Cast-iron protection racket

Little is known about how the haem group that carries oxygen in blood and is the active centre of several enzymes is transported from where it is made in the cell to its host protein assembly. Now, UV-Vis spectroscopy has helped identify an enzyme that also functions as a haem transporter as well as protecting the fragile iron(II).

Iron is the most abundant transition metal in the human body. Its intriguing reduction-oxidation properties endow it with the active role as an essential cofactor in countless proteins. Some of these are involved in oxygen transport (haemoglobin in the blood), electron exchange for powering biochemical reactions and energy release (cytochromes), and the control of potentially harmful free radicals.

We can absorb iron from our diets in the form of inorganic iron from plant source and as the haem found in animal haemoglobin and myoglobin. Researchers have spent many years attempting to unravel the mechanism by which absorbed iron is transported to where it is needed for biosynthesis of cofactors and iron-bearing protein production.

Now, Elaine Frawley and Robert Kranz of the Department of Biology, at Washington University, St Louis, Missouri, have shed new light on the trafficking of haem from its sites of synthesis to sites of haem-protein assembly.

For animals, haem biosynthesis takes place inside mitochondria, in plants it is constructed in the chloroplasts, and bacteria, of course, have their own systems for its production too. "There is emerging evidence that organisms use specific transporters to traffic haem and porphyrins into and between cells," the researchers explain. However, because haem has not been trapped in the integral membrane transporter proteins, the trafficking process to heme-bearing proteins was still an enigma.

Cytochrome c enzymes are synthesised and function in the prokaryotic periplasm, the mitochondrial intermembrane space, and the chloroplast lumen. This means that the haem group (iron at the centre of a porphyrin type chemical ring) must somehow be delivered direct to these sites ready for use. "Yet the molecular mechanisms of delivery are unknown," the teams says.

They explain that cytochrome c synthesis follows one of three pathways (systems I, II, and III) and itself requires the formation of two bonds between the sulfur-containing amino acids of the protein awaiting the haem, the apoprotein, and two so-called vinyl groups on the haem. For this bonding to occur, the iron ion in its oxidation III state (Fe³⁺) must be reduced to the Fe²⁺ state. How this happens is another mystery.

To unravel the mechanism of haem trafficking and iron reduction, the team has studied an integral membrane protein that allows trapping of haem. Using a variety of techniques, including ultraviolet spectroscopic studies, the team has shown that the protein, "CcsBA", a representative of a superfamily of integral membrane proteins involved in cytochrome c biosynthesis, exports and protects haem from oxidation.

The study shows that CcsBA has ten transmembrane domains (TMDs), its presence allows cytochrome c synthesis to take place in the periplasm of the laboratory technician's favourite bacterium, Escherichia coli. In other words, CcsBA itself is a cytochrome c synthetase enzyme.

The team analysed purified CcsBA and found that it contains haem in an "external haem binding domain" on the protein. Two external histidine amino acids act as the anchors for the haem group in this case and also protect the Fe²⁺ ion from being oxidised back to the Fe³⁺ state. The team suggests that this anchored haem is in the active site of the synthetase.

The researchers have also carried out genetic studies involving the formation of mutant versions of CcsBA. Those studies reveal that two conserved histidine residues in TMDs are needed in order to carry the haem to the external haem binding domain. A surprising discovery with the genetic studies is that if these histidines are missing, the functioning of CcsBA can be fixed by adding imidazole to the reaction mixture. Overall, the results suggest that CcsBA has a haem binding site within the bilayer and that CcsBA acts as a haem-transporting channel.

The team will soon publish details in a major journal on purification, trapping, and spectrally characterization of the system I pathway complexes, Kranz told SpectroscopyNOW.