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Cytochrome c oxidase (CcO) 5 is the terminal enzyme of the energytransducing, electron transfer chain within the mitochondrial inner membrane. Mammalian CcO consists of 13 polypeptide subunits, three of which (CoxI-CoxIII) are encoded by the mitochondrial genome and the remaining 10 of which are encoded by the nuclear genome (1, 2). An additional 30 proteins are believed to be required for the assembly of the CcO complex (3). A number of these accessory proteins are important in the processing and translation of COXI-COXIII mRNA transcripts, in membrane insertion of subunits, and in either the synthesis or delivery of cofactors. The cofactors in CcO include two copper sites (Cu A and Cu B ), two heme A moieties, and a magnesium and zinc ion (4).The Cu A site is a binuclear, mixed valent copper center localized in the CoxII subunit, whereas the Cu B site consists of a single copper ion within a Cu-heme A binuclear center in the CoxI subunit (4). The CoxI and CoxII subunits are synthesized within the mitochondrion, so copper site insertion must occur during insertion of the nascent polypeptides into the inner membrane.Several proteins, including Cox11, Cox17, Cox19, Cox23, and Sco1, have been implicated in the assembly of the copper centers in CcO in yeast and all have human homologs (5). Cox17 is a soluble copper metallochaperone within the mitochondrial intermembrane space (IMS). Sco1 and Cox11 are inner membrane proteins tethered by a single transmembrane helix and are implicated in the assembly of the Cu A and Cu B centers, respectively (6, 7). Both proteins are copper-binding proteins, and mutations that abrogate Cu(I) binding attenuate in vivo assembly of . Recently, we demonstrated that Cox17 is capable of donating Cu(I) to both Sco1 and Cox11 (12). The prediction is that copper ions are only transiently bound to Sco1 and Cox11 in yeast prior to donation to CoxII and CoxI, respectively. Yeast Sco1 was shown to form a transient complex with CoxII (13).Assembly of CcO in mammalian cells has additional components, since two distinct Sco-like molecules are involved in the assembly process. Mutations in both human genes, SCO1 and SCO2, have been identified, and these result in respiratory chain deficiency associated with CcO assembly defects (14 -16). Although yeast also has a second Sco protein, designated Sco2, it has no function in CcO assembly (17). The human Sco1 and Sco2 molecules share the Cu(I) binding motif of yeast Sco1; however, nothing is currently known about the copper binding by human Sco1, whereas limited data exist on copper binding by human Sco2 (18,19). Neither human Sco1 nor Sco2 is able to rescue the respiratory defect of yeast sco1 null cells (20). However, a chimera containing the N-terminal segment of yeast Sco1 fused to the C-terminal segment of human Sco1 is functional in sco1 null cells (20). Two lines of evidence suggest that both human Sco1 and Sco2 proteins probably function in copper metallation of CcO. First, overexpression of COX17 partially rescues the CcO deficiency of SCO2, ...

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Methyl-coenzyme M reductase (MCR) catalyzes methane formation from methyl-coenzyme M (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoBSH). MCR contains a nickel hydrocorphin cofactor at its active site, called cofactor F(430). Here we present evidence that the macrocyclic ligand participates in the redox chemistry involved in catalysis. The active form of MCR, the red1 state, is generated by reducing another spectroscopically distinct form called ox1 with titanium(III) citrate. Previous electron paramagnetic resonance (EPR) and (14)N electron nuclear double resonance (ENDOR) studies indicate that both the ox1 and red1 states are best described as formally Ni(I) species on the basis of the character of the orbital containing the spin in the two EPR-active species. Herein, X-ray absorption spectroscopic (XAS) and resonance Raman (RR) studies are reported for the inactive (EPR-silent) forms and the red1 and ox1 states of MCR. RR spectra are also reported for isolated cofactor F(430) in the reduced, resting, and oxidized states; selected RR data are reported for the (15)N and (64)Ni isotopomers of the cofactor, both in the intact enzyme and in solution. Small Ni K-edge energy shifts indicate that minimal electron density changes occur at the Ni center during redox cycling of the enzyme. Titrations with Ti(III) indicate a 3-electron reduction of free cofactor F(430) to generate a stable Ni(I) state and a 2-electron reduction of Ni(I)-ox1 to Ni(I)-red1. Analyses of the XANES and EXAFS data reveal that both the ox1 and red1 forms are best described as hexacoordinate and that the main difference between ox1 and red1 is the absence of an axial thiolate ligand in the red1 state. The RR data indicate that cofactor F(430) undergoes a significant conformational change when it binds to MCR. Furthermore, the vibrational characteristics of the ox1 state and red1 states are significantly different, especially in hydrocorphin ring modes with appreciable C=N stretching character. It is proposed that these differences arise from a 2-electron reduction of the hydrocorphin ring upon conversion to the red1 form. Presumably, the ring-reduction and ligand-exchange reactions reported herein underlie the enhanced activity of MCR(red1), the only form of MCR that can react productively with the methyl group of methyl-SCoM.