There is a challenge for metalloenzymes to acquire their correct metals because some inorganic elements form more stable complexes with proteins than do others. These preferences can be overcome provided some metals are more available than others. However, while the total amount of cellular metal can be readily measured, the available levels of each metal have been more difficult to define. Metal-sensing transcriptional regulators are tuned to the intracellular availabilities of their cognate ions. Here we have determined the standard free energy for metal complex formation to which each sensor, in a set of bacterial metal sensors, is attuned: The less competitive the metal, the less favorable the free energy and hence greater availability to which the cognate allosteric mechanism is tuned. Comparing these free energies with values derived from the metal affinities of a metalloprotein reveals the mechanism of correct metalation exemplified here by a cobalt-chelatase for vitamin B12.

Protein metal-occupancy (metalation) in vivo has been elusive. To address this challenge, the available free energies of metals have recently been determined from the responses of metal sensors. Here, we use these free energy values to develop a metalation-calculator which accounts for inter-metal competition and changing metal-availabilities inside cells. We use the calculator to understand the function and mechanism of GTPase CobW, a predicted CoII-chaperone for vitamin B12. Upon binding nucleotide (GTP) and MgII, CobW assembles a high-affinity site that can obtain CoII or ZnII from the intracellular milieu. In idealised cells with sensors at the mid-points of their responses, competition within the cytosol enables CoII to outcompete ZnII for binding CobW. Thus, CoII is the cognate metal. However, after growth in different [CoII], CoII-occupancy ranges from 10 to 97% which matches CobW-dependent B12 synthesis. The calculator also reveals that related GTPases with comparable ZnII affinities to CobW, preferentially acquire ZnII due to their relatively weaker CoII affinities. The calculator is made available here for use with other proteins.

No abstract

Summary1. Endangered species subjected to reintroduction programmes often occur as small and isolated populations with local high density and depressed fecundity. Variation in territory quality may lead to this low fecundity owing to increasing occupation of suboptimal territories as population density grows, known as the habitat heterogeneity hypothesis (HHH). In this context, food supplementation in poor territories may be used to produce extra young which could be allocated to reintroduction programmes. 2. We analyse the density-dependent fecundity pattern and the underlying mechanism in a small population of bearded vultures Gypaetus barbatus in Arag on (northeast Spain). We then use population simulations to examine the viability of a hypothetical reintroduction programme using extra young produced by supplementary feeding on poor-quality territories and the effect on the donor population. We also compare the economic cost of such a reintroduction programme in relation to the cost of a traditional captive breeding programme. 3. The wild population showed clear negative, density-dependent fecundity regulation driven by the HHH mechanism. Simulations showed that extractions for translocations had no relevant long-term effects on the donor population viability, but a marked population reduction during the extraction period. However, the implementation of supplementary feeding to produce extra young for translocation lessened significantly this expected initial population reduction. 4. Analyses showed that the annual budget of a captive breeding programme for this species could be seven times more expensive than the translocation of extra young produced by food supplementation. 5. Synthesis and applications. Reintroduction programmes based on translocation of wildreared individuals, after a supplementary feeding programme oriented to poor-quality territories, provide a source of young at least seven times cheaper than those from captive breeding programmes. The use of this approach would decrease initial effects on donor population avoiding public criticism. Increasing the number of young released during the first years of the reintroduction decreases total financial cost and increases the final population size in the new area.