6. Marco Referencial
6.2 Marco conceptual
6.2.2 Rol del Trabajador Social
binding to MTs
The concept of metal binding cooperativity to MTs is significantly different from the traditional biochemical view of cooperativity, such as the binding of oxygen to the four domains of hemoglobin. Due to the lack of preformed binding sites, metal-induced folding of the protein strand, and numerous possible intermediate structures, the simple model of traditional biochemical cooperativity becomes much more complex when discussing the cooperativity of MT metal binding. However, the overall thermodynamic driving force remains the same. A multistep process is cooperative when the successive reactions become more thermodynamically favourable rather than less favourable, as expected theoretically.119 In the case of oxygen binding to tetrameric hemoglobin, for example, binding of one molecule of oxygen in one of the four domains triggers a conformational change in the other three domains that increases their oxygen binding affinities and promotes the formation of fully oxygenated (O2)4-hemoglobin.120 Thus, only deoxy- and fully oxygenated hemoglobin states are predominant, resulting in a fully cooperative mechanism.
In the cooperative mechanism of the reactions shown in Scheme 1.1, the successive K values increase, that is Ki+1 > Ki, promoting the formation of the fully metal saturated product. In this mechanism, the only species that are present in solution at equilibrium are the fully metallated end point of the reaction, which is formed from any amount of added metal, and the fully unsaturated apoMT. Since the reaction equilibrium constants increase for cooperative systems, the intermediate metallation states are thermodynamically
unfavoured and formation of saturated MT is promoted over partial metallation states. For example, if zinc binding to mammalian MT was fully cooperative, addition of 3 equiv of Zn to a solution of apoMT would result in the formation of ~3/7 Zn7MT leaving ~4/7 apoMT with little to no intermediate metallation states.
The biophysical rationale for cooperativity in MTs is justified by the preferential clustering (over beading) of metals formed from metal templated folding and formation of transiently empty metal binding sites. Thus, reorganization of the apopeptide upon binding of one zinc ion preforms subsequent zinc binding sites, likely near the first binding site, promoting clustering. These reactions – metal binding leading to formation of a new metal binding site – cascade, one after another, which drives the reaction to completion and forming the fully metal-saturated MTs.
In contrast, in a non-cooperative process, the successive K values decrease such that Ki+1 < Ki, following the theoretically expected trend. This means that, for example, for the set of successive reactions shown in Scheme 1.1, the reactions become less favoured with each additional metallation step. In this mechanism, all of the intermediate metallation states are formed statistically and the distribution of Znn-MT species will depend on the mole equivalents of zinc added. For example, if zinc binding to MT was fully non- cooperative, addition of 3 equiv of Zn to a solution of apoMT would result in formation of an approximately normal distribution of Zn1-5-MT, where the average zinc load would equal Zn3-MT. The actual speciation distribution that would be centered on Zn3-MT would depend on the ratios of the K values for the reactions describing the formation of Zn3-MT from Zn2-MT and the reaction of Zn3-MT forming Zn4-MT.
The structural implications of non-cooperativity in the metallation reactions of MTs result in the preferential formation of (MIIS4)2- beads over clusters. As each metal is added, there is no significant change to subsequent metallation reactions, with the exception that there are four fewer free thiols available to form the next beaded binding site. Thus, in a non-cooperative pathway, subsequent reactions become less favoured largely due to there being less open coordination sites. In fact, as was shown for arsenic metallation of MTs121 (noting that arsenic binds in a trigonal pyramidal geometry,
forming (AsIIIS3)-beads and does not form clusters), the decrease in reaction rate constants was approximately linear with remaining open metal binding sites.122, 123 A further corollary of non-cooperative binding is that cluster formation involving bridging thiols occurs only for the 6th and 7th divalent metals bound (Figure 1.3).
1.5.1
Experimental evidence of metal binding cooperativity in MTs
Numerous studies have suggested that zinc and cadmium bind to MTs in a cooperative fashion,111 though more recent results from ESI-MS experiments, specifically on MT1116, 124and MT3,125 have suggested the operation of a non-cooperative metal binding mechanism. There has been considerable debate on whether the binding of zinc and cadmium to mammalian MT was cooperative or non-cooperative. Results from CD, UV, NMR, and other experimental techniques have suggested that cadmium and zinc binding to MTs was cooperative and domain-specific. Analysis of optical spectral data led to the conclusion that the first four metals bound cooperatively to apoMT and were located specifically in the α domain.126 For the intact 20 cysteine protein, this implies that only the apo [(SH)9β-(SH)11α-MT] and (MII)4 [(SH)9β-(MII)4S11-αMT] species would be stable for up to four equivalents of divalent metals added. However, these experimental techniques provide the average metallation state of the solution species and are unable to resolve multiple metallation states. Recently, ESI-MS techniques have been applied to the study of metallation reactions of MT. As described above, ESI-MS is able to identify the stoichiometry of bound metals, including multiple metallation states, in addition to structural and other quantitative and qualitative information, demonstrating the power of this technique for studying the multiple metal binding events in MTs.