Characterisation of a haem-binding role for the CydDC transporter of Escherichia coli

The ABC transporter CydDC of E. coli has previously been reported to be involved in maintaining correct redox poise in the periplasm and to be essential for the assembly of various haem-binding cytochromes. Furthermore, purified CydDC is also known to bind a b-type haem, but a functional role for this interaction remains elusive. This project aimed to investigate the haem-binding role for this membrane transporter. Firstly, a new continuous assay was successfully implemented to facilitate future kinetic investigation of CydDC, and a Vmax of 415 ± 40 nmoles/min/mg protein was recorded that was comparable to previous work. In addition, a Km value for ATP was recorded for the first time (10.2 ± 2 µM). As a b-type haem is known to bind to CydDC, a histidine ligand must therefore be available for haem binding. Hence, potential histidine candidates for haem ligation were investigated using multiple sequence alignments and structural modelling. As the possibility of haem-transport is an exciting prospect, haem was also docked into the central channel of CydDC structural models in the inward open and outward open conformations. Together with new structural insights from collaborators and the findings of our analyses, histidine 85 on CydC was chosen as a potential ligand for haem. Site directed mutagenesis was then successfully undertaken to generate an expression plasmid to make a H85A(CydC) variant of CydDC. This work has future implications for studying a potential haem transport role for CydDC.