International
Tables for
Crystallography
Volume F
Crystallography of biological macromolecules
Edited by M. G. Rossmann and E. Arnold

International Tables for Crystallography (2006). Vol. F. ch. 12.1, pp. 251-253   | 1 | 2 |

Section 12.1.5.3. B-metal reagents

D. Carvin,a S. A. Islam,b M. J. E. Sternbergb and T. L. Blundellc*

aBiomolecular Modelling Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Field, London WC2A 3PX, England,bInstitute of Cancer Research, 44 Lincoln's Inn Fields, London WC2A 3PX, England, and cDepartment of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, England
Correspondence e-mail:  tom@cryst.bioc.cam.ac.uk

12.1.5.3. B-metal reagents

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The most useful members of the B-metal group, platinum, gold and mercury, give rise to an extensive range of heavy-atom compounds which form covalent, electrostatic and van der Waals complexes with proteins. Some compounds can bind to the protein molecule in different ways; for example, [\hbox{PtCl}_{4}^{2-}] can bind either covalently to the thioether group of methionine or electrostatically with positively charged residues.

Mercury compounds have proved very successful for preparing heavy-atom derivatives of protein crystals (Table 12.1.5.1[link]), mainly due to the ease of formation of covalent bonds with cysteine residues. An example is given in Fig. 12.1.5.2[link] in which mercuric chloride has been used to replace zinc in thermolysin. Hg2+ complexes are commonly two-coordinate linear and four-coordinate tetrahedral. The most popular mercury reagents are given in Table 12.1.5.3[link]. The covalent character in Hg—L bonds, especially in the two-coordinate complexes, can cause solubility problems in aqueous solutions. However, an excess of an alkali metal salt (e.g. [\hbox{HgI}_{2} + 2\hbox{KI} \rightarrow \hbox{K}_{2}\hbox{HgI}_{4}]) will often convert the compound to a more soluble anionic complex of the type [\hbox{Hg}X_{4}^{2-}], where X = Cl, Br, I, SCN, NCS, CN, [\hbox{SO}_{4}^{2-}], oxalate2−, [\hbox{NO}_{3}^{-}] or [\hbox{NO}_{2}^{-}]. In the presence of ammonium salts at high pH values, the cationic tetraammine complex, [\hbox{Hg(NH}_{3})_{4}^{2+}], tends to form. Variation in the charge on the aromatic groups of organomercurials can give rise to different substitution patterns.

Table 12.1.5.3| top | pdf |
The five most popular mercury derivatives

The first column gives the number of times the reagent has been used in the analyses included in the heavy-atom data bank.

No.Compound
101 Mercury(II) acetate
98 Mercury(II) chloride
85 Ethylmercurythiosalicylate (EMTS)
82 Potassium tetraiodomercurate(II)
81 para-Chloromercuriobenzenesulfonate (PCMBS)
[Figure 12.1.5.2]

Figure 12.1.5.2 | top | pdf |

Mercuric ions replace zinc in thermolysin (3TLN). The mercuric ion is shown superposed on the parent crystal structure; notice that the mercuric ion is slightly displaced from the zinc position due to its larger ionic radius.

Silver, used as the nitrate, tends to interact with cysteine or histidine (see Fig. 12.1.5.3[link]). In the presence of ammonium sulfate, it probably reacts as the ammonia complex, [\hbox{Ag(NH}_{3})_{4}^{+}]. Silver ions are less polarizing and less reactive than Hg2+ ions; thus they give similar derivatives but often with less disorder, as in glucagon (Sasaki et al., 1975[link]). Where the metal ion displaces a proton, Ag+ will need to react at a higher pH than Hg2+.

[Figure 12.1.5.3]

Figure 12.1.5.3 | top | pdf |

The binding of a silver ion to immunoglobulin Fab (2FB4). The positions of the ligands in the parent crystals are shown, and these must move in the complex to coordinate the silver ion.

The class B metals palladium, platinum and gold form stable covalent complexes with soft ligands, such as chloride, bromide, iodide, ammonia, imidazole and sulfur groups. The stereochemistry of their complexes depends on the number of d electrons present. For instance, the d10 ion of Au(I) gives a linear coordination of two [e.g. [\hbox{Au(CN)}_{2}^{-}]], whereas d8 ions of Pd(II), Pt(II) and Au(III) are predominantly square planar, giving cationic [e.g. [\hbox{Pt(NH}_{3})_{4}^{2+}]], anionic [e.g. [\hbox{Au(CN)}_{4}^{-}], [\hbox{PtCl}_{4}^{2-}] and [\hbox{PdCl}_{4}^{2-}]] or neutral [e.g. Pt(NH3)2Cl2] complexes. These may accept an additional ligand to give square pyramidal coordination or two ligands to give octahedral coordination. The additional ligands are normally more weakly bound. Pt(IV) has a d6 configuration and forms stable octahedral complexes, such as [\hbox{PtCl}_{6}^{2-}], with six equivalent covalently bound ligands.

The kinetic and thermodynamic stability of these complexes depends on the protein ligands, buffer, pH and salting in/out agent (Petsko et al., 1978[link]). Anionic groups do not readily react with anionic reagents, such as RS, but are attacked more readily by neutral nucleophiles such as RSH, R-imidazole or RNH2. The inert cationic group [\hbox{Pt(NH}_{3})_{4}^{2+}] is most likely to form electrostatic complexes with anionic groups, such as carboxylate. The neutral Pt(NH3)2Cl2 molecule, however, can penetrate into hydrophobic areas but requires a stronger nucleophile such as RS. In acidic and neutral solutions, [\hbox{PtCl}_{4}^{2-}] reacts most commonly with methionine (Figs. 12.1.5.4[link] and 12.1.5.5[link]), cystine (disulfide) (Fig. 12.1.5.6[link]), N-termini and histidine to form stable complexes. However, methionine reacts faster than histidine. Thus, it is possible to use time as a variable to define specificity. The most popular platinum reagents are listed in Table 12.1.5.4[link].

Table 12.1.5.4| top | pdf |
The five most popular platinum derivatives

The first column gives the number of times the reagent has been used the analyses included in the heavy-atom data bank.

No.Compound
287 Potassium tetrachloroplatinum(II)
61 Potassium tetranitritoplatinum(II)
58 Potassium tetracyanoplatinum(II)
57 Dichlorodiammineplatinum(II)
51 Potassium hexachloroplatinum(IV)
[Figure 12.1.5.4]

Figure 12.1.5.4 | top | pdf |

The binding of [\hbox{PtCl}_{4}^{2-}] through a methionine in azurin (1AZU).

[Figure 12.1.5.5]

Figure 12.1.5.5 | top | pdf |

The relative positions of methionine side chains (carbon: green; sulfur: yellow) in the parent crystals to the binding of platinum (pink) of [\hbox{PtCl}_{4}^{2-}]. The methionine side chains have been least-squares fitted.

[Figure 12.1.5.6]

Figure 12.1.5.6 | top | pdf |

The relative positions of cystine disulfide bridges (carbon: green; sulfur: yellow) in the parent crystals to the binding of platinum (pink) of [\hbox{PtCl}_{4}^{2-}]. The cystine side chains have been least-squares fitted, and only those with torsion angles in the range [99.7 \pm 8.3^{\circ}] have been used.

In aqueous solution, the square-planar complex [\hbox{AuCl}_{4}^{-}] is hydrolysed to [\hbox{Au(OH)}_{4}^{-}] in about one hour, or in the presence of a protein, reduced to Au(I) by methionine. In ammonium sulfate it probably exists as AuCl3(NH3), [\hbox{AuCl}_{2}(\hbox{NH}_{3})_{2}^{+}] and [\hbox{Au(NH}_{3})_{4}^{3+}]. In contrast, [\hbox{Au(CN)}_{2}^{-}] is more stable and normally binds electrostatically. However, on occasions at pH > 6.0, the [\hbox{Au(CN)}_{2}^{-}] complex has bound to cysteine residues by nucleophilic displacement reactions.

Osmium resembles platinum in many ways and typically acts as a class B metal. It occurs in all oxidation states from 0 to VIII, but most usually in III, as in K3OsCl6; in IV, as in K2OsCl6; in VI, as in K2OsO2(OH)4; and in VIII, as in osmium tetraoxide, OsO4. Higher-oxidation-state compounds tend to be reduced to OsO2(OH)2 in most crystallization solutions and in the presence of ammonia or halide ion they can become further reduced to cationic or anionic complexes, such as [\hbox{Os}(\hbox{NH}_{3})_{6}^{3+}] or [\hbox{Os}\hbox{Cl}_{6}^{2-}] . Anionic complexes may be substituted by histidine residues at pH > 7.0 or bound as ion pairs by histidine at pH < 7.0 or protonated amino groups. Cationic complexes tend to bind to negatively charged residues via electrostatic interactions.

Iridium is found in all oxidation states from II to VI but commonly exists in III, as in K3IrCl6, and IV, as in (NH4)2IrCl6. Ir(III) is similar to rhodium(III) and is found in a variety of cationic, uncharged and anionic complexes. All Ir(III) complexes are kinetically inert, whereas most anionic complexes of Rh(III) are labile. Ir(IV) is commonly found as the hexahalo complexes [\hbox{Ir}X_{6}^{2-}] (except iodine), which are also fairly kinetically inert. Cationic [e.g. [\rm{Ir(NH}_{3})_{6}^{3+}]], neutral (i.e. IrCl3) and anionic (i.e. [\hbox{IrCl}_{6}^{2-}]) species have proved useful in forming derivatives of protein crystals.

References

First citationPetsko, G. A., Phillips, D. C., Williams, R. J. P. & Wilson, I. A. (1978). On the protein crystal chemistry of chloroplatinite ions: general principles and interactions with triose phosphate isomerase. J. Mol. Biol. 120, 345–359.Google Scholar
First citationSasaki, K., Dockerill, S., Adamiak, D. A., Tickle, I. J. & Blundell, T. L. (1975). X-ray analysis of glucagon and its relationship to receptor binding. Nature (London), 257, 751–757.Google Scholar








































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