Iron deficiency afflicts more than three billion people worldwide, and plants are the principal source of iron in most diets. Low availability of iron often limits plant growth because iron forms insoluble ferric oxides, leaving only a small, organically complexed fraction in soil solutions. The enzyme ferric-chelate reductase is required for most plants to acquire soluble iron. Here we report the isolation of the FRO2 gene, which is expressed in iron-deficient roots of Arabidopsis. FRO2 belongs to a superfamily of flavocytochromes that transport electrons across membranes. It possesses intramembranous binding sites for haem and cytoplasmic binding sites for nucleotide cofactors that donate and transfer electrons. We show that FRO2 is allelic to the frd1 mutations that impair the activity of ferric-chelate reductase. There is a nonsense mutation within the first exon of FRO2 in frd1-1 and a missense mutation within FRO2 in frd1-3. Introduction of functional FRO2 complements the frd1-1 phenotype in transgenic plants. The isolation of FRO2 has implications for the generation of crops with improved nutritional quality and increased growth in iron-deficient soils.

Almost half of all enzymes must associate with a particular metal to function. An ambition is to understand why each metal-protein partnership arose and how it is maintained. Metal availability provides part of the explanation, and has changed over geological time and varies between habitats but is held within vital limits in cells. Such homeostasis needs metal sensors, and there is an ongoing search to discover the metal-sensing mechanisms. For metalloproteins to acquire the right metals, metal sensors must correctly distinguish between the inorganic elements.

Protein metal-coordination sites are richly varied and exquisitely attuned to their inorganic partners, yet many metalloproteins still select the wrong metals when presented with mixtures of elements. Cells have evolved elaborate mechanisms to scavenge for sufficient metal atoms to meet their needs and to adjust their needs to match supply. Metal sensors, transporters and stores have often been discovered as metal-resistance determinants, but it is emerging that they perform a broader role in microbial physiology: they allow cells to overcome inadequate protein metal affinities to populate large numbers of metalloproteins with the right metals.

The current state of knowledge on how copper metallochaperones support the maturation of cuproproteins is reviewed. Copper is needed within mitochondria to supply the CuA and intramembrane CuB sites of cytochrome oxidase, within the trans-Golgi network to supply secreted cuproproteins and within the cytosol to supply superoxide dismutase 1 (Sod1). Subpopulations of copper-zinc superoxide dismutase also localize to mitochondria, the secretory system, the nucleus and, in plants, the chloroplast, which also requires copper for plastocyanin. Prokaryotic cuproproteins are found in the cell membrane and in the periplasm of gram-negative bacteria. Cu(I) and Cu(II) form tight complexes with organic molecules and drive redox chemistry, which unrestrained would be destructive. Copper metallochaperones assist copper in reaching vital destinations without inflicting damage or becoming trapped in adventitious binding sites. Copper ions are specifically released from copper metallochaperones upon contact with their cognate cuproproteins and metal transfer is thought to proceed by ligand substitution.

The metal binding preferences of most metalloproteins do not match their metal requirements. Thus, metallation of an estimated 30% of metalloenzymes is aided by metal delivery systems, with ∼25% acquiring preassembled metal cofactors. The remaining ∼70% are presumed to compete for metals from buffered metal pools. Metallation is further aided by maintaining the relative concentrations of these pools as an inverse function of the stabilities of the respective metal complexes. For example, magnesium enzymes always prefer to bind zinc, and these metals dominate the metalloenzymes without metal delivery systems. Therefore, the buffered concentration of zinc is held at least a million-fold below magnesium inside most cells.

Metals are needed by at least one-quarter of all proteins. Although metallochaperones insert the correct metal into some proteins, they have not been found for the vast majority, and the view is that most metalloproteins acquire their metals directly from cellular pools. However, some metals form more stable complexes with proteins than do others. For instance, as described in the Irving-Williams series, Cu(2+) and Zn(2+) typically form more stable complexes than Mn(2+). Thus it is unclear what cellular mechanisms manage metal acquisition by most nascent proteins. To investigate this question, we identified the most abundant Cu(2+)-protein, CucA (Cu(2+)-cupin A), and the most abundant Mn(2+)-protein, MncA (Mn(2+)-cupin A), in the periplasm of the cyanobacterium Synechocystis PCC 6803. Each of these newly identified proteins binds its respective metal via identical ligands within a cupin fold. Consistent with the Irving-Williams series, MncA only binds Mn(2+) after folding in solutions containing at least a 10(4) times molar excess of Mn(2+) over Cu(2+) or Zn(2+). However once MncA has bound Mn(2+), the metal does not exchange with Cu(2+). MncA and CucA have signal peptides for different export pathways into the periplasm, Tat and Sec respectively. Export by the Tat pathway allows MncA to fold in the cytoplasm, which contains only tightly bound copper or Zn(2+) (refs 10-12) but micromolar Mn(2+) (ref. 13). In contrast, CucA folds in the periplasm to acquire Cu(2+). These results reveal a mechanism whereby the compartment in which a protein folds overrides its binding preference to control its metal content. They explain why the cytoplasm must contain only tightly bound and buffered copper and Zn(2+).

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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.