The chemistry of the CuB site in cytochrome c oxidase and the importance of its unique His–Tyr bond

Helsinki Bioenergetics Group, Programme of Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 05/2009; 1787(4):221-33. DOI: 10.1016/j.bbabio.2009.01.002
Source: PubMed
ABSTRACT
The CuB metal center is at the core of the active site of the heme-copper oxidases, comprising a copper atom ligating three histidine residues one of which is covalently bonded to a tyrosine residue. Using quantum chemical methodology, we have studied the CuB site in several redox and ligand states proposed to be intermediates of the catalytic cycle. The importance of the His-Tyr crosslink was investigated by comparing energetics, charge, and spin distributions between systems with and without the crosslink. The His-Tyr bond was shown to decrease the proton affinity and increase the electron affinity of both Tyr-244 and the copper. A previously unnoticed internal electronic equilibrium between the copper atom and the tyrosine was observed, which seems to be coupled to the unique structure of the system. In certain states the copper and Tyr-244 compete for the unpaired electron, the localization of which is determined by the oxygenous ligand of the copper. This electronic equilibrium was found to be sensitive to the presence of a positive charge 10 A away from the center, simulating the effect of Lys-319 in the K-pathway of proton transfer. The combined results provide an explanation for why the heme-copper oxidases need two pathways of proton uptake, and why the K-pathway is active only in the second half of the reaction cycle.

Full-text

Available from: Dage Sundholm, Apr 24, 2014
The chemistry of the Cu
B
site in cytochrome c oxidase and the importance of its
unique HisTyr bond
Ville R.I. Kaila
a,
, Mikael P. Johansson
b
, Dage Sundholm
c
, Liisa Laakkonen
d
, Mårten Wikström
a,
a
Helsinki Bioenergetics Group, Programme of Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, P.O. Box 65, FI-00014 Helsinki, Finland
b
Lundbeck Center for Theoretical Chemistry, Aarhus University, DK-8000 Århus C, Denmark
c
Department of Chemistry, University of Helsinki, P.O. Box 55, FI-00014 Helsinki, Finland
d
Division of Biochemistry, Department of Biological and Environmental Sciences, Faculty of Biosciences, University of Helsinki, FIN-00014, Helsinki, Finland
abstractarticle info
Article history:
Received 13 August 2008
Received in revised form 7 January 2009
Accepted 9 January 2009
Available online 20 January 2009
Keywords:
Oxygen reduction
Proton transfer
DFT
Quantum chemistry
The Cu
B
metal center is at the core of the active site of the hemecopper oxidases, comprising a copper atom
ligating three histidine residues one of which is covalently bonded to a tyrosine residue. Using quantum
chemical methodology, we have studied the Cu
B
site in several redox and ligand states proposed to be
intermediates of the catalytic cycle. The importance of the HisTyr crosslink was investigated by comparing
energetics, charge, and spin distributions between systems with and without the crosslink. The HisTyr bond
was shown to decrease the proton afnity and increase the electron afnity of both Tyr-244 and the copper. A
previously unnoticed internal electronic equilibrium between the copper atom and the tyrosine was
observed, which seems to be coupled to the unique structure of the system. In certain states the copper and
Tyr-244 compete for the unpaired electron, the localization of which is determined by the oxygenous ligand
of the copper. This electronic equilibrium was found to be sensitive to the presence of a positive charge 10 Å
away from the center, simulating the effect of Lys-319 in the K-pathway of proton transfer. The combined
results provide an explanation for why the hemecopper oxidases need two pathways of proton uptake, and
why the K-pathway is active only in the second half of the reaction cycle.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
Cytochrome c oxidase (CcO) is the terminal enzyme in the
respiratory chain of mitochondria and several bacteria. By reducing
molecular oxygen to water CcO drives the respiratory chain and
contributes to the generation of an electrochemical proton gradient
across the membrane, which is used for example to drive the synthesis
of ATP by F
o
F
1
-ATPase [13]. High resolution X-ray structures of CcO
have been available for over ten years [4,5], and details of the
mechanism of proton pumping and oxygen reduction have been
studied in a multitude of both experimental [610] and theoretical
works [1116]. Although many mechanistic principles are already
understood, some crucial questions still remain; e.g. why are two
pathways required for proton uptake from the negatively charged N-
side of the membrane (Fig. 1)?
Electrons are fed stepwise to CcO from the soluble cytochrome c
(cyt c). The electrons are rst accepted by the dinuclear Cu
A
site
(Fig. 1), which passes them further to a six-coordinated low-spin
heme a center in a process that is followed by loading of a pump
site with a proton taken up from the proton-conducting D-channel
[9,10] (see Fig. 1). A conserved glutamic acid, Glu-242
1
at the end of
the D-channel [1720], has recently been suggested to work as a
valve in minimizing leakage of the pumped proton back to the D-
channel [21,22]. Protonation of the pump site raises the mid-point
potential of the binuclear site, heme a
3
/Cu
B
, which accepts the
electron from heme a together with a second proton from Glu-242
[23]. Uptake of the latter proton produces the equivalent of water at
the binuclear site and repels the proton at the pump site, which is
ejected to the P-side of the membrane [23,24].
The reduction of molecular oxygen to water takes place at the
binuclear site, the active site of CcO, which is composed of a high-spin
heme, heme a
3
, and a copper center, Cu
B
, which has three histidine
ligands (His-240, His-290, and His-291, see Fig. 1). A tyrosine residue
Biochimica et Biophysica Acta 1787 (2009) 221233
Abbreviations: pmf, proton motive force; DFT, density functional theory; E
m,7
, mid-
point redox potential at pH 7 relative to NHE; CcO, cytochrome c oxidase; cyt c,
cytochrome c; RMSD, root mean square deviation; wt, the model system of the wild
type structure of the Cu
B
site; HisTyr mutant, the in silico mutated model system of
the Cu
B
site, where the HisTyr bond has been cut; MD, molecular dynamics; His, H
histidine; Tyr, Y tyrosine; Lys, K lysine; YOH, protonated Tyr; YO
, neutral Tyr-
radical; YO
, anionic Tyr (tyrosinate); PA, proton afnity; EA, electron afnity; HOMO,
highest occupied molecular orbital; LUMO, lowest unoccupied molecular orbital
Corresponding authors.
E-mail addresses: ville.kaila@helsinki. (V.R.I. Kaila), marten.wikstrom@helsinki.
(M. Wikström).
1
All amino acid numbers refer to the amino acid sequence of Bos taurus subunit I.
0005-2728/$ see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbabio.2009.01.002
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbabio
Page 1
(Tyr-244) is post-translationally crosslinked to one of the histidines
(His-240), as shown by protein chemical and structural studies
[4,25,26]. During the catalytic cycle of CcO the binuclear center cycles
through different redox and ligand states. The redox cycle can be
divided in an oxidative and a reductive phase (Fig. 2, Table 1). The
oxidative phase starts by irreversible cleavage of the OO bond,
transferring four electrons and a proton to the oxygen molecule. Two
electrons are taken from heme a
3
, one from the copper, and one
electron together with a proton are thought to derive from Tyr-244
[27]. This yields a species with ferryl iron, Fe[IV]fO, cupric copper and
a neutral tyrosine radical, a state which for historical reasons is known
as P
M
. However, the fourth electron in the oxygen cleavage reaction
has recently been suggested to originate from a conserved tryptophan
(Trp-126) producing a tryptophan radical that was suggested to relax
in the P
M
state [28,29]. In the next step of the cycle, P
M
is reduced by
an electron from heme a, yielding the P
R
state. Due to the magnetic
coupling between Cu
B
and heme a
3
, the Cu
B
site is usually spectro-
scopically invisible. In P
R
, however, Cu
B
shows a unique cupric EPR
signal with a peak at g 2.24 [30] and unchanged optical properties of
the ferryl heme, relative to P
M
. It may therefore be concluded that the
electron has been transferred to the tyrosine radical, producing a
tyrosin ate. Recent infrared studies have directly supported this
conclusion [31]. Protonation of P
R
yields the F state, in which the
hydroxide ligand of Cu
B
, which points towards the distal axial oxygen
of heme a
3
, is presumably protonated to water since the visible
spectrum of the ferryl heme is strongly perturbed. Cu
B
is EPR-silent
[30] in this state, and the infrared feature of the tyrosinate is
unchanged [31].
The protons consumed in oxygen reduction to water (chemical
protons) are trans ferred to th e binuc lear site by two proton-
conducting pathways, the D- and K-channels (named after Asp-91
and Lys-319, respectively). In the oxidative phase, the D-channel is
used for conducting the chemical protons while in the reductive
phase, starting at O
H
, the K-channel is employed for this purpose
[32,33]. Whereas this holds for the members of the aa
3
-type heme
copper oxidases, recent experiments have indicated that members of
the ba
3
-type family may employ only one proton conducting channel,
and pump protons with only half of the proton-pumping stoichio-
metry of the aa
3
-type oxidases [3436].
The K-channel ends at residue Tyr-244. Reduction of O
H
yields the
E
H
state, in which the charge transfer band of heme a
3
at 660 nm is
shifted [9], a signal which has been suggested to be due to the
interaction between ferric iron and cupric copper [37].
The Cu
B
center is unique for the hemecopper oxidases and is
different from other commonly found copper sites, e.g. type IIII
copper centers [38,39], although the type II copper centers found in,
e.g., galactose oxidase, dopamine β-monooxygenase, and laccase,
also have three histidine ligands [38,39]. The importance of the
interesting HisTyr crosslink in the Cu
B
center has raised much
discussion, and it has been suggested to lower the proton afnity of
the tyrosine [26,40]. Studies on organic model compounds, such as 2-
imidazol-1-yl-4-methylphenol, revealed that the covalent bond
between the imidazol and phenol rings down-shifts the pK
a
of the
phenolic proton by 1.6 pK-units to 8.6, and increases the redox
potential of the radical form by 66 mV to 750 mV (E
m,7
) [41].Ina
recent quantum chemical study on a phenolimidazol model system,
the bond between the two aromatic moieties was also found to
contribute to the proton and electron afnities; however, a copper
atom, simulated as a point charge, was suggested to strongly
contribute to the thermodynamic properties of the phenol [42].
Based on DFT studies, Daskalakis et al. recently suggested, that the
importance of the HisTyr crosslink is to x the Cu
B
center to a certain
conguration in relation to heme a
3
[43]. However, this study focused
on the CO-bound state of the Cu
B
center and its IR-vibrations, without
considering structural constrains from the protein backbone.
The effects of the different functionalities of the Cu
B
center are
difcult to decipher experimentally because amino acid substitutions
at the center render the enzyme inactive. Here, we have therefore
approached this problem by quantum-chemical methods employing a
large ( 110 atoms) realistic model of the Cu
B
center that includes the
crosslinked tyrosine. In the spirit of biological experiments, we call the
original model with the HisTyr crosslink a wild type system,
whereas the model where the HisTyr bond has been cut will be called
a mutation. The results conform well with some of the known
Fig. 1. The structure and location of redox groups in CcO. The electrons are transferred from cytochrome c (not shown) via Cu
A
and heme a to the binuclear heme a
3
/Cu
B
site (dashed
line). Protons are conducted via two proton conducting channels; the D-channel, ending up at Glu-242, and the K-channel ending up at Tyr-244. The protons are conducted from Glu-
242 either to: 1) the pump site and further to the P-side of the membrane, or 2) to the binuclear heme a
3
/Cu
B
site. Inset: the structure of the Cu
B
site.
222 V.R.I. Kaila et al. / Biochimica et Biophysica Acta 1787 (2009) 221233
Page 2
properties of this site, and bring forward new features that might
elucidate the importance of the HisTyr crosslink and the role of the
K-channel in proton transfer.
2. Models and methods
Density functional theory (DFT) [44,45] has proven very suitable for
the study of biological metal centers [4653]. We have used the B3LYP
hybrid functional [54,55] as the main methodology in this study, due to
its established good performance. Benchmark calculations suggest that
the B3LYP functional generally reproduces geometries with an accuracy
of 0.040.05 Å and energetics with a mean error on the order of 3 kcal/
mol [56]. For transition metal complexes, B3LYP calculations have
yielded a somewhat larger error [5762]. The Cu
B
model was built from
the structure of cytochrome c oxidase from Bos taurus, PDB entry 1V54
[63]. The effect of heme a
3
on Cu
B
was tested in our previous study
concerning the derivation of point charges for the metal centers in CcO,
and was found to have only a minor contribution on the charge
distribution of the Cu
B
system [64]. Therefore, the heme was not
included in the current calculations. It should also be pointed out that
the effect of heme a
3
is expected to be the same on the wild type
enzyme (wt) as on a variant system where the HisTyr crosslink was
abolished (see below), with the exception of the interaction to the
farnesyl group of heme a
3
. The structures of the Cu
B
center in different
ligand and redox states were optimized using a split-valence basis set
augmented with polarization functions [65],onallatomsexceptcopper,
for which a triple-zeta valence basis set augmented with polarization
functions [66] was used. The complete backbone of each amino acid
was included in the model. In addition, each amino acid was capped
with N-terminal methyl groups and C-terminal acetyl groups to
simulate the continuation of the peptide chain. The effect of the protein
surroundings was modeled by using the electrostatic continuum model
COSMO [67] with a dielectric constant ɛ =4. Furthermore, the restraints
imposed by the protein backbone were incorporated into the models
Fig. 2. Proposed reaction cycle of CcO. The cycle starts by cleavage of the OO bond in the A P
M
transition. In each proton pumping step (P F, F O, O
H
E
H
, and E
H
R) one
proton is transferred from Glu-242 to the proton loading site (blue arrow, H
+
), an electron is transferred to the binuclear site (red arrow, e
), a second proton is transferred to the
binuclear site (red arrow, red or green H
+
), and the proton is released from the proton loading site to the P-side of the membrane. The chemical protons are taken from D-channel in
the oxidative phase (P O
H
, red H
+
), and from the K-channel in the reductive phase (O
H
R , green H
+
).
Table 1
Proposed intermediates of the catalytic cycle and their possible structures
State Structure of BNC Q
tot
(Cu
B
)S
tot
(Cu
B
)
heme a
3
Cu
B
A Fe[II] Cu[I] YOH +1 0
P
M
Fe[IV]fO Cu[II]OH YO +1 1
P
R
Fe[IV]fO Cu[II]OH YO
0 1/2
F Fe[IV]fO Cu[II]H
2
OYO
+1 1/2
O
H
Fe[III]OH Cu[II]H
2
OYO
+1 1/2
E
H
Fe[III]OH Cu[I]H
2
OYO
00
R Fe[II] Cu[I] YOH +1 0
223V.R.I. Kaila et al. / Biochimica et Biophysica Acta 1787 (2009) 221233
Page 3
via structural constraints; the carbons of the terminal methyl groups of
the peripheral peptide bonds of the amino acid residues surrounding
the metal centers were xed to their position in the crystal structure.
The effect of the covalent bond between His-240 and Tyr-244 was
studied by optimizing the Cu
B
system in the various redox and ligand
states but without the crosslink, i.e., by protonating the N
ɛ
and C
ɛ
atoms
of His-240 and Tyr-244, respectively. The same optimization protocol
was used for these in silico HisTyr mutant systems as for the wild
type. Energetics, spin, and charge densities were evaluated by single
point calculations using a TZVP basis set on all atoms.
Along the reaction cycle, two charges separate from the Cu
B
center
can be assumed to perturb the spin and charge distributions of the
system: a negatively charged oxygenous ligand of heme a
3
, and the
positively charged protonated Lys-319 located in the K-channel. Their
effects on the electronic structure of the state Cu[II]H
2
OYO
were
simulated by using a point charge model. The heme a
3
ligand was
simulated by placing a point charge approximately 1.5 Å from the
water ligand of copper, its magnitude being scaled from 0.0e to 1.0e.
Protonated Lys-319 was simulated by a point charge located 10 Å from
the phenolic oxygen of Tyr-244 in the direction of the K-channel.
Single point energy calculations were performed, from which
Mulliken spin and point charges were calculated.
A value of 109.5 kcal/mol was used as the energy of an electron
reducing the Cu
B
system. This energy corresponds to the redox
difference between reduced and oxidized heme a [68]. A vacuum
energy of 270.6 kcal/mol was used for the proton [69].
All quantum chemical calculations were performed with the
T
URBOMOLE v5.9 quantum chemistry package [70]. VMD [71] and
gOpenMol [72,73] were used for visualization.
3. Results
3.1. Geometry of the Cu
B
site during the catalytic cycle
The optimized Cu
B
systems in different redox and ligand states
closely resemble the available X-ray structures of CcO. For example,
the root-mean-square deviation (RMSD) between all non-hydrogen
atoms of Cu[II] YOH and the fully oxidized X-ray structure is 0.3 Å. For
comparison, the RMSD between the fully oxidized and reduced X-ray
structures of the Cu
B
center in B. taurus CcO is 0.1 Å, while the RMSD
between the X-rays structures of the Cu
B
center from four different
organisms is 0.3 Å [63,7476].
Although there are so me interesting redox- state dep endent
structural variations in the coordination of the copper (see below),
the overall structural features of the optimized Cu
B
systems are very
similar to each other. The RMSD between all optimized structures is
0.18 Å (without hydrogens); if Cu[I] and Cu[II] states are compared
separately, the RMSD is 0.06 Å and 0.15 Å, respectively.
The structural features of the Cu
B
system are summarized in
Table 2. For the Cu[I] states, the bond to His-290 is on average 0.23 Å
longer than the two other HisCu bonds (2.182.26 Å vs. 1.962.01 Å).
For the corresponding Cu[II] structures, the histidines bind more
symmetrically (all bonds 1.942.08 Å). This is in agreement with studies
on inorganic cupric and cuprous complexes, in which the former prefer
a trigonal coordination while the latter prefer tetragonal geometry
[77]. EXA FS studies on the Cu
B
center in CcO have also suggested a
dif ferent coordinatio n between the cuprous and cupric states [78].A
dif ference between Cu[I] and Cu[II] can also be obse rved in the
angles of the bound li gand; the angle between His-290, Cu, and its
OH or H
2
O ligand is 10 in the cuprous states while it is 150° in
the cupric states. A water ligan d binds to Cu[I] and Cu[II] sta tes with
bond lengths of 2.36 Å and 2.08 Å, respectively, indicati ng that in the
cup rous state the water molecule is more weakly bonded to the
copper compared to the cupric state. The hydroxyl ligan d has a
characteristic bond length of 1.851.87 Å, which does not change
signicantly between the different states. The Cu[I]OH TyrOH
structure was found to be unstable, i.e., the structure dissociates
upon structure optimization, possible re asons f or which will be
discussed below. Interestingly, the Cu[II]H
2
OYO
state seems to be
an outlier among the Cu[II] states sh own in Table 2, since its bond
lengths are more reminiscent of a Cu[I] state (see below).
Apart from the dissociation of His-240 from Tyr-244 in the HisTyr
mutant systems, there are otherwise only minor structural changes
in the coordination of the copper due to this modication (Table 2). In
the Cu[II]H
2
OYO
state, the bond between His-290 and Cu
B
is 0.06 Å
longer in the mutant compared to the wild type. The Cu[I]H
2
OY
OH state has also a somewhat longer Cuwater bond length in the
mutant (0.05 Å), which is even more pronounced in Cu[II]H
2
OY
OH (0.28 Å) (Table 2). The largest structural variation in the HisTyr
mutant takes place in Cu[I]H
2
OYO
where the backbone
hydrogen bonding between His-240 and Tyr-244 is somewhat longer
compared to the other states, but is compensated by hydrogen
bonding between the aquo ligand of the copper and Tyr-244. This
structural variation is probably due to the lack of the HisTyr bond, but
does not affect the general conclusions of this work, as it is only found
to take place in this structure.
3.2. Comparison of HOMOLUMO gaps
The energy gap between highest occupied (HOMO) and lowest
unoccupied molecular orbitals (LUMO) is a measure of the excitability,
but gives also indication on the stability and chemical reactivity of the
molecule. A low HOMOLUMO gap usually indicates that the molecule
is chemically less inert. HOMOLUMO gaps of the different redox and
ligand states from both the wild type and HisTyr mutant systems
are summarized in Table 3.TheHOMOLUMO gap for the Cu[I]
structures are in general higher than for Cu[II] structures, indicating
that cuprous structures are in general more inert than the cupric
structures, which is typical for closed-shell molecules. As the Cu[I]OH
YOH dissociates upon structure optimization, the value reported in the
table is calculated using the Cu[II]OH YOH structure in a cuprous state.
Table 2
Geometrical properties of the wild type (rst number) and the HisTyr mutant
(second number) in different redox and ligand states
[Å] [°]
D(Cu-H240) D(Cu-H290) D(Cu-H291) D(Cu-L) ϕ(H290-Cu-L)
Cu[I]YOH 1.96/1.96 2.18/2.22 1.98/1.96 /––/
Cu[I]H
2
O YOH 2.00/1.99 2.19/2.26 2.01/1.99 2.36/2.41 104.62/95.11
Cu[I]H
2
OYO
1.97/2.02 2.26/2.28 1.99/2.03 2.43/2.34 95.08/110.64
Cu[II]YOH 1.94/1.96 1.99/2.06 1.95/1.96 /––/
Cu[II]OHYOH 2.06/2.05 2.07/2.07 2.05/2.06 1.85/1.86 150.14/149.91
Cu[II]H
2
OYOH 1.98/2.00 2.00/2.12 1.98/1.99 2.08/2.37 146.95/99.99
Cu[II]OH YO 2.08/2.05 2.07/2.07 2.04/2.05 1.85/1.86 151.48/166.90
Cu[II]OH YO
2.04/2.02 2.08/2.08 2.05/2.04 1.87/1.86 151.60/164.13
Cu[II]H
2
OYO
1.98/1.98 2.19/2.27 2.00/1.98 2.40/2.39 99.64/94.6
Table 3
HOMOLUMO gaps of the different redox and ligand states of Cu
B
for the wild type
and HisTyr mutant systems
HOMOLUMO gaps [eV] wild type”“HisTyr mutant ΔE
HOMOLUMO
Cu[I]YOH +4.73187 +5.30707 0.57520
Cu[I]OH YOH
a
+1.98565 +2.14017 0.15452
Cu[I]H
2
O YOH +4.33454 +4.90886 0.57432
Cu[I]H
2
OYO
+3.62566 +3.73859 0.11293
Cu[II]YOH +0.58433 +0.38431 +0.20000
Cu[II]OH YOH +3.04741 +1.73419 +1.31322
Cu[II]H
2
OYOH +1.32188 +0.34084 +0.98104
Cu[II]OH YO +2.15990 +2.32996 0.17006
Cu[II]OH YO
+1.08038 +0.87172 +0.20866
Cu[II]H
2
OYO
+1.20223 +1.64323 0.44100
a
calculated in the Cu[II]OH YOH structure.
224 V.R.I. Kaila et al. / Biochimica et Biophysica Acta 1787 (2009) 221233
Page 4
For the cuprous structures the systems without the HisTyr crosslink
have larger gaps than the wild type structures, whereas the opposite
holds for the cupric structures. The only exceptions in this trend are Cu
[II]OH YO and Cu[II]H
2
OYO
, which may be due an increased radical
character in systems without the HisTyr bond (see Section 3.3). The
HOMOLUMO gap for Cu[II]H
2
O YOH in the HisTyr mutant is very
similar to the HOMOLUMO gap of the unligated model, which is in
agreement with the long watercopper distance (2.4 Å, Table 2).
3.3. Spin distributions
The spin populations of the cupric states are shown in Fig. 3 and
summarized in Table 4. In the Cu[II]OH YOH, Cu[II]H
2
O YOH, and Cu
[II]OH YO
states, the unpaired electron is delocalized over the
copper atom, the histidine nitrogens and the oxygenous ligand, and
approximately 60% of the spin resides on the copper atom itself (Table
4). The Cu[II]OH Y-O state with S =1 shows a similar s pin
distribution around the copper as in the S=1/2 systems, but in
addition, 95% of the spin of the second electron is localized at the
tyrosine ring, as expected. The spin of an unpaired electron also
delocalizes somewhat ( 15%) to th e tyrosine moiety when no
oxygenous ligand is bound to the copper (Cu[II] YOH). In general, if
a system has no natural location for excess spin, it will lead to
scattering of the spin distribution over a large area (see Section 3.4).
The Cu[II]H
2
OYO
state shows an aberrant spin distribution, as
already indicated by its geometrical properties (Fig 3). The spin of the
unpaired electron in this S= 1/2 system resides to 95% on the tyrosine,
which indicates that the Cu[I]H
2
OY-O resonance form is dominant.
Note that this is not the case for Cu[II]OH Y-O
where the spin lies
Fig. 3. Spin distributions of the wild type system. Blue regions represent excess α spin density and red regions excess β spin density. Isocontour values of 0.01e and 0.01e have been
used for the α and β spin densities, respectively.
Table 4
Comparison of the spin densities between wild type and HisTyr mutant systems
H240 Y244 H290 H291 Cu L Gross-SD
Cu[II] YOH 9.6/3.0 15.2/54.6 13.5/6.3 6.4/2.8 55.3/33.2 0.0/0.0 1.14/1.12
Cu[II]OH
YO
9.4/6.8 95.6/100 4.4/5.4 7.2/7.8 61.6/58.8 21.8/21.2 2.50/2.57
Cu[II]OH
YO
7.0/8.4 0.0/0.0 4.1/5.1 6.9/7.2 62.4/57.8 19.7/21.4 1.09/1.12
Cu[II]H
2
O
YO
3.7/0.0 95.0/100 0.0/0.0 0.0/0.0 1.4/0.0 0.0/0.0 1.42/1.46
Cu[II]H
2
O
YOH
9.2/4.3 0.0/33.7 12.6/7.6 8.9/4.1 66.1/50.3 3.2/0.0 1.09/1.10
Cu[II]OH
YOH
6.4/6.9 0.0/0.0 4.4/4.3 7.0/6.8 61.2/58.1 21.1/23.9 1.10/1.11
The amount of (Mulliken) spin density is divided into subsystems: The histidine
residues H-240, H-290 and H-291; the tyrosine Y-244; the copper ion Cu; the ligand to
copper, L. The integrated gross spin density is also reported (gross SD). The rst and
second numbers indicate [%] of total spin on wild type and HisTyr mutant systems,
respectively.
225V.R.I. Kaila et al. / Biochimica et Biophysica Acta 1787 (2009) 221233
Page 5
entirely on the copper and its immediate surroundings. However, in
Cu[II]H
2
OY-O
a small fraction of the spin ( 1%) is nevertheless on
the copper and some on His-240 ( 4%), which indicates that this state
is not a pure Cu[I] state either. The half-and-half functional, BHLYP
[54,55], which contains 50% HF-exchange, gives qualitatively a similar
description as obtained with the B3LYP functional, although at the
BHLYP level, the spin distribution is somewhat more localized on Tyr-
244 (97%). This is reassuring, as it has been suggested that B3LYP in
some cases tends to underestimate the spin populations of copper
[79]; here, this is not the case.
The corresponding spin populations of the HisTyr mutant are
shown in Fig. 4 (see also Table 4). Compared to the wild type, the
spin is now generally more localized, either to the region around the
copper or to the tyrosine ring. This trend may be expected as the
HisTyr bond connects the electronic systems of the aromatic
phenol and imidazole rings. Comparison of Cu[II]H
2
OYO
in the
wild type and HisTyr mutant indicates another function of the
bond: while an electronic equilibrium between the two resonance
forms (Cu[I]/Tyr-O Cu[II]/Tyr-O
) can be observed in the wild
type, the radical form is more dominant in the mutant in which
the complete spin of the unpaired electron resides on the phenol
moiety. Hence, the HisTyr bond destabilizes the radical of Tyr, and
therefore increases the relative electron afnity of Tyr (cf. section V).
The spin of the second electron in Cu[II]OH Y-O of the mutant is
similarly completely localized to the phenol. Finally, the spin
distribution in the Cu[II]H
2
OYOH state is very different in the
HisTyr mutant compared to the wild type; the unpaired
electron in the mutant delocalizes all over the system, resembling
the spin distribution in the unligated state. This is probably due to
the relatively long bond distance between the copper and its water
ligand when the HisTyr bond is absent (Table 2). This is also
supported by a similar HOMOLUMO gap (Table 3) of this structure
compared to the unligated structures. Care was taken to assure that
this unexpected spin distribution is not, for example, due to
convergence to an excited state. Tighter convergence thresholds
and denser grids for the DFT quadrature lead to the same state. Also,
the lowest excitation energy out of this state was conrmed to be
positive. Although convergence to the global minimum can never be
rigorously ensured, we nd no indication of the opposite.
Further insight into the spin distributions was obtained by
studying the amount of spin polarization in the different ligand states.
This was done via the gross spin density, that is, the sum of unpaired α
and β spin densities in the systems, as reported in Table 4. A few points
are worth noting. First, all open-shell systems exhibit spin-polariza-
tion to a certain degree. For most systems, the gross spin density is ca.
10% higher than the net spin density, much smaller than what is found
Fig. 4. Spin distributions of the HisTyr mutant system. Blue regions represent excess α spin density and red regions excess β spin density. Isocontour values of 0.01e and 0.01e
have been used for the α and β spin densities, respectively.
226 V.R.I. Kaila et al. / Biochimica et Biophysica Acta 1787 (2009) 221233
Page 6
in, for example, low-spin iron porphyrins [51]. Two of the systems
behave differently. The (formal) Cu[II]H
2
OYO
state and the Cu[II]
OH Y-O radical both contain an excess of roughly half an unpaired
electron. As seen in Figs. 3 and 4, most of the polarization, expressed as
presence of β-spin, manifests itself around the tyrosine carbons. This
further corroborates the view that here, Cu[II]H
2
OYO
is more
correctly described as a Cu[I]H
2
OY-O radical.
Comparing the wild type and mutant systems, some differ-
ences in the degree of spin polarization are seen, but none are very
large. We also emphasize that spin contamination is insignicant, and
not the source of the polarization. At the B3LYP level, the doublet
states have bS
2
N values of 0.750.77, and the triplet states a value of
2.03, while the pure, non-polarized expectation values are 0.75 and 2,
respectively.
3.4. Charge density differences
Fig. 5 shows the difference in charge densities between cuprous and
cupric states for the wild type system, calculated in the geometries of
the cupric states. In Fig. 6, the corresponding differences are shown in
the absence of the HisTyr crosslink (see also Table 5). When compared
to Figs. 3 and 5, and as observed previously [50,51], it is seen that the
localization of charge and spin densities do not always coincide.
In the P
M
P
R
transition i.e. Cu[II]OH Y-O Cu[II]OH YO
, the
charge of the incoming electron spreads according to the population
analysis as follows: 82% to Tyr-244, 12% to His-240, and the remaining
6% to the copper and its two other histidine ligands. In the
corresponding HisTyr mutant, 98% of the additional charge upon
reduction is found on the tyrosine ring, indicating that charge ows
through the HisTyr bond from Tyr-244 to His-240.
The Cu[II]H
2
OYO
Cu[I]H
2
OYO
transition may occur in the
O
H
O
H,R
step of the catalytic cycle (Fig. 2). In accordance with the
aberrant spin distribution in Cu[II]H
2
OYO
, where most of the
unpaired spin is on the tyrosine (see above), Tyr-244 accepts 79% of
the charge of the electron while His-240 accepts 14%. The HisTyr
bond seems to play a similar role as in the previous transition; in the
HisTyr mutant, 83% and 7% of the added charge localize on Tyr-244
and His-240, respectively.
In the Cu[II]H
2
OYOH Cu[I]H
2
OYOH transition, the charge
of the incoming electron localizes quite symmetrically around the
copper atom (Table 5), while only 8% of the charge localizes on Tyr-
244. In the HisTyr mutant, 30% of the charge, which in the wild
type system resides on His-290, His-291 and the water ligand, is
transferred to Tyr-244. Also here the HisTyr bond seems to work as a
wire for the spreading out charge.
Cu[II]OH YOH Cu[I]OH YOH is a hypothetical transition as
the latter state was found to be unstable. The instability can be
understood from the charge density difference plot; the hydroxy
ligand accumulates extensive negative charge around the copper,
which cannot be stabilized by the cuprous metal. The charge spreads
Fig. 5. Charge density differences in the wt system. Blue regions represent excess positive charge (less electron density) and red regions excess negative charge (higher electron
density) between reduced and oxidized structures. Isocontour values of 0.01 e and 0.01 e have been used for the low and high electron density, respectively.
227V.R.I. Kaila et al. / Biochimica et Biophysica Acta 1787 (2009) 221233
Page 7
quite symmetrically around the copper atom and its ligands, and is
similar in the wild type (Fig. 5) and HisTyr mutant (Fig. 6) systems.
3.5. Thermodynamic importance of the HisTyr bond
Energy level diagrams of the different redox and ligand states of
Cu
B
are summarized in Fig. 7a for the wild type and in Fig. 7b for
the HisTyr mutant. All transitions are relative to the energy of Cu
[II]OH Y-O. In addition, the energetics discussed below represent
only the internal energetics of the Cu
B
center, without heme a
3
.In
many transitions of the binuclear site, e.g. P
M
P
R
, P
R
F, and
O E, the structure of heme a
3
remains unchanged (within the
current model, Fig. 2). Therefore it seems justied to draw parallels
between the properties of the Cu
B
site and the binuclear site. In
addition, we consider that the contribution of heme a
3
would be
the same on the Cu
B
center for the wild type and the HisTyr
mutant systems.
The rst transition shows that for the wild type system the
reduction of the Tyr-radical in Cu[II]OH Y-O (the P
M
state) is slightly
endergonic (+2.8 kcal/mol). The prior A P
M
transition (Fig. 2)was
not considered in this work, but it is known experimentally to go to
completion. Using an experimental error marginal of 1%, this means
that this transition must be downhill by at least 2.8 kcal/mol.
Blomberg et al. have estimated the A P
M
transition to be exergonic
by 5.0 kcal/mol [80], which implies that the A P
R
transition has a
total energy drop of 2.2 kcal/mol ( 5.0+2.8 kcal/mol).
In contrast, reduction of P
M
is far more costly in the HisTyr
mutant (+12.0 kcal/mol; Fig. 7b), so that the P
R
state is energetically
much more difcult to reach without the HisTyr bond. This suggests
that the HisTyr crosslink increases the electron afnity of Tyr-244 by
ca. 0.4 eV in the P
M
state (12.02.8 kcal/mol=9.2 kcal/mol 0.4 eV).
The overall energetics of A P
R
is also changed. During A P
M
the
Cu
B
center undergoes a Cu[I] YOH Cu[II]OH Y-O transition. In the
HisTyr mutant this transition is 3.3 kcal/mol more stable compared
to the wild type system. Furthermore, if it is assumed that the
contribution of heme a
3
would be the same for both wild type and
Fig. 6. Charge density differences in the HisTyr mutant system. Blue regions represent excess positive charge (less electron density) and red regions excess negative charge (higher
electron density) between reduced and oxidized structures. Isocontour values of 0.01e and 0.01e have been used for the low and high electron density, respectively.
Table 5
Comparison of the charge density differences between oxidized and reduced systems
([II]/[I] or TyrO/TyrO
) for the wild type and HisTyr mutant, respectively
H240 Y244 H290 H291 Cu L
Cu[II]OH Y-O/ 12.0/ 0.9 82.0/97.6 1.4/1.1 2.0/0.6 1.7/1.0 0.9/0.5
Cu[II]/[I]H
2
OY
OH
14.5/15.4 7.8/32.4 31.9/22.5 26.3/16.0 6.3/10.3 13.2/3.4
Cu[II]/[I]H
2
OY
O
14.4/6.9 79.3/82.8 3.2/1.6 0.2/5.5 3.7/ 2.9 0.8/6.1
Cu[II]/[I]OH Y
OH
15.0/19.6 5.6/1.4 20.2/19.5 21.4/20.5 12.8/12.7 25.0/26.3
Cu[II]/[I] YOH 18.3/9.8 20.5/53.3 30.6/17.5 20.0/10.7 10.6/8.7 0.0/0.0
The amount of charge is divided into subsystems: The histidine residues H-240, H-290
and H-291; the tyrosine Y-244; the copper ion Cu; the ligand to copper, L. The rst and
second numbers indicate the distribution of one electron charge on the subsystems in
[%] for the wild type and HisTyr mutant systems, respectively.
228 V.R.I. Kaila et al. / Biochimica et Biophysica Acta 1787 (2009) 221233
Page 8
mutant system during A P
M
, this suggest that the overall A P
R
is
endergonic by 3.7 kcal/mol ( 5.0 3.3+12.0 kcal/mol) in the mutant,
whereas this transition is exergonic by 2.2 kcal/mol in the wild type
(see above). Thus the HisTyr bond has an important function in
favoring OO bond cleavage, and in formation of the P
R
state.
Protonation of the P
R
state (Cu[II]OH YO
) yields state F (Fig. 2).
Here, protonation of the hydroxyl ligand of copper is favored over
protonation of Tyr-244 by 2.1 kcal/mol (27.625.5 kcal/mol, Table 6), in
agreement with the difference in optical spectrum between the two
species (see Introduction), and recent infrared data, which suggest
that Tyr-244 is anionic in state F [31]. The observed energetic
preference for the Cu[II]H
2
OYO
structure over Cu[II]OH YOH
also circumvents formation of the unstable Cu[I]OH YOH species
upon reduction by the next electron. As shown in Fig. 7A,B, Tyr-244
has a weak proton afnity in the preferred F state (Cu[II]H
2
OYO
;
Fig. 7A,B, 24.8 ( 8.6) kcal/mol=16.2 kcal/mol). However, when this
state is reduced to Cu[I]H
2
OYO
, the tyrosinate acquires a very high
proton afnity ( 43.5 ( 21.9) kcal/mol= 21.6 kcal/mol), and will
easily attract a proton via the K-channel (see Discussion).
In the F state of the mutant system, the energetic preference for
protonating Tyr-244 over the hydroxyl ligand of the copper is lowered
to 1.2 kcal/mol (32.130.9 kcal/mol, Fig. 7b, Table 6). The reason is that
the proton afnities of both Tyr-244 and the oxygenous copper ligand
increase compared to the wild type system, but the proton afnity
(PA) of Tyr-244 increases somewhat more. The proton afnity of Tyr-
244 in the preferred F state (Cu[II]H
2
OYO
) is even lower than in
the wild type system (20.8 kcal/mol vs. 16.2 kcal/mol in the wild
type), probably due to the very weak bond between the water and
Fig. 7. Energy level diagrams for (A) the wt system, and (B) the HisTyr mutant system.
229V.R.I. Kaila et al. / Biochimica et Biophysica Acta 1787 (2009) 221233
Page 9
copper in Cu[II]H
2
OYOH (see Table 2). As for the P
M
state, the
reduction of F (Cu[II]H
2
OYO
) is more costly without a HisTyr
bond (6.5 kcal/mol vs. 2.9 kcal/mol in wild type). However, as for the
wild type system, Cu[I]H
2
OYO
is a state with a very high proton
afnity (Fig. 7a,b, 40.3 ( 13.6) kcal/mol= 26.7 kcal/mol).
Table 6 summarizes the differences in proton and electron
afnities (EA) for the wild type and the HisTyr mutant systems.
The PA and EA values involving the Cu[II]H
2
OYO
state are
complicated by the energetics of the Cu[II] Cu[I] transition. For
example, reduction of Cu[II]H
2
OYO
to Cu[I]H
2
OYO
,reects
both the reduction of the Tyr-radical to a tyrosinate, and the partial
reduction of a cupric state to a cuprous state. In contrast, in the His
Tyr mutant, this transition is a rather pure reduction of a tyrosine
radical (see Section 3.3, and Fig. 4). Interestingly, in the mutant the
reduction of the Tyr-radical requires signicantly more energy if the
copper binds a hydroxyl instead of a water molecule (+12.0 kcal/mol,
and 6.5 kcal/mol, respectively). This indicates that without the HisTyr
bond, the electrostatic effect of the copper ligand is rather large.
However, in the wild type system the energy for reduction of the Tyr
radical is nearly the same whether the copper binds a water molecule
or a hydroxyl group (2.8 kcal/mol, and 2.9 kcal/mol, respectively). This
indicates that the negative charge of the phenolate delocalizes to a
larger area due to the crosslink, decreasing the electrostatic repulsion
between the hydroxyl and phenolate (Figs. 3 and 4).
3.6. Environmental effects on the electronic structure of Cu[II] H
2
OYO
The analysis carried out above has been restricted to an isolated
albeit relatively large system around the Cu
B
site. One unexpected
nding was the spin and charge distribution of the formal Cu[II]H
2
O
YO
state, in which the spin was found to be largely localized to the
tyrosine with predominantly cuprous Cu
B
. This structure of the Cu
B
system is expected for both states F and O
H
in the catalytic cycle
(Fig. 2). It was therefore of interest to test how this state might be
perturbed by an oxygenous axial ligand on heme a
3
iron, and a
positive charge on the Lys-319 in the K-channel of proton transfer that
eventually leads to the crosslinked Tyr-244. The effect of a negatively
charged distal axial ligand of heme a
3
on the spin and charge
distribution of the Cu[II]H
2
OYO
state is shown in Fig. 8. When the
magnitude of the point charge is scaled from 0.0e to 1.0e, the spin
distribution between the copper and Tyr-244 changes from 1/95 to 17/
73. Similarly, the negative charge on the Tyr increases from 0.0 e to
0.19e, indicating stabilization of the Cu[II]/Tyr-O
resonance form.
The aim with this point charge perturbation is not as such to model
the effect of heme a
3
, but rather to study the maximal perturbation
effect heme a
3
might have on the spin and charge distributions of Cu
[II]H
2
OYO
.
If a positive charge is placed 10 Å away from Tyr-244 (see Models
and methods), simulating a positively charged lysine-319 in the K-
pathway, the spin and charge distributions are perturbed dramatically
(Fig. 8). Even without the negative charge near the Cu, 62% of the spin
resides on the copper, and the charge of Tyr is 0.87e indicating a clear
phenolate character (for comparison, the charge of Tyr is 0.9e in Cu
Table 6
The effect of the HisTyr bond on electron and proton afnities of the system
Electron afnities
Tyr: wild type”“HisTyr
mutant
ΔEA[kcal/
mol]
Cu[II]OHYO
Cu[II]OHYO +e
2.8 12.0 +9.2
Cu[I]YOH Cu[II]OHYO +e
–– +3.3
Cu:
Cu[I]H
2
OYOH Cu[II]H
2
OYOH+e
+34.9 +41.0 6.1
Cu[I]H
2
OYO
Cu[II]H
2
OYO
+e
2.9 6.5 +3.6
Cu[I] YOH Cu[II] YOH+e
+48.4 +48.7 0.3
Proton afnities
Tyr: wild type”“HisTyr
mutant
ΔPA[kcal/
mol]
Cu[II]OH YOH Cu[II]OH YO
+H
+
+25.5 +30.9 5.4
Cu[II]H
2
OYOH Cu[II]H
2
OYO
+H
+
16.2 20.8 +4.6
Cu[I]H
2
OYOH Cu[I]H
2
OYO
+H
+
+21.6 +26.7 5.1
CuL:
Cu[II]H
2
OYOH Cu[II]OH YOH+ H
+
14.1 19.6 +5.5
Cu[II]H
2
OYO
Cu[II]OH YO
+H
+
+27.6 +32.1 4.5
Fig. 8. Spin (A), and Mulliken charges (B) of Cu and Tyr-244 when the point charge of the negative oxygenous ligand of heme a
3
is scaled from 0.0e to 1.0e with (+LYS) and without
( LYS) Lys-319.
230 V.R.I. Kaila et al. / Biochimica et Biophysica Acta 1787 (2009) 221233
Page 10
[II]OH YO
and 0.0e in Cu[II]OH YO), and in this case the negative
charge near the copper does not enhance this effect much further (Fig
8A and B). This point charge effect indicates that the relative redox
potentials of the copper and the Tyr may be easily perturbed by an
electrostatic eld which alters the electronic state of the Cu
B
site (for
comparison, two unit point charges 10 Å apart have an interaction
energy of 8 kcal/mol if the dielectric constant is 4).
From Table 4 it may be roughly estimated that the midpoint redox
potential (E
m
) of the copper is 190 mV higher than the E
m
for tyrosine
in the formal Cu[II]H
2
OYO
state (which makes it appear more as a
Cu[I]H
2
OY-O state). A positive charge at lysine-319 changes the ΔE
m
to 80 mV the tyrosine now having the higher potential (Fig. 8), an
effect of 270 mV. This is a reasonable electrostatic effect over 10 Å as it
predicts a mean effective dielectric constant of 5 between the lysine
and the tyrosine.
4. Discussion
The HisTyr crosslink works as a wire for spreading out the charge
in the Cu
B
system, as indicated by the charge density analysis (see Figs.
5 and 6, and Table 5). Some of the charge of Tyr-244 is transferred to
His-240, which causes Tyr-244 to become less negative and weakens
the electrostatic attraction between the phenolic proton of Tyr and the
ring, lowering its proton afnity. For the same reason there is no
energetic difference with respect to reduction of the Tyr radical in the
wild type system, whether the copper binds a hydroxyl or a water
molecule (see above). The increase in electron afnity of Tyr-244 due
to the cros slink can also be understood in terms of electron
delocalization; the repulsion between a negatively charged surface
and a negatively charged particle is stronger if the surface charge is
concentrated to a smaller area. The removal of an electron from a
system with a high charge delocalization, such as the wild type
system, requires more energy compared to a situation where the
charge is more localized, as in the HisTyr mutant. The increased
electron delocalization facilitated by the HisTyr crosslink thus leads
to a larger electron afnity and a smaller proton afnity of the Tyr
moiety than for the HisTyr mutant without the connecting bond.
The energetics of protonating Tyr-244 can be understood from the
charge density distribution of the system. When the Cu[II]H
2
OYO
state is reduced, negative charge accumulates on Tyr, which drastically
increases its proton afnity. This could explain why the K-channel is
not utilized for proton uptake prior to the E
H
state, in which cuprous
copper appears for the rst time (see Fig 2). In the E
H
E
H,R
transition,
reduction of Fe[III]OH yields a highly unstable Fe[II]OH species,
which must immediately accept a proton. Since the aqueous ligand of
Cu[I] cannot provide this proton due to the instability of the cuprous
hydroxide (see above), the proton is donated by Tyr-244, which
reforms state Cu[I]H
2
OYO
. The observed high pK
a
of Tyr-244 in
this state attracts a proton from the K-channel, producing the R state,
thus closing the reaction cycle. These ndings suggest the following
function of the proton-conducting K-channel. Analogously to the
proposed function of Tyr-244 as the proton donor in the A P
R
transition where the OO bond is broken, Tyr-244 donates a proton to
the oxygenous ligand of the heme a
3
iron in the reductive phase of the
catalytic cycle. Moreover, when the proton afnity of Tyr-244 is high,
it can only be (re)-protonated from the K-channel. As suggested by
MD-studies [11], Glu-242 is the proton donor for the oxygenous ligand
of the copper in the oxidative phase, where the K-channel is not used
for proton uptake. This suggests that the D-channel conducts the
chemical proton for the oxygenous copper ligand, whereas the K-
channel conducts protons to the distal heme a
3
ligand.
Recent FTIR experiments have suggested that Tyr-244 is deproto-
nated in the P
R
and F states and at least partially in the O
H
state [31].
The 1308 cm
1
vibration seen in these experiments indicates that Tyr-
244 is in the phenolate and not the radical form, which seemingly
contradicts our observation that the structure Cu[II]H
2
OYO
(in
states F and O
H
) favors the cuprous-tyrosine radical resonance form.
However, the observed perturbation of the electronic equilibrium
between the copper and Tyr by protonation of Lys-319 may explain
this discrepancy. The point charge effect of Lys-319 on the electronic
structure of Cu
B
may be of central importance in introducing coupling
between the protonation state of the K-channel and the thermo-
dynamic properties of Cu
B
. Lepp et al. recently reported that mutation
of Lys-319 to a methionine slows down the A P
R
transition from 30
to 90 μs [81]. Our analysis showed that the positively charged Lys-319
stabilizes the anionic tyrosinate, which may explain this observation.
Although we have shown how different redox and ligand states
inuence the electronand proton afnities of the Cu
B
site, andsuggested
a possible explanation to why the K-channel is activated in the second
half of the reaction cycle, there are still many open questions concerning
the Cu
B
site, for example, the difference between the O
H
and O states
[82,83]. So far, no spectroscopic differences have been found between
these states [84], although reduction of the former is coupled to proton-
pumping while the latter is not. The relaxation of O
H
to O occurs
spontaneously, in the absence of an external electron donor. Since the
Cu
B
center is spectroscopically invisible in most states, the difference
between O
H
and O might be found in the structure of this center. It has
been suggested that the redox potential of Cu
B
is too low in the O state to
drive proton translocation [3]. One possible explanation might be the
coordination of the Cu
B
site; cuprous complexes prefer a trigonal
coordination, whereas cupric complexes prefer a tetragonal one.
Therefore, a cupric complex of trigonal geometry is expected to have a
high E
m
. The (formal) Cu[II]H
2
OYO
state was shown to have a
geometry more reminiscent of Cu[I] complexes due to the dominant Cu
[I]H
2
OY-O resonance form. It was also shown that the electronic
structure of this state is affected by environmental effects, such as a
positive charge in the K-channel, which might also change the
coordination of the copper. It is therefore possible that the O
H
to O
transition is linked to the protonation state of the K-channel. Calcula-
tions are currently in progress to study this connection more thoroughly.
5. Conclusions
Quantum chemical theory has been applied to describe the
electronic structure and energetics of the Cu
B
site in cytochrome c
oxidase when cycling through different redox and ligand states. By
energetic considerations, it may be understood why two different
proton-conducting channels are used in the rst and latter part of the
cycle. It was suggested that the appearance of a high proton afnity of
the tyrosinate in the Cu[I]H
2
OYO
state triggers proton transfer from
the K-channel to Tyr-244 in the O
H
O
H,R
transition. The K-channel is
not used in proton transfer prior to the O
H
state because the proton
afnity of Tyr-244 is very low as long as Cu
B
is in the cupric form. In silico
mutations were done to reveal the importance of the unique HisTyr
bond. It was shown that this bond, which links the aromatic systems of
His-240and Tyr-244, hasa crucialeffect on the functionof the enzyme. It
lowers the proton afnity of Tyr-244 and increases its electron afnity,
thereby favoring OO bond scission during catalysis. The bond also
affects the copper, decreasing the protonafnity of its oxygenous ligand.
Acknowledgements
Prof. P.R. Taylor and Dr. Michael I. Verkhovsky are acknowledged
for critical comments and enlightening discussion. The authors wish
to acknowledge Prof. Margareta Blomberg for helpful advice. This
work was supported by grants from the Sigrid Juselius Foundation,
Biocentrum Helsinki, HENAKOTO and the Academy of Finland. It is
also supported by the Academy of Finland through its Centers of
Excellence Programme 2006-2011. The research collaboration is
supported by the Nordic Center of Excellence in Computational
Chemistry (NCoECC) project funded by NordForsk (070253). CSC The
Finnish IT Center for Science provided computational resources. V.R.I.
231V.R.I. Kaila et al. / Biochimica et Biophysica Acta 1787 (2009) 221233
Page 11
K. is supported by the Finnish Cultural Foundation, and the Graduate
School of Biotechnology and Molecular Biology. M.P.J. is supported by
the Lundbeck Foundation.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bbabio.2009.01.002.
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  • Source
    • "Electrons donated by the reducing substrates are transferred intra-molecularly from heme a to heme a 3 and Cu B . In the fully reduced state, at 1.9 Å resolution, CcOX displays a trigonal planar coordination of Cu B by three histidine residues, one of which is covalently linked to a tyrosine residue of subunit I (Y244) thereby taking an important part in the O 2 reduction cycle [67] [68]. CcOX is targeted and inhibited by a number of small molecules/ions, such as CN "
    [Show abstract] [Hide abstract] ABSTRACT: Background: The reactions between Complex IV (cytochrome c oxidase, CcOX) and nitric oxide (NO) were described in the early 60's. The perception, however, that NO could be responsible for physiological or pathological effects, including those on mitochondria, lags behind the 80's, when the identity of the endothelial derived relaxing factor (EDRF) and NO synthesis by the NO synthases were discovered. NO controls mitochondrial respiration, and cytotoxic as well as cytoprotective effects have been described. The depression of OXPHOS ATP synthesis has been observed, attributed to the inhibition of mitochondrial Complex I and IV particularly, found responsible of major effects. Scope of review: The review is focused on CcOX and NO with some hints about pathophysiological implications. The reactions of interest are reviewed, with special attention to the molecular mechanisms underlying the effects of NO observed on cytochrome c oxidase, particularly during turnover with oxygen and reductants. Major conclusions and general significance: The NO inhibition of CcOX is rapid and reversible and may occur in competition with oxygen. Inhibition takes place following two pathways leading to formation of either a relatively stable nitrosyl-derivative (CcOX-NO) of the enzyme reduced, or a more labile nitrite-derivative (CcOX-NO(2)(-)) of the enzyme oxidized, and during turnover. The pathway that prevails depends on the turnover conditions and concentration of NO and physiological substrates, cytochrome c and O(2). All evidence suggests that these parameters are crucial in determining the CcOX vs NO reaction pathway prevailing in vivo, with interesting physiological and pathological consequences for cells.
    Full-text · Article · Sep 2011 · Biochimica et Biophysica Acta
  • Source
    • "When deprotonated, the copper binds water whereas the iron binds a hydroxide and vice versa when Tyr-244 is protonated. This effect is probably due to the increased Cu[I]/Tyr-O ⁎ character of the Cu[II]-H 2 O structure when Tyr-244 is deprotonated [10]. When two hydroxides are bound to the BNC, the O–O distance increases to 2.65–2.75 "
    [Show abstract] [Hide abstract] ABSTRACT: Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain. By reducing oxygen to water, it generates a proton gradient across the mitochondrial or bacterial membrane. Recently, two independent X-ray crystallographic studies ((Aoyama et al. Proc. Natl. Acad. Sci. USA 106 (2009) 2165-2169) and (Koepke et al. Biochim. Biophys. Acta 1787 (2009) 635-645)), suggested that a peroxide dianion might be bound to the active site of oxidized CcO. We have investigated this hypothesis by combining quantum chemical calculations with a re-refinement of the X-ray crystallographic data and optical spectroscopic measurements. Our data suggest that dianionic peroxide, superoxide, and dioxygen all form a similar superoxide species when inserted into a fully oxidized ferric/cupric binuclear site (BNC). We argue that stable peroxides are unlikely to be confined within the oxidized BNC since that would be expected to lead to bond splitting and formation of the catalytic P intermediate. Somewhat surprisingly, we find that binding of dioxygen to the oxidized binuclear site is weakly exergonic, and hence, the observed structure might have resulted from dioxygen itself or from superoxide generated from O(2) by the X-ray beam. We show that the presence of O(2) is consistent with the X-ray data. We also discuss how other structures, such as a mixture of the aqueous species (H(2)O+OH(-) and H(2)O) and chloride fit the experimental data.
    Full-text · Article · Jul 2011 · Biochimica et Biophysica Acta
  • Source
    • "Mutagenesis studies show that the active-site histidine–tyrosine crosslink is crucial for catalytic activity in the -aa 3 and type-ba 3 oxidases [4, 7,23242526272829. The cross-link has been proposed to maintain the structure of the active site [7, 29]. In previous publications, Y288F mutant in cytochrome bo 3 from E. coli [23] and Y280F mutant in cytochrome aa 3 from P. denitrificans [26] reveal complete loss of CuB and propose tyrosine is essential element forming the CuB site. "
    [Show abstract] [Hide abstract] ABSTRACT: The cbb 3-type oxidases are members of the heme-copper oxidase superfamily, distant by sequence comparisons, but sharing common functional characteristics. The cbb 3 oxidases are missing an active-site tyrosine residue that is absolutely conserved in all A and B-type heme-copper oxidases. This tyrosine is known to play a critical role in the catalytic mechanisms of A and B-type oxidases. The absence of this tyrosine in the cbb 3 oxidases raises the possibility that the cbb 3 oxidases utilize a different catalytic mechanism from that of the other members of the superfamily, or have this conserved residue in different helices. Recently sequence comparisons indicate that, a tyrosine residues that might be analogous to the active-site tyrosine in other oxidases are present in the cbb 3 oxidases but these tyrosines originates from a different transmembrane helix within the protein. In this research, three conserved tyrosine residues, Y294, Y308 and Y318, in helix VII were substituted for phenylalanine. Y318F mutant in the Rhodobacter capsulatus oxidase resulted in a fully assembled enzyme with nativelike structure and activity, but Y294F mutant is not assembled and have a catalytic activity. On the other hand, Y308F mutant is fully assembled enzyme with nativelike structure, but lacking catalytic activity. This result indicates that Y308 should be crucial in catalytic activity of the cbb 3 oxidase of R. capsulatus. These findings support the assumption that all of the heme-copper oxidases utilize the same catalytic mechanism and provide a residue originates from different places within the primary sequence for different members of the same superfamily.
    Full-text · Article · Nov 2010 · Molecular Biology Reports
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