International Journal of
Molecular Sciences
Article
ns-µs Time-Resolved Step-Scan FTIR of ba3
Oxidoreductase from Thermus thermophilus:
Protonic Connectivity of w941-w946-w927
Antonis Nicolaides 1 , Tewfik Soulimane 2 and Constantinos Varotsis 1, *
1
2
*
Department of Environmental Science and Technology, Cyprus University of Technology, P.O. Box 50329,
3603 Lemesos, Cyprus; ag.nicolaides@edu.cut.ac.cy
Chemical and Environmental Science Department and Materials & Surface Science Institute,
University of Limerick, V94 T9PX Limerick, Ireland; tewfik.soulimane@ul.ie
Correspondence: c.varotsis@cut.ac.cy; Tel.: +357-2500-2451
Academic Editor: Samuel De Visser
Received: 15 August 2016; Accepted: 21 September 2016; Published: 29 September 2016
Abstract: Time-resolved step-scan FTIR spectroscopy has been employed to probe the dynamics of
the ba3 oxidoreductase from Thermus thermophilus in the ns-µs time range and in the pH/pD 6–9
range. The data revealed a pH/pD sensitivity of the D372 residue and of the ring-A propionate of
heme a3 . Based on the observed transient changes a model in which the protonic connectivity of
w941-w946-927 to the D372 and the ring-A propionate of heme a3 is described.
Keywords: cytochrome ba3 ; ns time-resolved step-scan FTIR; heme-copper oxidoreductases
1. Introduction
The electron and proton transfers in conjunction with the protonic connectivity between the
environments sensed by key residues play a vital role in the biological function of proteins [1].
The conformational rigidity of Thermophilic enzymes against heat denaturation has attracted the
biotechnological research community because of the molecular events associated with enzymatic
catalysis. Based on the crystal structure cytochrome ba3 from Thermus thermophilus contains
a homodinuclear copper center (CuA ), a low-spin heme b, and a heme a3 -CuB center [2–24].
Cytochrome ba3 catalyzes the reductions of oxygen (O2 ) to water (H2 O) and of nitric oxide (NO)
to nitrous oxide (N2 O), as well and the oxidation of carbon monoxide (CO) to carbon dioxide
(CO2 ) [2–24]. The photolyzed ba3 -CO species is an excellent model for time-resolved spectroscopic
studies [7–9,13,15,18,24]. In the past, we used time-resolved Raman and step-scan FTIR (TRS2 -FTIR)
spectroscopy to probe the binding of CO to CuB and the structural changes of the ring-A propionate of
heme a3 and D372 [7–10]. It was concluded that the trans/cis isomerization of the ring-D propionate
plays a crucial role in controlling the orientations of “docked” CO between the heme rings-A and -D
propionates and that the protein environment removes the barrier to the two orientations of CO [10].
The role of the heme a3 -D372-H2 O site and of ring-A propionate as proton carriers to the H2 O pool,
which is conserved among all structurally-known heme-copper oxidases, were reported [11,16,18,23].
The observation of deprotonated and protonated forms of heme a3 rings-A and -D propionate and
D372 indicated a protonic connectivity between the ring-A propionate, a H2 O molecule and D372. It
was proposed that the environment of the ring-A heme a3 propionate-D372-H2 O moiety can contribute
to proton motion [18,23].
Time-resolved Raman and step-scan FTIR are powerful structure-sensitive techniques for
exploring changes that occur to metal centers and individual amino acids as a result of changes
in the ligation state of the metal centers and/or redox and conformational changes induced by
Int. J. Mol. Sci. 2016, 17, 1657; doi:10.3390/ijms17101657
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2016, 17, 1657
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the changes in the coordination of the metal centers [24–27]. The temperature dependency of
these changes is expected to give insight into the thermostability of the thermophilic enzymes.
Ligand photodissociation can also induce protonation/deprotonation reactions of key residues
and the pH dependency of the photodynamic/protonation/deprotonation can contribute towards
the elucidation of events not previously reached by other spectroscopic techniques. Furthermore,
the detection of protonation/deprotonation of ionizable groups is important towards the elucidation
of the proton motions that take place in cytochrome c oxidases. The dynamics of the protein cavities in
controlling the motion of O2 migration to the binuclear heme Fe-CuB center is also important towards
the elucidation of ligand binding since the enzyme operates at high temperature/low O2 concentration.
To address these issues the Time Resolved Step-Scan Fourier Transform Infrared (TRS2 -FTIR) studies
of the fully-reduced CO complex in the pH/pD 6–9 range were examined and compared to determine
the conformations of the key residue D372 and those of the heme a3 ring-A propionate. The main goal
was to compare the pH/pD results in a time-resolved approach for the protonated and deprotonated
forms of ba3 . The effect of H/D exchange and the dynamic behavior of the 1749/1743 and 1723 cm−1
modes which have been assigned to ν(COO(H)) of two conformations of the protonated forms of D372,
as well as the coupling of the protonic connectivity of w941-w946-w927 to the ring-A propionate of
heme a3 and D372 are discussed.
2. Results
Figure 1 shows the Time Resolved Step-Scan Fourier Transform Infrared (TRS2 -FTIR) difference
spectra (td = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced ba3 -CO at pH 7.0 subsequent
to CO photolysis by a 7 ns 532 nm laser pulse. At td = 100 ns, the spectra show a peak at 1697 cm−1 ,
“W” shape troughs at 1706 and 1724 cm−1 , and also features at 1717(+), 1733(+), 1738 (−), 1744(−),
and 1749(+) cm−1 . The peak/trough at 1697/1706 cm−1 is characteristic of the perturbation of the
C=O stretching band exhibiting stronger H-bonding interaction to surrounding groups in the transient
spectra that we have assigned to the ring-A propionate of heme a3 . [7]. At td = 500–80,000 ns the
1706 cm−1 mode appears as a doublet with intensity and frequency changes. The 1717 and 1733 cm−1
modes, which are not exhibiting frequency shifts or intensity changes in the td = 100–80,000 ns range,
can be attributed to the C=O mode of either protonated aspartic or glutamic residues which are affected
by the induced perturbation of CO photodissociation. The 1749/1738, 1744 cm−1 modes have been
tentatively assigned to the ν(COO(H)) of two conformations of protonated D372 [7,18]. A broad
negative mode at 1548 cm−1 is also shown and is tentatively assigned, in agreement with previous
work, to originate from the coupled His-Tyr ring mode with large contributions from the C–N of the
covalent bond between both ring systems [28]. The 1541 cm−1 positive mode appeared as a broad
peak and it was attributed to amide II vibrations and remained unchanged in the td = 100–80,000 ns
range [29]. Features consisting of a negative peak at 1530 and positive peak at 1559 cm−1 are present at
td = 100 ns, and were previously assigned to the νas (COO− ) of the deprotonated form of the ring-A
propionate of heme a3 [11,18,23]. This is evidence that there is equilibrium between the protonated and
deprotonated forms of the ring-A propionate of heme a3 . It should be noted that the transient binding
of CO to CuB in aa3 oxidase is dynamically linked to structural changes around a protonated carboxyl
group [30]. Finally, a peak/trough at 1506/1513 cm−1 is present and exhibits small intensity changes,
but the ratio of the 1506/1513 cm−1 modes remained unchanged. This derivative form feature has
been attributed to tyrosinate/tyrosine vibrations [31].
Figure 2 shows the Time Resolved Step-Scan Fourier Transform Infrared (TRS2 -FTIR) difference
spectra of 1500–1760 cm−1 region (td = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced
ba3 -CO at pH 6.0 subsequent to CO photolysis by a 7 ns 532 nm laser pulse. The Time Resolved
Step-Scan (TRS2 ) FTIR difference spectra in the 1690–1760 cm−1 region show the following changes
when compared with those obtained at pH 7. At td = 100 ns, the protonated form of D372 is observed
at 1742 cm−1 , showing a 7 cm−1 downshift, which is representative of a weaker C=O bond exhibiting
stronger H-bonding interaction to surrounding groups. The 1733 cm−1 mode has gained intensity,
Int. J. Mol. Sci. 2016, 17, 1657
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whereas that of the 1717 cm−1 remained the same. The 1697 cm−1 mode is not altered in intensity
and/or frequency shifts; however, there are two weak negative peaks located at 1706 and 1714 cm−1 ,
which at td = 80,000 ns have gained intensity and appeared as a single mode at 1714 cm−1 . Compared
to pH 7, we conclude that there is a pH sensitivity of the protonated forms of D372 and the ring-A
Int. J. Mol. Sci. 2016, 17, 1657
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propionate of heme a3 . The observed 1728(−), 1733(+), and 1742(+) cm−1 modes do not present
Int. J. Mol. Sci. 2016, 17, 1657
3 of 10
to pH 7,
we conclude
that there isshifts
a pH sensitivity
protonated ns
forms
of D372
and the ring-A
any intensity
changes
or frequency
in the tdof=the
100–80,000
range.
Compared
to the pH 7
−1 modes do not present any
−
1
propionate
of
heme
a
3. The observed 1728(−), 1733(+),
and
1742(+)
cm
spectra, there
is
also
a
frequency
shift
of
the
1723
cm
mode,
which
has
been
attributed
to
one of the
to pH 7, we conclude that there is a pH sensitivity of the protonated forms of D372 and the ring-A
intensity changes or frequency shifts −
in1 the td = 100–80,000 ns range. Compared
to the pH 7 spectra,
−1
two conformations
D372,
to 1728
cm 1728(−),
. This1733(+),
indicates
the second
propionate ofof
heme
a3. The
observed
andsensitivity
1742(+) cm upon
modesprotonation
do not presentofany
there is also a frequency shift
of the 1723 cm−1 mode, which has been−attributed
to one of the two
−1 shifts
1 mode
intensity
changes
or
frequency
inis
the
td = 100–80,000
ns1541
range.cm
Compared
toat
thepH
pH67isspectra,
conformer
of
D372.
The
1559
cm
mode
broader
and
the
similar
−1
conformations of D372, to 1728 cm . This indicates
sensitivity upon protonation of the second to that
is also a frequency shift of−1the−1723
cm−1 mode, which has been
attributed to one of the two
1 observed
at pH 7. there
The
negative
peak
at
1548
cm
at
pH
becomes
a doublet
the
appearance
conformer of D372. The 1559 cm mode
is broader and the 7,
1541
cm−1 mode
at pH 6 iswith
similar
to that
−1. This indicates sensitivity upon protonation of the second
conformations of D372, to 1728−cm
1
−
1
−1 observed at pH 7, becomes
at
pH
7.
The
negative
peak
at
1548
cm
a
doublet
with
the
appearance
of
of a newconformer
negativeofpeak
at 1554 cm . The trough at 1530 cm , which was previously assigned
to
D372. The 1559 cm−1−1mode is broader and the −11541 cm−1 mode at pH 6 is similar to that
−
a
new
negative
peak
at
1554
cm
.
The
trough
at
1530
cm
,
which
was
previously
assigned
to
the
the deprotonated
form
of
ν
(COO
)
of
the
ring-A
propionate
of
heme
a
,
is
present
at
pH
6
without
−1
as
at pH 7. The negative peak at 1548
cm observed at pH 7, becomes a doublet 3with the appearance of
deprotonated form of νas(COO−)−1of the ring-A propionate−1 of heme a3, is present at pH 6 without
a new
peak
at 1554 cm
. The
trough
at 1530 cm , which was previously assigned to the
presenting
anynegative
changes
regarded
to the
pH
alteration.
presenting any changes regarded to the pH alteration.
deprotonated form of νas(COO−) of the ring-A propionate of heme a3, is present at pH 6 without
presenting any changes regarded to the pH alteration.
Figure 1. Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR) difference spectra of
Figure 1.1500–1760
Time Resolved
Step-Scan Fourier Transform Infrared (TRS2 -FTIR) difference spectra of
cm−1 region (td = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced ba3-CO
2-FTIR) difference spectra of
−
1
−1 spectral
Figure
1.
Time
Resolved
Fourier Transform
Infrared (TRS
1500–1760
cm region
(td = Step-Scan
100–80,000
cmpulse
subsequent
to CO photolysis
by a 7 ns 532ns,
nm4laser
at pH 7.0.resolution) of fully-reduced ba3 -CO
1500–1760 cm−1 region (td = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced ba3-CO
subsequent to CO photolysis by a 7 ns 532 nm laser pulse at pH 7.0.
subsequent to CO photolysis by a 7 ns 532 nm laser pulse at pH 7.0.
Figure 2. Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR) difference spectra of
1500–1760 cm−1 region (td = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced ba3-CO
Figure 2. Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR)
difference spectra of
to CO photolysis
by a 7Fourier
ns 532 nmTransform
laser pulse at
pH 6.0. (TRS2 -FTIR) difference spectra of
Figure 2.subsequent
Time Resolved
Step-Scan
Infrared
1500–1760
cm−1 region (td = 100–80,000 ns, 4 cm−1−1spectral resolution) of fully-reduced ba3-CO
−
1
1500–1760
cm region (td = 100–80,000
ns, 4 cm spectral resolution) of fully-reduced ba3 -CO
subsequent to CO photolysis
by a 7 ns 532 nm laser pulse at pH 6.0.
subsequent to CO photolysis by a 7 ns 532 nm laser pulse at pH 6.0.
Int. J. Mol. Sci. 2016, 17, 1657
4 of 10
Figure 3 shows the Time Resolved Step-Scan Fourier Transform Infrared (TRS2 -FTIR) difference
−1 region (t = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced
Mol. Sci. 2016, 17,
1657
4 of 10
spectra Int.
ofJ.1500–1760
cm
d
ba3 -CO at pH 9.0 subsequent to CO photolysis by a 7 ns 532 nm laser pulse. At td2 = 100 ns, the observed
Figure 3 shows the Time Resolved
Step-Scan Fourier Transform Infrared (TRS -FTIR) difference
peak/trough
feature of 1697/1706 cm−1 is similar to that observed at pH 7. This is in contrast to the pH
spectra of 1500–1760 cm−1 region (td = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced
−1 were observed indicating the pH sensitivity
6 data where
negative
peaks at
and 1714bycm
ba3-CO two
at pH
9.0 subsequent
to 1706
CO photolysis
a 7 ns
532 nm laser pulse. At td = 100 ns, the
−1 exhibits a
−1
of the ring-A
propionate
hemeofa31697/1706
. The protonated
form
of D372
observed
1749
observed
peak/troughoffeature
cm is similar
to that
observed
at pH 7.at
This
is incm
contrast
−1
−
1
−
1
the pHwhen
6 datacompared
where two negative
peaks
at 1706 at
and
1714
cm awere
observed
indicating
the pH
7 cm to
upshift
with that
observed
pH
6, and
3 cm
downshift
when
compared
sensitivity
of
the
ring-A
propionate
of
heme
a
3. The protonated form of D372 observed at 1749 cm−1
with that observed at pH 7, confirming the pH sensitivity of the protonated D372. In addition, the
exhibits a 7 cm−1 upshift when compared with that observed at pH 6, and a 3 cm−1 downshift when
negative peak at 1739 cm−1 , which has been assigned to D372, has gained intensity at td = 100 ns
compared with that observed at pH 7, confirming the pH sensitivity of the protonated D372. In
when compared
tonegative
that at pH
td−1,=which
80 µshas
has
lostassigned
almosttoall
of its
This observation
addition, the
peak9,atbut
1739atcm
been
D372,
hasintensity.
gained intensity
at td
indicates
thatnsthe
dynamics
ofto
the
D372
are9,linked
dynamics
of theall
photodissociated
CO [7,23].
= 100
when
compared
that
at pH
but at tto
d = the
80 μs
has lost almost
of its intensity. This
−1 mode
observation forms
indicates
thatring-A
the dynamics
of the
D372significant
are linkedchanges
to the as
dynamics
ofcm
the
The deprotonated
of the
propionate
exhibit
the 1530
−1
[7,23]. The
forms of the ring-A propionate exhibit significant
appearsphotodissociated
as a doublet. InCO
addition,
at tdeprotonated
d = 100 ns, there are two trough at 1548 and 1554 cm . The latter
changes as the 1530 cm−1 mode appears as a doublet. In addition, at td = 100 ns, there are two trough
trough loses intensity at−1times longer than 100 ns and disappears at td = 80 µs, indicating that its
at 1548 and 1554 cm . The latter trough −
loses intensity at times longer than 100 ns and disappears at
behavior
is coupled to that of the 1739 cm 1 trough.
td = 80 μs, indicating that its behavior is coupled to that of the 1739 cm−1 trough.
Figure 3. Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR) difference spectra of
Figure 3. Time Resolved
Step-Scan Fourier Transform Infrared (TRS2 -FTIR) difference spectra of
1500–1760
cm−1 region (td = 100–80,000 ns, 4 cm−1
spectral resolution) of fully-reduced ba3-CO
−
1
1500–1760
cm region (td = 100–80,000
ns, 4 cm−1 spectral resolution) of fully-reduced ba3 -CO
subsequent to CO photolysis
by a 7 ns 532 nm laser pulse at pH 9.0.
subsequent to CO photolysis by a 7 ns 532 nm laser pulse at pH 9.0.
Figure 4 presents the Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR)
difference spectra of the fully-reduced ba3-CO complex in D2O. The experiments were performed
in
Figure 4 presents the Time Resolved Step-Scan Fourier Transform Infrared (TRS2 -FTIR) difference
D2O in order to study the behavior of the protein upon H/D exchange. The amide I band arises 80%
spectra from
of the
ba3 -CO
complex
in D2 O. group
The experiments
performed
in D2 O in
thefully-reduced
C=O stretching mode
of the
amide functional
and 20% from were
C–N stretching
[8–13].
order toThe
study
the
behavior
of
the
protein
upon
H/D
exchange.
The
amide
I
band
arises
80%
−1
protein secondary structure consists of a-helix (1648–1660 cm ), β-sheet (1625–1640 andfrom the
C=O stretching
the(1660–1685
amide functional
andstructures
20% from
C–N stretching
[8–13]. The protein
1672–1694mode
cm−1), of
turns
cm−1), andgroup
unordered
(1640–1650
cm−1) [32,33].
−
1
Figure
5
presents
the
pH
sensitivity
of
Propionate
A
and
aspartic
acid
residue
D372.
Features at cm−1 ),
secondary structure consists of a-helix (1648–1660 cm ), β-sheet (1625–1640 and 1672–1694
1736(+)/1744(−),
1729(−),
1697(+)/1706(−),
1686(+),
1668(+)/1675(−),
1652(+)/1660(−),
1638(+)/1644(−),
−
1
−
1
turns (1660–1685 cm ), and unordered structures (1640–1650 cm ) [32,33].
1630(−), 1559(+), 1541(+)/1548(−) 1519(−)/1527(−), 1535(−), and 1506(+)/1513(−) at 100 ns, subsequent to
Figure 5 presents the pH sensitivity of Propionate A and aspartic acid residue D372.
CO photolysis, remained unchanged in the td = 100–8000 ns range. The 1736(+)/1744(−) and 1729(−)
Features
at 1736(+)/1744(
−), 1729(−), 1697(+)/1706(
−),
1686(+),
1668(+)/1675(
), 1652(+)/1660(
−),
features
are slightly pH/pD-dependent
since they show
small
frequency
shifts, but the−absence
of the
−1
1638(+)/1644(
−
),
1630(
−
),
1559(+),
1541(+)/1548(
−
)
1519(
−
)/1527(
−
),
1535(
−
),
and
1506(+)/1513(
−)
1750 cm in the pD spectra demonstrates the sensitivity of the protonated form of D372 to pH/pD
The 1697(+)/1706(−)
feature, which
has been
attributed tointhethe
protonated
form of the
at 100 exchanges.
ns, subsequent
to CO photolysis,
remained
unchanged
td = 100–8000
ns range.
The 1736(+)/1744(−) and 1729(−) features are slightly pH/pD-dependent since they show small
frequency shifts, but the absence of the 1750 cm−1 in the pD spectra demonstrates the sensitivity of
the protonated form of D372 to pH/pD exchanges. The 1697(+)/1706(−) feature, which has been
Int. J. Mol. Sci. 2016, 17, 1657
Int. J. Mol. Sci. 2016, 17, 1657
5 of 10
5 of 10
attributed
to the protonated form of the ring-A propionate, is insensitive to pD exchanges. 5Aofgroup
of
Int. J. Mol. Sci. 2016, 17, 1657
10
ring-A
is insensitive
to pD exchanges.
A to
group
of vibrations
at 1668(+)/1675(−)
are −) to
vibrations
at propionate,
1668(+)/1675(
−) are tentatively
assigned
protein
turns, those
at 1652(+)/1660(
tentatively
assigned isto insensitive
protein turns,
those
at 1652(+)/1660(−)
group
of vibrations and,
ring-A
propionate,
to pD
exchanges.
A group toofα-helical
1668(+)/1675(−)
are All of
α-helical
group
of
vibrations
and, finally,
those
at 1638(+)/1644(
−vibrations
), 1630(
−at) to
β-sheet [29,32].
finally,
those
at 1638(+)/1644(−),
1630(−)
to at
β-sheet
[29,32]. Allto of
the abovementioned
vibrations
tentatively
assigned
to
protein
turns,
those
1652(+)/1660(−)
α-helical
group
of
vibrations
and,
the abovementioned vibrations remained unchanged in the td = 100–80,000 ns range. The behavior of
remained
unchanged
in the td = 100–80,000
range.[29,32].
The behavior
of the vibrational
features
finally, those
at 1638(+)/1644(−),
1630(−) to ns
All of of
theallabovementioned
vibrations
all of the
vibrational
observed
pDpD
7β-sheet
are
similarS1atand
at pD
observed
at pD 7features
are similar
at at pD 6atand
9 (Figures
S2). 6 and pD 9 (Figures S1 and S2).
remained unchanged in the td = 100–80,000 ns range. The behavior of all of the vibrational features
observed at pD 7 are similar at at pD 6 and pD 9 (Figures S1 and S2).
Figure 4. Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR) difference spectra of
Figure 4. Time Resolved
Step-Scan Fourier Transform
Infrared (TRS2 -FTIR) difference spectra of
1500–1760
cm−1 Resolved
region (tdStep-Scan
= 100–80,000
ns, Transform
4 cm−1 spectral
resolution)
of fully-reduced
ba3-CO
Figure
4.
Time
Fourier
Infrared
(TRS2-FTIR)
difference
spectra
ofba -CO
−
1
−
1
1500–1760
cm toregion
(td = 100–80,000
ns,
4 cmpulse
spectral
resolution)
of fully-reduced
3
subsequent
CO
photolysis
a 7 ns 532 ns,
nm laser
at pD resolution)
7.0.
−1 region
1500–1760 cm
(td =by
100–80,000
4 cm−1 spectral
of fully-reduced ba3-CO
subsequent to CO photolysis by a 7 ns 532 nm laser pulse at pD 7.0.
subsequent to CO photolysis by a 7 ns 532 nm laser pulse at pD 7.0.
Figure 5. Time Resolved Step-Scan Fourier Transform Infrared (TRS2-FTIR) difference spectra of
1690–1760
cm−1 Resolved
region (tdStep-Scan
= 100–80,000
ns, Transform
4 cm−1 spectral
resolution)
of fully-reduced
ba3-CO
Figure 5. Time
Fourier
Infrared
(TRS2-FTIR)
difference spectra
of
2 -FTIR)
Figuresubsequent
5. Time Resolved
Step-Scan
Fourier
Transform
Infrared
(TRS
difference
spectra of
to
CO
photolysis
by
a
7
ns
532
nm
laser
pulse
at
pH
6.0,
7.0,
and
9.0.
1690–1760 cm−1 region (td = 100–80,000 ns, 4 cm−1 spectral resolution) of fully-reduced ba3-CO
1 spectral resolution) of fully-reduced ba -CO
1690–1760
cm−1 toregion
(td = 100–80,000
cm−
3
subsequent
CO photolysis
by a 7 ns 532ns,
nm4laser
pulse
at pH 6.0, 7.0, and 9.0.
subsequent to CO photolysis by a 7 ns 532 nm laser pulse at pH 6.0, 7.0, and 9.0.
Int. J. Mol. Sci. 2016, 17, 1657
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Int. J. Mol. Sci. 2016, 17, 1657
6 of 10
3. Discussion
The Time Resolved Step-Scan FTIR data have already proven to be a very powerful for
3. Discussion
understanding the transient changes during protein action. The intensity/frequency changes
The Time
Step-Scan
FTIR
data is
have
to be a very
powerful
for
observed
in theResolved
TRS2-FTIR
difference
spectra
the already
result ofproven
the perturbation
induced
by the
understanding
the
transient
changes
during
protein
action.
The
intensity/frequency
changes
observed
photodissociation of CO from heme a3 and its subsequent binding to CuB and to the docking site,
2 -FTIR difference spectra is the result of the perturbation induced by the photodissociation
in the TRS
which
consists
of the ring-A propionate heme a3-D372-H2O moiety. The presence of
of CO from heme a3 and itsforms
subsequent
binding
to Cu
and to the
docking site,
which consists
of the
the
protonated/deprotonated
of D372
and of
theB ring-A
propionate,
in association
with
ring-A
propionate
heme
a
-D372-H
O
moiety.
The
presence
of
protonated/deprotonated
forms
of
3
2forms on the environment, indicates a protonic connectivity
dependence of their deprotonated
D372 andthe
of the
ring-A
in association
witha3the
dependence
of water
their deprotonated
formsand
on
between
D372,
the propionate,
ring-A propionate
of heme
, and
the pair of
molecules w941
the
environment,
indicates
a
protonic
connectivity
between
the
D372,
the
ring-A
propionate
of
heme
a3 ,
w927. To account for the presence of the observed pH/pD changes and the presence of protonated
and
the
pair
of
water
molecules
w941
and
w927.
To
account
for
the
presence
of
the
observed
pH/pD
and deprotonated forms, we present, in Figures 6 and 7, a scheme that includes the ring-A
changes and the pair
presence
of protonated
and
deprotonated
forms,phase,
we present,
in Figures
6 and 7,
propionate/D372
and w927/941.
In the
oxidative
or reductive
a proton
can be accepted
a
scheme
that
includes
the
ring-A
propionate/D372
pair
and
w927/941.
In
the
oxidative
or
reductive
by the ring-A propionate/D372 pair, which influences the release of a proton to the H2O pool
phase, a The
proton
can is
be not
accepted
by the ring-A
propionate/D372
pair,
influencesofthethe
release
[34–36].
w941
exchangeable;
however,
it contributes
towhich
the dynamics
ring
of
a
proton
to
the
H
O
pool
[34–36].
The
w941
is
not
exchangeable;
however,
it
contributes
the
2
A-D372-w927. In the scheme, states B and D, in which a single proton is shared between the to
D372
dynamics
of
the
ring
A-D372-w927.
In
the
scheme,
states
B
and
D,
in
which
a
single
proton
is
shared
and the heme a3 ring A-propionate, can accept a single proton. We propose that this is not operative
between
the D372(A)
andorthe
heme a3 ring
A-propionate,
can accept
single
proton.pH/pD
We propose
that
in
the protonated
deprotonated
(C)
states. We postulate
thatathe
observed
changes
in
this
is
not
operative
in
the
protonated
(A)
or
deprotonated
(C)
states.
We
postulate
that
the
observed
2
the TRS -FTIR data are due to the exchangeable w927 that provides the H-bonded connection in the
pH/pD
changes
theD372
TRS2 -FTIR
data are
due ato
the exchangeable w927 that provides the H-bonded
local
moieties
ofinthe
and ring-A
heme
3 propionate, and has activation energy for proton
connection
in the local
of the D372
and ring-A
heme
a3 propionate,
andduring
has activation
energy
motion
connecting
the moieties
ligand docking
site with
the water
pool.
Consequently,
the formation
for
proton
motion
connecting
the
ligand
docking
site
with
the
water
pool.
Consequently,
during
the
of the chemical and pumped H+, the H2O pool may serve as a primary acceptor for the water
+
formation of
thedata
chemical
and pumped
H , the
H2labile
O pool
may serve
a primary
the
molecules.
The
reported
here indicate
that
protons
and as
w927
are theacceptor
source for
of the
water
molecules.
The
data
reported
here
indicate
that
labile
protons
and
w927
are
the
source
of
the
observed changes to D372, whereas w946-w941-w942, with prop-A-D372 and His-376, form the
observed
changes
to D372,
whereas w946-w941-w942,
with prop-A-D372
and His-376,
the proton
proton
loading
site.
The observation
of H217O as a product
in the reduction
of the Oform
2 reaction near
17
loadingwhich
site. The
observation
of H2 O with
as a product
the reduction of the O2 reaction
H376,
H376,
is located
in a complex
several in
crystallographically-detected
H2O near
molecules,
which
is
located
in
a
complex
with
several
crystallographically-detected
H
O
molecules,
implies
2
implies a unique H2O exit pathway [34]. At this point it should be noted that
the mobility of H2Oa
unique H2 O
pathway [34].
At this
point
it should
be noted
thecrystallography.
mobility of H2 OThe
molecules
molecules
in exit
hydrophobic
cavities
makes
them
undetectable
bythat
X-ray
ability
in
hydrophobic
cavities
makes
them
undetectable
by
X-ray
crystallography.
The
ability
of
D
2 O to
of D2O to access the propionate-A-D372 moiety in the pD has been demonstrated by the observed
access
the
propionate-A-D372
moiety
in
the
pD
has
been
demonstrated
by
the
observed
changes
to
the
changes to the frequencies of the protonated forms of propionate-A and D372 in the pD 6–9 range.
frequencies
of thethat
protonated
formsinofconjunction
propionate-Awith
and D372
in the+ pD
It is suggested
that
It
is suggested
w941/w946
Prop-A-H
acts6–9
as range.
the Zundel
cation that
+ acts as the Zundel cation that forms the loading proton
w941/w946
in
conjunction
with
Prop-A-H
forms the loading proton site (Figures 6 and 7) [18]. In the absence of water molecules in the
site (Figurescenter
6 and 7)
In the absence
of water
molecules
in the
centerinwethe
conclude
binuclear
we[18].
conclude
that the
proton
loading
sitebinuclear
is located
hemethat
a3
the
proton
loading
site
is
located
in
the
heme
a
Prop-A-w946-w941w927-D372
moiety
[37].
3
Prop-A-w946-w941w927-D372 moiety [37].
Figure
The
binuclear
heme
B center and region of the heme a3 propionates of ba3
Figure 6.6.The
binuclear
heme
a3 -CuaB3-Cu
center
and region of the heme a3 propionates of ba3 oxidoreductase
oxidoreductase
from Thermus
thermophilus
illustrating
the residues
interest
yellow
and
from Thermus thermophilus
illustrating
the residues
of interest
[6]. Red, of
yellow
and[6].
blueRed,
colors
represent
blue
colors
represent
the
oxygen,
carbon
and
nitrogen
atoms,
respectively.
The
blue
sphere
the oxygen, carbon and nitrogen atoms, respectively. The blue sphere represents the CuB atom. In w941,
represents
Cuthe
B atom. In w941, w946 and w927 the red and blue colors represent the oxygen and
w946 and the
w927
red and blue colors represent the oxygen and hydrogen atoms, respectively.
hydrogen
atoms,
respectively.
highlighted
molecules
are conserved
in heme-copper
The highlighted water
moleculesThe
are conserved
in water
heme-copper
oxidases
[18].
oxidases [18].
Int. J. Mol. Sci. 2016, 17, 1657
Int. J. Mol. Sci. 2016, 17, 1657
7 of 10
7 of 10
Figure 7. Protonic connectivity between the ring-A propionate of heme a3, the D372, and the water
Figure 7. Protonic connectivity between the ring-A propionate of heme a3 , the D372, and the water
molecule w927. Blue, red and yellow colors represent protons, oxygen and carbon atoms,
molecule w927. Blue, red and yellow colors represent protons, oxygen and carbon atoms, respectively.
respectively. In states B and D, a single proton is shared between ring-A propionate of heme a3 and
In states B and D, a single proton is shared between ring-A propionate of heme a3 and D372, while in
D372, while in state A, ring-A propionate of heme a3 and D372 are protonated and in state C, ring-A
state A, ring-A propionate of heme a3 and D372 are protonated and in state C, ring-A propionate of
propionate
hemeare
a3 and
D372 are deprotonated.
heme a andofD372
deprotonated.
3
4. Materials and Methods
4. Materials and Methods
4.1.
4.1. Sample
Sample Preparation
Preparation
Cytochrome
Cytochrome ba
ba33 was
was isolated
isolated from
from Thermus
Thermus thermophilus
thermophilus HB8
HB8 cells
cells according
according to
to previously
previously
published
procedures.
The
ba
3 samples were placed in a desired 0.1 M buffer, pH/pD 7.0, HEPES
published procedures. The ba3 samples were placed in a desired 0.1 M buffer, pH/pD 7.0,
(4-(2-hydroxyethyl)
piperazine-1-ethanesulfonic
acid), pH/pDacid),
6.0, MES
hydrate6.0,
(2-(N-morpholino)
HEPES (4-(2-hydroxyethyl)
piperazine-1-ethanesulfonic
pH/pD
MES hydrate
ethanesulfonic
acid
hydrate, 4-morpholineethanesulfonic
acid) and acid)
pH/pD
CHES
(2-(N-morpholino)
ethanesulfonic
acid hydrate, 4-morpholineethanesulfonic
and 9.0,
pH/pD
9.0,
(2-(cyclohexylamino)ethanesulfonic
acid).
The
buffers
prepared
for
the
D
2O experiments were
CHES (2-(cyclohexylamino)ethanesulfonic acid). The buffers prepared for the D2 O experiments were
measured
concentration
of of
thethe
samples
waswas
determined
by
measured assuming
assuming pD
pD==pH(observed)
pH(observed)+ +0.4.
0.4.The
The
concentration
samples
determined
UV-VIS
measurements
performed
on a on
Lambda
25 UV-VIS
spectrometer
(Perkin
Elmer,Elmer,
Italy), using
by UV-VIS
measurements
performed
a Lambda
25 UV-VIS
spectrometer
(Perkin
Italy),
εusing
416,ox = 152 mM−1·cm−1, and
was
~700
μM.
The
fully-reduced
CO
bound
form
of
the
enzyme
3-CO)
−
1
−
1
ε416,ox = 152 mM ·cm , and was ~700 µM. The fully-reduced CO bound form of the(ba
enzyme
was
prepared
by using by
sodium
as a reducing
agent andagent
subsequently
exposed to
1 atm of
(ba3 -CO)
was prepared
usingdithionite
sodium dithionite
as a reducing
and subsequently
exposed
to
CO
under
anaerobic
conditions.
The
final
samples
were
transferred
to
an
air-tight,
sealed
FTIR
cell,
1 atm of CO under anaerobic conditions. The final samples were transferred to an air-tight, sealed
composed
by two CaFby
2 windows. The path length was 6 μm for the samples in H216O and 15 μm 16
FTIR cell, composed
two CaF2 windows. The path length was 6 µm for the samples in H2 for
O
the
samples
in
D
2O. The total enzyme volume used for the experiments was ~1.5 mL. The 12CO gas
and 15 µm for the samples in D2 O. The total enzyme volume used for the experiments was ~1.5 mL.
was
obtained
Messer (Germany)
and
D2O was and
purchased
from
Sigma-Aldrich
(Taufkirchen,
The 12
CO gas from
was obtained
from Messer
(Germany)
D2 O was
purchased
from Sigma-Aldrich
Germany).
(Taufkirchen, Germany).
4.2. ns-μs
ns-µs Time-Resolved
Time-Resolved Step-Scan FTIR Spectroscopy
The
Resolved Step-Scan
Step-Scan Fourier
Infrared measurements
measurements (TRS
(TRS22-FTIR)
The ns-μs
ns-µs Time
Time Resolved
Fourier Transform
Transform Infrared
-FTIR)
were
70 v
v FTIR
FTIR spectrometer
spectrometer (Bruker,
(Bruker, Karlsruhe,
Karlsruhe, Germany)
were performed
performed on
on aa Vertex
Vertex 70
Germany) fitted
fitted with
with aa
liquid
Mercury-Cadmium-Telluride (MCT)
(MCT) detector
detector (Figure
(Figure 8).
8). The
liquid nitrogen-cooled
nitrogen-cooled fast
fast Mercury-Cadmium-Telluride
The optical
optical
bench
and
thethe
sample
compartment
waswas
purged
withwith
N2. The
bench was
was kept
keptunder
undervacuum
vacuumconditions
conditions
and
sample
compartment
purged
N2 .
−1the
spectral
resolution
was was
4 cm4−1cm
and
resolution
was 100
spectral
rangerange
was
The spectral
resolution
andtime
the time
resolution
wasns.
100The
ns. covered
The covered
spectral
−1an
1200–2400
cm−1 cm
and
Infrared
filter 4200
US INC.,
Mountain
Lakes,
NJ, USA)
was
was 1200–2400
and
an Infrared
filter nm
4200(Spectrogon
nm (Spectrogon
US INC.,
Mountain
Lakes,
NJ, USA)
used.
The total
number
of time
slicesslices
was 800;
of50
them
werewere
takentaken
before
the laser
triggering
and
was used.
The total
number
of time
was 50
800;
of them
before
the laser
triggering
were
used
as
a
background
reference
for
the
data
analysis,
and
750
time
slices
were
taken
after
laser
and were used as a background reference for the data analysis, and 750 time slices were taken after
triggering.
A 532A nm
pulse
(second
harmonic)
from
a aContinuum
laser triggering.
532 laser
nm laser
pulse
(second
harmonic)
from
ContinuumMinilite
MiniliteNd-YAG
Nd-YAG laser
laser
(Continuum,
ns width,
width, 5–8
5–8 mJ/pulse,
mJ/pulse, 88 Hz)
(Continuum, San
San Jose,
Jose, CA,
CA, USA)
USA) (7
(7 ns
Hz) was
was used
used to
to photolyze
photolyze the
the heme
heme
aa33-CO
were
used
to to
direct
thethe
532532
nmnm
laser
beam
inside
the spectrometer
and
-COcomplex.
complex.Two
Twomirrors
mirrors
were
used
direct
laser
beam
inside
the spectrometer
through
the sample.
A Quantum
Composers
Plus
pulse
delay
and through
the sample.
A Quantum
Composers
Plus
pulse
delaygenerator,
generator,Model
Model9514
9514 (Quantum
(Quantum
Composers Inc., Bozeman Montana, MT, USA) was used to synchronize the spectrometer with the
laser. A total of 10 coadditions per retardation data point were collected and 35 measurements of
Int. J. Mol. Sci. 2016, 17, 1657
8 of 10
Composers
Inc.,17,Bozeman
Int.
J. Mol. Sci. 2016,
1657
Montana, MT, USA) was used to synchronize the spectrometer with
8 ofthe
10
laser. A total of 10 coadditions per retardation data point were collected and 35 measurements
of single-sided
interferograms
collected
and averaged
in to
order
to improve
S/N
single-sided
interferograms
werewere
collected
and averaged
in order
improve
the S/N the
ratio.
Theratio.
AC
The DC
AC measurements
and DC measurements
were
taken separately
the sameThe
sample.
The AC
was
and
were taken
separately
using the using
same sample.
AC signal
wassignal
amplified
amplified
byof
a factor
of two ausing
a Model
Low-Noise
preamplifier
(Stanford
researchsystems,
systems,
by
a factor
two using
Model
SR560SR560
Low-Noise
preamplifier
(Stanford
research
Sunnyvale, CA,
CA, USA).
USA). The
The phase
phase from
from DC
DC measurements
measurements was
was used
used for
for phase
phase correction
correction of
of the
the AC
AC
Sunnyvale,
−
1 phase resolution
−1
measurements.
The
Blackman–Harris
three-term
apodization
function
with
32-cm
measurements. The Blackman–Harris
function with 32-cm phase resolution
and the
the Mertz/No
Mertz/No Peak
Peak search
search phase
phase correction
correction algorithm
algorithm were used. Difference
Difference spectra
spectra were
were
and
calculated using
using ΔA
∆A ==−log
−log(I(I
calculated
S/I
).R ).
SR/I
Figure
setup
forfor
thethe
ns ns
Time-Resolved
Step-Scan
Fourier
Transform
Infrared
(ns
Figure 8.8. Experimental
Experimental
setup
Time-Resolved
Step-Scan
Fourier
Transform
Infrared
2−FTIR).
2
−
TRS
The
red
and
green
arrows
represent
the
infrared
beam
and
the
532
nm
photolysis
beam,
(ns TRS FTIR). The red and green arrows represent the infrared beam and the 532 nm photolysis
respectively.
beam, respectively.
Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/17/10/1657/s1.
Supplementary Materials:
Supplementary
materials
can be
found
www.mdpi.com/1422-0067/17/10/1657/s1.
Acknowledgments:
This work
was supported
by funds
from
theatCyprus
Research. Promotion Foundation to
C.V.
(TEXNOLOGIA/THEPIS/0609(BE)/05.
Acknowledgments:
This work was supported by funds from the Cyprus Research. Promotion Foundation to C.V.
(TEXNOLOGIA/THEPIS/0609(BE)/05.
Author Contributions: Antonis Nicolaides performed the experiments and analyzed the data; Tewfik Soulimane
Author Contributions:
Antonis Nicolaides
performed
thepaper.
experiments and analyzed the data; Tewfik Soulimane
provided
materials and Constantinos
Varotsis
wrote the
provided materials and Constantinos Varotsis wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
Conflicts of Interest: The authors declare no conflict of interest.
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