The general questions: Heme is found in many different locations including the mitochondrion (respiratory cytochromes), the chloroplast (photosynthetic cytochromes), the endoplasmic reticulum (P450 cytochromes), the nucleus (heme-responsive transcription factors), the cytosol (catalases), the peroxisomes (catalases) and extracellular compartments (peroxidases). Its synthesis is restricted to the mitochondrion in a typical eukaryotic (non-plant) cell. How is heme transported from the mitochondrion? How is it delivered from its site of synthesis to the site where heme proteins are made? What is the mechanism of cofactor protein association? Is heme allocated preferentially to particular pathways in response to metabolic demand? The occurrence of two pathways (plastidic and mitochondrial) adds an extra layer of complexity in a plant cell. Which one provides heme to extra-organellar proteins? Is there a mechanism to control the flux out of the plastid vs. the mitochondrion?

        Our discoveries: The genetic dissection of specific assembly pathways was envisioned as a first step towards the discovery of heme chaperones and transporters in the expected regulatory targets. We chose the cytochromes of the b6/f complex as our model for the study of heme protein assembly in chloroplasts. The b6/f complex contains two types of cytochromes cytochrome b6, an integral membrane protein with two bis-histidyl liganded b-hemes (bH and bL), and cytochrome f, a membrane-anchored protein with a single covalently attached c-heme.

        In the c-cytochromes, heme is covalently attached to the polypeptide through thioether linkages. Thus, the fact that assembly is catalyzed was appreciated decades ago. Both biochemical and genetic approaches have been applied to the study of cytochrome c assembly in bacteria, mitochondria and chloroplasts, and common principles have emerged (reviewed in Howe and Merchant, 1994). In general, the c-cytochromes are synthesized as precursor proteins that are targeted post-translationally to their site of function. Heme attachment is a terminal (or near terminal) step in their maturation, occurring on the p-side of the energy transducing membrane where the cytochromes function. This demands a mechanism for heme delivery from its site of synthesis to its site of utilization for holocytochrome c formation.

        The pathway for pre-apocytochrome c6 and f maturation in the chloroplast was deduced by the application of pulse-chase methods (27,30). A subset of cytochrome-deficient mutants was found to be blocked specifically at the terminal step of heme attachment (22,43). These mutants display a pleiotropic deficiency in c-type cytochromes and define a minimum of 7 loci, ccsA in the plastid and CCS1 through CCS6 in the nucleus. Ccs1 and ccsA encode multi-spanning proteins in the thylakoid membrane (36,41). The proteins are found in a 200 kD complex, probably in association with the products of other Ccs genes. We predict that CcsA functions in a novel heme delivery pathway, while Ccs1 might be an apoprotein chaperone.

        In collaboration with the Wollman group (Institut de Biologie Physico-Chimique, Paris), we deduced a pathway for the stepwise association of the bL and bH hemes of  holocytochrome b6 in vivo (40). Genetic analysis of a subset of cytochrome b6/f-deficient strains indicates a minimum of 4 CCB loci that function solely at the bH-heme insertion step. These results dispelled the long-standing dogma that b cytochrome assembly occurred without catalysis in vivo. It is our view that specific mechanisms exist for each type of metalloprotein to ensure selective cofactor ligation in vivo; these will undoubtedly be discovered through the more detailed characterization of assembly-defective respiratory and photosynthetic mutants.

        Immediate plans: The Ccs factors appear to be novel proteins whose biochemical functions cannot be ascertained through sequence analysis. A key objective is to deduce their specific catalytic roles in heme and apoprotein metabolism in the thylakoid lumen. Eventually, we would like to identify all Ccs and Ccb factors and reconstitute the assembly of cytochromes b and c in vitro.