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: g||=
2.18; g^
= 2.03. g||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. The spectral properties of the
H252N
variant (histidine at position 252 replaced by asparagine) and the
high-pH
form of P. denitrificans CuA were
similar,
showing that one copper ion had lost one histidine ligand in the latter
form. The dimer was converted into a valence trapped [Cu(+1)-Cu(+2)]
state,
which may retain the metal-metal interaction.