change is the factor governing the elevation of phytochelatins’ stability and illuminates the importance of the chelate effect in shifting the free Gibbs energy.

The Bioinorganic Chemistry of Mammalian Metallothioneins

“…Thus, entropy is a determining effect in the thermodynamics of the binding process. 154 In T, there are no preorganized coordination environments, and protein folding, that is, the spatial organization of the protein's main chain, is coupled to the metal binding with a potentially very large conformational space in the process. There will be significant differences between the binding of the first couple of metal ions, which serve a role in nucleation of protein folding, and the binding of the last metal ions, which bind to an already mostly folded protein.…”

“…190−193 However, there is a caveat: the Cd(II) metalation process differs from that for Zn(II), and despite the chemical similarity of the two cations information obtained for one cannot be transferred readily to the other. 98,154,163 Important steps in the characterization of MTs were the determination of NMR structures of isolated domains of human and rabbit MT2 as well as the solution of the crystal structure of rat MT2. 44,46−48 The MT structures characterized are the ones for the fully metal-saturated proteins.…”

“…The significance of entropy is even more obvious, as the enthalpy of binding of each metal ion is very similar in the same coordination spheres. Thus, entropy is a determining effect in the thermodynamics of the binding process . In T, there are no preorganized coordination environments, and protein folding, that is, the spatial organization of the protein’s main chain, is coupled to the metal binding with a potentially very large conformational space in the process.…”

“…Structures of partially metal-depleted species are not available. Our knowledge about them is based only on computational methods and a characterization of structure at low resolution by mass spectrometry and pertain almost exclusively to Cd­(II) species. − Importantly, all discussed energetic effects that control metal-to-protein affinity can be different in states with a different metal load and, therefore, should be considered separately when discussing specific metal association or dissociation processes. Overall, despite formally being in the same tetrahedral coordination sphere, the metal ions in MT have various affinities.…”

“…Since Zn­(II) has no signatures in most spectroscopic methods, other metal ions such as Cd­(II) or Co­(II) were used as probes for the Zn­(II) binding processes and coordination modes . Cd­(II) complexes with Cys-rich proteins have conspicuous ligand-to-metal charge transfer bands with lower energy when compared to Zn­(II), facilitating the interpretation of conformational changes. − Spectra of Co­(II) complexes allow a direct observation of d-d bands and conclusions about coordination environment and geometries of the metal ions in partially and fully saturated MT species. , Fluorimetric methods were applied for structural studies of MT with attached FRET probes and to examine the metal association and dissociation and the determination of metal affinities. ,,,, 113 Cd and 111 Cd-NMR spectroscopy was critical for the elucidation of the cluster formation in the α- and β-domains, cluster dynamics, and the kinetics of Cd­(II) transfer. − However, there is a caveat: the Cd­(II) metalation process differs from that for Zn­(II), and despite the chemical similarity of the two cations information obtained for one cannot be transferred readily to the other. ,, Important steps in the characterization of MTs were the determination of NMR structures of isolated domains of human and rabbit MT2 as well as the solution of the crystal structure of rat MT2. ,− The MT structures characterized are the ones for the fully metal-saturated proteins. 3D structural investigations on partially metalated species were never performed.…”