Probing the activities of the Ccm proteins of the cytochrome c biosynthesis system

Proteins are complex biomolecules that are essential to the structure and function of all living cells. In order to diversify their functions, proteins use additional molecules called cofactors, of which there are many types. Heme is one such cofactor; it is an organic chemical with an iron atom in the centre and has many essential functions in different cellular processes. Many proteins that contain heme undergo a process after they are made in the cell in which bonds are formed between the protein and the heme; these are called cytochromes c and are essential in respiration and in reactions that involve the transfer of electrons. In fact, cytochromes c are believed to be one of the earliest classes of protein to have appeared in evolution. In other cases, heme associates with proteins without forming bonds with them, for example in hemoglobin, which uses the heme to bind oxygen in blood. In the case where the heme cofactor becomes bonded to the cytochrome, other proteins are needed to assist in this reaction, which is not spontaneous. Using the prototypical laboratory bacterium E. coli as a model system, we study this process of heme attachment, which is complex and involves a large number of proteins. Unexpectedly, it has been found that these systems differ in different types of cells. Our proposed research seeks to understand how these proteins function, using a variety of experimental approaches and techniques. Once we have elucidated the cellular mechanisms of attaching heme to cytochromes in bacteria, we might have the possibility of designing drugs to target these proteins. This would be possible because a completely different system exists in humans to attach heme to our cytochromes, allowing us to selectively target disease-causing bacteria, for example. It is also important to understand the process of making cytochromes c as a fundamental and central biological process, in order to complete a description of the essential biochemical reactions that constitute life.

A c-type cytochrome has one or more hemes covalently attached to a polypeptide chain via thioether bonds that are formed between sulphur atoms of a CXXCH motif in the protein and the vinyl groups of heme. The best-known c-type cytochromes are monoheme proteins that occur in mitochondria and are involved in electron transfer as part of respiration. In bacteria there are similar proteins but also multi-heme examples, some of which catalyse a reaction at one of the hemes. These cytochromes are made by a post-translational system, the type of which (there are four) varies depending upon cell type. The intention here is to learn more about the eight proteins of the Ccm system that is operative in E. coli and plant mitochondria. A variety of approaches will be used to define those parts of individual Ccm proteins that interact with their substrate, the apo form of a c-type cytochrome. We shall in particular exploit the recent introduction of a peptide substrate (microperoxidase) for the Ccm system for recognition and interaction. A second principal focus will be study of how heme is maintained in the appropriate oxidation state while it is bound to the CcmE heme chaperone protein and to further investigate the covalent binding of heme in this protein. We will also use novel screening methods to identify proteins involved in producing c-type cytochromes that have not yet been identified, and explore whether the proteins from alternative biogenesis systems can function in E. coli.