Spectroscopic and mutagenesis studies on the CuA centre from the cytochrome-c oxidase complex of Paracoccus denitrificans.
Cytochrome-c oxidase contains an unusual copper centre (CuA) located in subunit II. This centre mediates one-electron transfer from cytochrome c to low-spin heme a. Recent spectroscopic and biochemical studies have shown that this centre is a valence delocalised dinuclear [Cu(+1.5)-Cu(+1.5)] centre. We have measured the absorption, EPR and variable-temperature magnetic circular dichroism spectra of the CuA-binding domain isolated from Paracoccus denitrificans cytochrome aa3. The EPR spectrum showed the following signals: gparallel = 2.18; gperpendicular = 2.03. gparallel exhibited a seven-line hyperfine splitting pattern, with an intensity ratio showing that the single unpaired electron interacted equally with two copper nuclei. The magnetic circular dichroism spectrum was identical to those from CuA in bovine heart cytochrome-c oxidase and centre A of nitrous-oxide reductase, showing the close structural similarity between the three centres. To identify the ligands of CuA, all the conserved putative ligands in the P. denitrificans CuA domain were substituted. Only five residues, Cys244, Cys248, His209, His252, and Met255, were required for correct assembly of the CuA centre. Replacement of Met255 caused protein misfolding. Hence, methionine may have a structural role for the folding of the protein rather than being a CuA ligand. Given that both copper ions must have identical coordination geometries, the number of possible structures is limited. Two models are proposed: one involves the thiolate side-chains of Cys244 and Cys248 bridging a pair of copper ions with one histidine coordinating each copper ion, and the other has terminal ligation of each copper ion by one cysteine and one histidine residue. In both models, the metal-metal distance can be sufficiently short to permit direct d-orbital overlap of the copper ions. The magnetic circular dichroism transitions at 475 nm and 525 nm are assigned to thiolate-to-copper charge-transfer processes polarised perpendicular to one another, although the magnetic circular dichroism intensities show that the excited states were heavily mixed with copper d-orbitals. These intensities can be interpreted in the thiolate bridged model in terms of transitions within a Cu2(SR)2 rhomb. In the model involving terminal cysteine ligation, exciton coupling of two thiolate-to-copper charge-transfer transitions of similar energy, polarised along the Cu-S bonds, would contribute two transitions perpendicular to one another. This requires that the cysteine ligands have a cis orientation relative to one another.(ABSTRACT TRUNCATED AT 400 WORDS)
Farrar
J A
JA
Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences, University of East Anglia, Norwich, England.
Lappalainen
P
P
Zumft
W G
WG
Saraste
M
M
Thomson
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry*; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010231:Paracoccus denitrificans / Q000201:enzymology*
D008024:Ligands; D003300:Copper; D003576:Electron Transport Complex IV