Structural studies of hemerythrin proteins in the human pathogen Campylobacter jejuni

Hemerythrins are oxygen-binding proteins, originally found in the body fluids and tissues of marine invertebrates such as the worms Sipunculids and Priapulida. In these organisms, a multimeric form of hemerythrin is found in their blood cells and a monomeric myohemerythrin in their muscles. Both, however, are very similar in function and structure and are often described according to the oxidation and ligation states of their iron centre. Hemerythrins differ from haemoglobin and haemocyanin in that they possess non-haem di-iron atom sites that bind one molecule of O2. Recently, they have been reported in a wide variety of bacteria particularly in microaerophilic and anaerobic bacteria. \nCampylobacter jejuni is one species in the family Campylobacteraceae, and it is one of the most common causes of gastroenteritis worldwide, but a commensal of chickens and other fowl. C. jejuni is a microaerophile and therefore needs a quite low oxygen environment (5-10% O2) for optimal growth. Throughout its life cycle, however, it is essential for it to be able to survive in the aerobic environment (21% O2), as well as the oxidative stress response of the human host. There is no clear molecular explanation for the microaerophilic nature of C. jejuni. The majority of Campylobacter species have been shown to have multiple hemerythrin-like proteins, many of which contain a C-terminal domain of unknown function but with high homology among strains. The analysis of the genome of NCTC11168 in C jejuni shows three different genes encoding proteins with iron and oxygen binding hemerythrin-like characteristics: cj0241c, cj1224 and cj0045c. Protein sequences of the Cj0241, Cj1224 and Cj0045 hemerythrins reveal that they all include conserved histidine motifs H, HxxE, HxxxH and HxxxxD, which are important to bind the di-iron centre critical for hemerythrin function. Therefore, the project aimed to resolve the question of the presence of these three genes, determine information on their different roles and the function of some the essential residues in these hemerythrins and their role in oxygen sensing and protection in Campylobacter jejuni. \nPlasmids for the overexpression of cj0241c, cj1224 and cj0045c were obtained from the laboratory of Prof David Kelly. A range of experiments were performed related to function and structure determination. The expression and purification of Cj0241 was very successful, and the structure was determined. The three-dimensional crystal structure was solved at 1.1 Å resolution using Single-wavelength Anomalous Dispersion (SAD) data sets. It showed a four alpha helix bundle with a central di-iron site bridged by an oxygen atom and coordinated by the conserved histidine residues. Analysis revealed a clear tunnel down the middle of the protein along which water and other solvent molecules could travel from the protein exterior to the di-iron site. The Cj1224 and Cj0045 proteins were successfully purified and analyses carried out on them although it was not possible to obtain crystal structures. Chemical, biochemical and biophysical experiments to study the properties of the proteins suggested that the C-terminal extensions in the Cj1224 and Cj0045 proteins might possess zinc binding properties. Attempts to measure the autoxidation rates of the hemerythrins implied a sensor or transport role for the proteins.