Jcb_201401009 1..14

Oxidative phosphorylation is a key process of cellular metabolism and takes place within mitochondria. The respiratory chain complexes in the inner membrane connect the transfer of electrons from reducing equivalents to the final electron acceptor oxygen with the establishment of a membrane potential, which is in turn used for ATP synthesis. Respiratory chain complexes thus contain sequentially acting subunits equipped with prosthetic groups that allow the acceptance or donation of electrons. In general, Fe/S clusters, FAD, and different hemes (a-, b-, and c-types) are used during electron transport. Fe/S cluster assembly and incorporation into respiratory chain complexes are partly understood (Lill, 2009), as well as certain aspects of heme attachment in c-type cytochromes (Steiner et al., 1996; Bernard et al., 2003, 2005; Kranz et al., 2009; Corvest et al., 2010). In contrast to c-type cytochromes, aand b-type hemes of cytochrome oxidase (COX) and complex III (bc1 complex) are not covalently linked but rather noncovalently coordinated by conserved residues. The mechanism by which aand b-type hemes are inserted is currently not known (Mick et al., 2011; Kim et al., 2012). Here, we used the central subunit of the bc1 complex, cytochrome b, as a model to understand how heme incorporation into respiratory chain subunits occurs in the mitochondrial inner membrane. Cytochrome b contains two heme bs, one low-potential heme bL located close to the intermembrane space side of the inner membrane and one high-potential heme bH at the opposite side of the membrane (Yun et al., 1991). Together with cytochrome c1 and the Rieske Fe/S protein, cytochrome b participates in the catalytic reactions of the bc1 complex, termed the Q cycle (Mitchell, 1975; Crofts, 2004; Osyczka et al., 2005). The hemes of cytochrome b are coordinated in a four-helix bundle by four conserved histidines, two located in the second transmembrane domain (H82 and H96 in baker’s yeast) and two in the fourth transmembrane helix (H183 and H197 in baker’s yeast; Yun et al., 1991; Hunte et al., 2000). We recently found that cytochrome b assembles through a series of four intermediates into the bc1 complex. Intermediate I, which is composed of cytochrome b, Cbp4, and the evolutionary conserved assembly factors Cbp3–Cbp6 (Tucker et al., 2013), accumulates at steady state in wild-type yeast cells and therefore likely represents a pool of cytochrome b ready to assemble Mitochondrial respiratory chain complexes convert chemical energy into a membrane potential by connecting electron transport with charge separation. Electron transport relies on redox cofactors that occupy strategic positions in the complexes. How these redox cofactors are assembled into the complexes is not known. Cytochrome b, a central catalytic subunit of complex III, contains two heme bs. Here, we unravel the sequence of events in the mitochondrial inner membrane by which cytochrome b is hemylated. Heme incorporation occurs in a strict sequential process that involves interactions of the newly synthesized cytochrome b with assembly factors and structural complex III subunits. These interactions are functionally connected to cofactor acquisition that triggers the progression of cytochrome b through successive assembly intermediates. Failure to hemylate cytochrome b sequesters the Cbp3–Cbp6 complex in early assembly intermediates, thereby causing a reduction in cytochrome b synthesis via a feedback loop that senses hemylation of cytochrome b. Assembly factors monitor sequential hemylation of cytochrome b to regulate mitochondrial translation