The sequential flow of electrons in the respiratory chain, from a low reduction potential substrate to O2, is mediated by protein-bound redox cofactors. In mitochondria, hemes—together with flavin, iron–sulfur, and copper cofactors—mediate this multi-electron transfer. Hemes, in three different forms, are used as a protein-bound prosthetic group in succinate dehydrogenase (complex II), in bc1 complex (complex III) and in cytochrome c oxidase (complex IV). The exact function of heme b in complex II is still unclear, and lags behind in operational detail that is available for the hemes of complex III and IV. The two b hemes of complex III participate in the unique bifurcation of electron flow from the oxidation of ubiquinol, while heme c of the cytochrome c subunit, Cyt1, transfers these electrons to the peripheral cytochrome c. The unique heme a3, with CuB, form a catalytic site in complex IV that binds and reduces molecular oxygen. In addition to providing catalytic and electron transfer operations, hemes also serve a critical role in the assembly of these respiratory complexes, which is just beginning to be understood. In the absence of heme, the assembly of complex II is impaired, especially in mammalian cells. In complex III, a covalent attachment of the heme to apo-Cyt1 is a prerequisite for the complete assembly of bc1, whereas in complex IV, heme a is required for the proper folding of the Cox 1 subunit and subsequent assembly. In this review, we provide further details of the aforementioned processes with respect to the hemes of the mitochondrial respiratory complexes. This article is part of a Special Issue entitled: Cell Biology of Metals

The cytochrome bc1 complex is an essential component of the electron transport chain in most prokaryotes and in eukaryotic mitochondria. The catalytic subunits of the complex that are responsible for its redox functions are largely conserved across kingdoms. In eukarya, the bc1 complex contains supernumerary subunits in addition to the catalytic core, and the biogenesis of the functional bc1 complex occurs as a modular assembly pathway. Individual steps of this biogenesis have been recently investigated and are discussed in this review with an emphasis on the assembly of the bc1 complex in the model eukaryote Saccharomyces cerevisiae. Additionally, a number of assembly factors have been recently identified. Their roles in bc1 complex biogenesis are described, with special emphasis on the maturation and topogenesis of the yeast Rieske iron–sulfur protein and its role in completing the assembly of functional bc1 complex. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

Although cells express hundreds of metalloenzymes, the mechanisms by which apoenzymes receive their metal cofactors are largely unknown. Poly(rC)-binding proteins PCBP1 and PCBP2 are multifunctional adaptor proteins that bind iron and deliver it to ferritin for storage or to prolyl and asparagyl hydroxylases to metallate the mononuclear iron center. Here, we show that PCBP1 and PCBP2 also deliver iron to deoxyhypusine hydroxylase (DOHH), the dinuclear iron enzyme required for hypusine modification of the translation factor eukaryotic initiation factor 5A. Cells depleted of PCBP1 or PCBP2 exhibited loss of DOHH activity and loss of the holo form of the enzyme in cells, particularly when cells were made mildly iron-deficient. Lysates containing PCBP1 and PCBP2 converted apo-DOHH to holo-DOHH in vitro with greater efficiency than lysates lacking PCBP1 or PCBP2. PCBP1 bound to DOHH in iron-treated cells but not in control or iron-deficient cells. Depletion of PCBP1 or PCBP2 had no effect on the cytosolic Fe-S cluster enzyme xanthine oxidase but led to loss of cytosolic aconitase activity. Loss of aconitase activity was not accompanied by gain of RNAbinding activity, a pattern suggesting the incomplete disassembly of the [4Fe-4S] cluster. PCBP depletions had minimal effects on total cellular iron, mitochondrial iron levels, and heme synthesis. Thus, PCBP1 and PCBP2 may serve as iron chaperones to multiple classes of cytosolic nonheme iron enzymes and may have a particular role in restoring metal cofactors that are spontaneously lost in iron deficient cells.diiron enzyme | iron regulatory protein T ransition metal ions, especially zinc, iron, copper, and manganese, serve as cofactors for hundreds of cellular proteins and play key roles in most biological processes (1). Coordination of metal centers within proteins typically involves a small number of amino acid side chains, and yet the identity and configuration of these side chains are frequently not sufficient to account for the incorporation of the native metal cofactor and the exclusion of nonnative cofactors. In vitro and in heterologous cells, many metalloproteins will bind nonnative metal cofactors with equal or greater affinity than the native metal. In both instances, misincorporation is likely to inactivate the enzyme. It is clear that cellular factors, particularly the subcellular compartment and the state of the cytosolic metal pools, may determine the species incorporated into a specific metal center. The pool of "free" metal ions in the cytosol is exceedingly low (2), because most metals are either tightly bound to proteins or coordinated by small molecules, such as glutathione (3). For most metalloproteins, the specific cellular factors contributing to metallation are unknown, but some proteins rely on metal chaperones: proteins that specifically bind metal ions and deliver them to target enzymes through direct protein-protein interactions (4).Iron is one of the most abundant metals in eukaryotic cells and cofactors in the form of heme, Fe-S clusters, a...

The assembly of the cytochrome bc 1 complex in Saccharomyces cerevisiae is shown to be conditionally dependent on a novel factor, Mzm1. Cells lacking Mzm1 exhibit a modest bc 1 defect at 30°C, but the defect is exacerbated at elevated temperatures. Formation of bc 1 is stalled in mzm1⌬ cells at a late assembly intermediate lacking the Rieske iron-sulfur protein Rip1. Rip1 levels are markedly attenuated in mzm1⌬ cells at elevated temperatures. Respiratory growth can be restored in the mutant cells by the overexpression of the Rip1 subunit. Elevated levels of Mzm1 enhance the stabilization of Rip1 through physical interaction, suggesting that Mzm1 may be an important Rip1 chaperone especially under heat stress. Mzm1 may function primarily to stabilize Rip1 prior to inner membrane (IM) insertion or alternatively to aid in the presentation of Rip1 to the inner membrane translocation complex for extrusion of the folded domain containing the iron-sulfur center.