Two variants of the assembly factor Surf1 target specific terminal oxidases in Paracoccus denitrificans

Abstract

Biogenesis of cytochrome c oxidase (COX) relies on a large number of assembly proteins, one of them being Surf1. In humans, the loss of Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder. In the soil bacterium Paracoccus denitrificans, homologous genes specifying Surf1 have been identified and located in two operons of terminal oxidases: surf1q is the last gene of the qox operon (coding for a ba(3)-type ubiquinol oxidase), and surf1c is found at the end of the cta operon (encoding subunits of the aa(3)-type cytochrome c oxidase). We introduced chromosomal single and double deletions for both surf1 genes, leading to significantly reduced oxidase activities in membrane. Our experiments on P. denitrificans surf1 single deletion strains show that both Surf1c and Surf1q are functional and act independently for the aa(3)-type cytochrome c oxidase and the ba(3)-type quinol oxidase, respectively. This is the first direct experimental evidence for the involvement of a Surf1 protein in the assembly of a quinol oxidase. Analyzing the heme content of purified cytochrome c oxidase, we conclude that Surf1, though not indispensable for oxidase assembly, is involved in an early step of cofactor insertion into subunit I.

Formation of the Redox Cofactor Centers during Cox1 Maturation in Yeast Cytochrome Oxidase

“…Formation of the two heme a centers may occur stepwise, with the heme a site being populated in a transition to the Shy1-containing complex whereas the heme a 3 center may be populated within the Shy1 complex. This would be consistent with studies of R. sphaeroides cells lacking the Shy1 homolog Surf1, which showed a selective depletion of heme a 3 (12,37). Alternatively, both heme a centers may be populated within the Shy1 complex, but in the absence of Shy1 only the heme a 3 center with a single axial ligand is labile.…”

“…This may account for the presence of the residual Shy1 BN complex in the Cox1 mutant lacking the heme a 3 single axial His. Mutations that stall CcO assembly at later steps, such as maturation of the Cu A site, result in hydrogen peroxide sensitivity of the mutant cells (26 (12,37). The stability of the Shy1-containing Cox1 complex is dependent on numerous factors.…”

“…The pro-oxidant heme a 3 :Cox1 intermediate is absent in cells lacking Shy1, Coa1, or Cox1 (35). Second, isolation of CcO in Rhodobacter or Paracoccus cells lacking Surf1 (a Shy1 ortholog) reveals an enzyme complex deficient in heme a 3 but not heme a (12,37). Shy1 is not likely a heme a 3 -insertase, since yeast shy1⌬ cells and mutant SURF1 human cells retain 10 to 15% residual CcO activity (17,36,47).…”

Deciphering protein–protein interactions during the biogenesis of cytochrome c oxidase from Paracoccus denitrificans

“…The insertion of the heme and copper cofactors into SUI may well be a mutually dependent process whereby only incorporation of both cofactors ensures complete stabilization of the a 3 ÁCu B center and terminal folding of this subunit. It is interesting to note that Surf1q [29], which is involved in delivering heme a exclusively to the ba 3 oxidase, was not observed in any of the antibody-mediated elution fractions, lending further weight to the specificity of the approach.…”

Dual Functions of Mss51 Couple Synthesis of Cox1 to Assembly of CytochromecOxidase inSaccharomyces cerevisiaeMitochondria

“…We found that a cox1 deletion mutation in mtDNA prevented coimmune precipitation of Shy1 with Mss51 (unpublished data), consistent with the dynamic assembly of Shy1-containing complexes nucleated by newly synthesized Cox1. Mutations inactivating Shy1 in yeast, and its orthologues in humans and bacteria, decrease but do not eliminate cytochrome c oxidase activity, apparently by decreasing the efficiency of heme a 3 insertion into Cox1 (Smith et al, 2005;Khalimonchuk et al, 2007;Bundschuh et al, 2008;Pierrel et al, 2008). Interestingly, respiratory growth of a yeast shy1⌬ mutant is improved by dominant suppressor mutations in MSS51 and by overexpression of wild-type MSS51 (Barrientos et al, 2002).…”