Although the thermodynamic binding features of ML1and ML2(Fig. 2.2) are very similar, a characteristic of transport systems, as opposed to those of retain- ing centres, is that the former must bind and release M reasonably quickly since transport must be continuous. The release of M may be achieved by lowering the oxidation state of the metal (to decrease the binding constant), by changes of pH (to decrease the effective binding constant), by changes in the conformation of the protein (to change the co-ordination site of the metal), or by hydrolysis of the ligand. We now know that certain metal ions are transport- ed in aqueous phases as well as in membranes, e.g. see copper chaperones in Chapter 15. As stated in Section 2.9.1, a special feature of proteins is that they may have ‘leader’ sequences which ensure that they can cross membranes. 2.12.3
Insertion in pre-formed holes or chelating rings
A particular method of selection by kinetic control may also be considered: that of insertion into a pre-formed hole or chelating ring, from which exchange of the metal ion concerned is virtually impossible. This reaction is not rapid, due to steric constraints, as mentioned before. Consider the case where, while the binding of M to L1is governed by thermodynamic factors, it could well be that the rates of transfer of M from ML1to L2are M-dependent and, in fact, L1could merely act to control those rates
An example makes clear the selectivity that can be introduced by this mechanism. The insertion of magnesium in the chlorin ring of chlorophyll cannot be made directly in vitro in the presence of other free metal ions, like copper, zinc, or even nickel and cobalt. It is hard to see how any reasonably devised intermediate step could prevent the latter ions from entering preferen- tially a strong porphyrin-type ligand like chlorin at equilibrium (see Fig. 2.8 for the relative binding strengths).
Now, let us suppose that the step ML1Æ ML2is irreversible and that the kinetic constants k are very different for different M. This could arise in part because the transition metals are complexed by other ligands (L4) and the con- centration of their free ions are low compared to that of magnesium, which is then complexed to a larger degree to a relatively poor ligand L1, e.g. an O-donor protein site such as ATP, or because the MgL1complex is specifically recognized by L2. It could even be that any ML1complex of the transition metals is not recognized due to conformational factors within a carefully modulated, even energized, protein transfer system. As a consequence of one or another of these factors, k would be much higher for the reaction MgL1Æ MgL2 than for any transition metal ion and, since the step is irreversible, this reaction would hinder the possibility of occurrence of all the other metal ions in ML2. In different words, those weak-binding metal ions, which are in excess over ligands but react rapidly, can block the entry of ions that bind better to L2in an absolute sense, provided the kinetics of transfer are suitably arranged. It is a striking observation that, while in vivo the insertion is performed correctly giving magnesium chlorophyll, in vitro it is very difficult to avoid the production of zinc chlorin when this metal is present even in trace amounts. M + L ML ML L initial final uptake uptake 1 1 2 2 1 ML ML L k k k æ Ææ ¨ æææ' æ Ææ + + .
In fact it seems likely that the correct insertion of each metal ion with less than 0.1 per cent error is achieved for magnesium in chlorin, iron in protopor- phyrin, cobalt in corrin, nickel in F-430 (nirrin), and copper in uroporphyrin (see Table 2.15). This result could only be achieved by kinetic control based on specific recognition of ML1complexes (Fig. 2.13). Thus the design of these ligands is to prevent easy access and so control associations.
Once in its new site, the complex ML2is recognized specifically and bound by a protein in such a way that it is still harder to exchange the metal M (see Fig. 2.7). In effect, ML2is almost a new ‘element’ in that it allows Fe, Co, Ni, Cu, and Mg to be used specifically in new ways. As proteins developed to bind these special complexes so the use of the elements expanded: it allowed mononuclear ring-chelate species of Fe2+(and Fe3+) in different spin states, of cobalt and nickel in at least two oxidation states and in low spin states, and of magnesium chlorophyll in membrane proteins, just as it was possible to retain copper, zinc, and molybdenum directly bound to the side-groups of the proteins. Previous to this development intracellular iron was overwhelmingly bound as FenSncomplexes, i.e. mixed valence clusters, cobalt might not have been cap- tured, and magnesium was associated only with rapidly equilibrating reactions of phosphate in water. Very large functional advantages accrue from the design of these small, relatively rigid molecule chelates, as will be seen later.
The selectivity conditions described in this section require that the different chelating rings produced for the different metal ions are synthesized in con- trolled amounts. Once more, there must be feedback from synthesized prod- uct to the genes.
Fig. 2.13 The diverse ring chelates produced for different metal ions from one precursor.
Table 2.15 Polypyrrole chelating agents and their selection of metal ions
Chelating agent Metal Binding site
Chlorin Mg Chlorophyll proteins Protoporphyrin Fe Haemoglobin, cytochromes Corrin Co Cobalamin (vitamin B12) proteins (Nirrin) F-430 Ni Factor F-430 proteins
2.12.4