Role and biogenesis of the heme ci' of bc complexes – Heme c'

Heme (Fe-protoporphyrin IX) is an essential cofactor for many fundamental biological processes. It is a hydrophobic and cytotoxic macrocycle, which therefore requires specific pathways to be safely delivered to its subcellular destinations. Prominent among the heme-binding proteins are cytochrome bc complexes, that associate c-type hemes, b-type hemes and iron-sulfur centres. They are ubiquitous in energy-transducing membranes, where they transfer electrons from a lipid-soluble transporter to a water-soluble protein transporter while building up a transmembrane proton gradient. Oxygenic photosynthesis, at the source of terrestrial life, is the most important bioenergetic pathway that poises the atmosphere on earth and allows carbon fixation. A most critical enzyme in this process, the cytochrome b6f complex, differs from other protein complexes of the same family by a very unusual and unique cofactor, heme ci' bound to one of its protein subunit cytochrome b6. To progress in our understanding of the role and biogenesis of heme ci', we propose to use the unicellular green alga Chlamydomonas reinhardtii as a model system because it is uniquely amenable to genetic, spectroscopic and biochemical studies. We have already selected several mutants with altered heme ci'. Nuclear ccb mutants preventing heme ci' binding and chloroplast mutants lacking the cysteine residue involved in covalent binding of heme ci' accumulate very little b6f complex subunits and are non phototrophic. Based on these mutants, we characterized four CCB proteins as integral membrane proteins required for covalent binding of heme ci' to cytochrome b6 (Kuras et al., submitted). None of these proteins had ever been assigned a biological function before. They define a completely new metabolic pathway (system IV), fully distinct from those previously implicated in cytochrome biogenesis in the past two decades. They are encoded by the genomes of all organisms performing oxygenic photosynthesis, whatever their phylogenetic distances. These proteins are thus among the few that distinguish photosynthetic cells evolving oxygen from other types of living cells. The interactions and specific function of these four factors will be characterized extensively by biochemical and enzymatic means and hybridization in various heterologous expression systems. Role of residues or domains will be tested by site–directed mutagenesis. New factors of biogenesis will be searched directly by random mutagenesis and indirectly by search of extragenic suppressors of previously obtained ccb mutants. We will perform genetic and phenotypic characterization for the phototrophic suppressors of the ccb mutants with the aim to reach an in depth understanding of the biogenesis and function of heme ci'. In preliminary studies we obtained an intriguing revertant that accumulates in vivo a b6f complex seemingly devoid of covalently-bound heme ci' that should offer a unique possibility to test the exact role of this heme. We will study the linear electron transfer, cyclic electron transfer, redox signalling, and state transitions properties in this mutant and in other suppressors that will be generated in this study. We assume that the tightly packed pair of redox centers heme ci'/ heme bh provides the two electrons required to reduce plastoquinone in plastoquinol, explaining the apparent absence of a semiquinone in the Qi site of wild-type b6f complexes at variance with what is observed in bacterial bc complexes performing anaerobic photosynthesis. We indeed observed that this revertant is very photosensitive in high light and only in presence of oxygen. We wish to further elucidate the mechanism for Reactive Oxygen Species (ROS) production in this revertant, identify which types of ROS are produced and how inhibitors of quinone binding sites affect this behaviour. Its photosensitivity will be used in a selection scheme to clone the gene(s) responsible for the phenotype. The Firmicute Bacillus subtilis is up to now the only non photosynthetic organism in which a cytochrome bc complex with heme ci has been biochemically characterized. The absence of most CCB homologues in Firmicutes indicates that Firmicutes require another novel mechanism for heme-binding on the n-side of their cytoplasmic membrane. Bacillus subtilis will be used to dissect the operon that is comprised of a ccb distant orthologue and other genes encoding putative additional factors as an eventual system V. Our laboratory groups genetic, genomic, biochemical and optical spectroscopy expertise. Previously, we have provided major contributions to the discovery, characterization and biogenesis of heme ci' in cytochrome b6f complexes. We have established the basis to succeed in our project to understand the role of this new heme and to characterize heme-binding pathways on the n-side of the membrane.