Molecular genetic identification of a pathway for heme binding to cytochrome b6

Heme binding to cytochrome b(6) is resistant, in part, to denaturing conditions that typically destroy the noncovalent interactions between the b hemes and their apoproteins, suggesting that one of two b hemes of holocytochrome b(6) is tightly bound to the polypeptide, We exploited this property to define a pathway for the conversion of apo- to holocytochrome b(6), and to identify mutants that are blocked at one step of this pathway, Chlamydomonas reinhardtii strains carrying substitutions in either one of the four histidines that coordinate the b(h) or b(1) hemes to the apoprotein were created, These mutations resulted in the appearance of distinct immunoreactive species of cytochrome b(6), which allowed us to specifically identify cytochrome b(6) with altered b(h) or b(1) ligation, In gabaculine-treated (i.e. heme-depleted) wild type and site-directed mutant strains, we established that (i) the single immunoreactive band, observed in strains carrying the b(1) site-directed mutations, corresponds to apocytochrome b(6) and (ii) the additional band present in strains carrying b(h) site-directed mutations corresponds to a b(1)-heme-dependent intermediate in the formation of holocytochrome b(6). Five nuclear mutants (ccb strains) that are defective in holocytochrome b(6) formation display a phenotype that is indistinguishable from that of strains carrying site-directed b(h) ligand mutants, The defect is specific for cytochrome b(6) assembly, because the ccb strains can synthesize other b cytochromes and all c-type cytochromes. The ccb strains, which define four nuclear loci (CCB1, CCB2, CCB3, and CCB4), provide the first evidence that a b-type cytochrome requires trans-acting factors for its heme association.