Abstract
Heme binding to cytochrome b6 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 b6 is tightly bound to the polypeptide. We exploited this property to define a pathway for the conversion of apo- to holocytochrome b6, 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 bh or bl hemes to the apoprotein were created. These mutations resulted in the appearance of distinct immunoreactive species of cytochrome b6, which allowed us to specifically identify cytochrome b6 with altered bh or bl 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 bl site-directed mutations, corresponds to apocytochrome b6 and (ii) the additional band present in strains carrying bh site-directed mutations corresponds to a bl-heme-dependent intermediate in the formation of holocytochrome b6. Five nuclear mutants (ccb strains) that are defective in holocytochrome b6 formation display a phenotype that is indistinguishable from that of strains carrying site-directed bh ligand mutants. The defect is specific for cytochrome b6 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.
Highlights
Quinol oxidizing complexes, the cytochrome bc1 complex of mitochondria and bacteria, and its chloroplast counterpart, the cytochrome b6 f complex, couple translocation of protons across the membrane to oxidation of lipophilic electron carriers and reduction of small hydrophilic proteins
Since cytochrome b6 did not stain with TMBZ more heavily than cytochrome f, which binds only one heme, we suggest that only part of the b heme complement of cytochrome b6 is tightly associated with the polypeptide
The unique denaturation-resistant association of heme with cytochrome b6 suggested an unusual mode of b heme binding, and it permitted us to dissect the b heme assembly pathway in vivo by exploiting genetic approaches in C. reinhardtii
Summary
The cytochrome bc complex of mitochondria and bacteria, and its chloroplast counterpart, the cytochrome b6 f complex, couple translocation of protons across the membrane to oxidation of lipophilic electron carriers (quinols) and reduction of small hydrophilic proteins (reviewed in Refs. 1 and 2). By chloroplast genes [5], petA (encoding cytochrome f, a c-type cytochrome), petB (encoding cytochrome b6), and petD (encoding subunit IV); whereas the Fe2S2 Rieske protein is encoded by a nuclear gene, PetC [6]. The cytochrome b6 f complex contains three transmembrane subunits of low molecular mass (Յ4 kDa), the products of the chloroplast genes petG [7,8,9] and petL [10] and the nuclear gene PetM [11, 12]. The cytochrome b6 f complex binds five cofactors: two b hemes (high potential bh and low potential bl), one c heme, a Fe2S2 cluster, and a chlorophyll a [13,14,15,16]. Cytochrome b6 and subunit IV are homologous, respectively, to the first four ␣-helices and three ␣-helices of cytochrome b of the cytochrome bc complex [17]. bh and bl hemes are associated with cytochrome b6, bl heme on the lumenal side of the thylakoid membrane (close to the quinol oxidizing site) and bh heme on the stromal side (close to the quinone reducing site)
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