Cytochrome oxidase (CcO) is a 13 subunit complex functioning as the terminal electron acceptor in the mitochondrial respiratory chain that functions in oxidative phosphorylation (picture shown below). The biogenesis of CcO is mediated by a myriad of assembly factors that function in the translation of the three mitochondrially encoded subunits Cox1-Cox3, insertion of the subunits into the inner membrane and the formation of redox cofactors copper and heme a moieties. Over 20 assembly factors are known in yeast that mediate CcO biogenesis. We use a combination of in vitro biochemical, in vivo cellular assays and genetic analyses to elucidate the mechanism and pathway by which a series of these assembly proteins mediate CcO biogenesis. The image below depicts the structure of CcO complex and the redox cofactors. Cox1 contains an isolated heme a center and the heme a3:CuB center where oxygen binds during catalysis. Cox2 contains the binuclear CuA center that is reduced by cytochrome c.

CcO deficiency is of the most frequent causes of OXPHOS respiratory chain defects in humans. Known human mitochondrial myopathies presenting with CcO deficiencies represent mutations in six distinct CcO assembly factors that function in either the synthesis or insertion of essential cofactors for CcO. We are actively studying the formation of the redox cofactor centers in CcO and attempting to understand how mutations in human assembly factors compromise CcO assembly. The two copper centers in CcO are formed by a series of proteins residing in the inner membrane space (IMS). A scheme for the copper metallation of Cox1 and Cox2 is shown below. Sco1 mediates formation of the Cu A center in Cox2, whereas Cox11 mediates Cu B site formation in Cox1. We are studying the mechanisms of these copper transfer reactions.

We are actively studying the maturation of the Cox1 and Cox2 subunits of CcO. Newly synthesized Cox1 is escorted through the maturation process by a series of inner membrane protein complexes shown below. One assembly intermediate contains Cox1 associated with Shy1. This is the stage in which the heterobimetallic CuB-heme a3 center is formed. The human Shy1 variant is SURF1. Human CcO deficiency patients often have mutations in SURF1. These patients present with a severe neurological disorder called Leigh Syndrome. We are attempting to gain further insight into Shy1/SURF1 by screening for suppressors of Shy1 mutants with human Leigh syndrome misssense mutations. A scheme for Cox1 maturation in yeast is shown below.

Mitochondria require a number of metal ions such as copper, iron and zinc for normal physiology. These metal ions are used as cofactors in metalloenzymes such as CcO. Proteins are imported into the mitochondria as unfolded metal-free polypeptides and metallation reactions occur within the organelle. We are studying how mitochondria acquire these metal ions for assembly of metalloproteins. We identified bioavailable pools of matrix zinc and copper in the matrix that are used for biosynthesis of mitochondrial zinc and copper proteins. We are taking a biochemical approaches to identify the ligands that stabilizes the metal pools and genetic approach to identify genes that modulate the concentration of these pools. We have identified a series of mitochondrial proteins important in the maintenance of matrix zinc pools and are working to elucidate the mechanism of this process.

One mitochondrial compartment important for CcO assembly reactions is the inner membrane space (IMS). Proteins are imported into the IMS by an oxidative folding pathway. The IMS is a unique cell compartment in which many proteins exist with stable disulfide bonds. We are interested in mechanisms of maintenance of the redox potential of the IMS and proper disulfide bond formation. Many Cys-containing IMS proteins are captured in the IMS by Mia40 in a transient intermolecular disulfide and is resolved with the imported protein containing the disulfide. Mia40 mediates the import of the Cu(I) metallochaperone for CcO biogenesis Cox17. Cox17 exists with two stable disulfides and two reduced Cys residues for Cu(I) coordination.