Electrochemically induced FT-IRdifference spectra after inhibition of the sample with cyanide allows assigning the formyl signals upon characteristic shifts of the mC¼O modes, which reflec
Trang 1Electrochemical, FT-IR and UV/VIS spectroscopic properties of the caa3
Petra Hellwig1, Tewfik Soulimane2* and Werner Ma¨ntele1
1
Institut fu¨r Biophysik der Johann-Wolfgang-Goethe-Universita¨t, Frankfurt/M., Germany;2Institut fu¨r Biochemie der Rheinisch-Westfa¨lischen-Technischen Hochschule, Aachen, Germany
The caa3-oxidase from Thermus thermophilus has been
studied with a combined electrochemical, UV/VIS and
Fourier-transform infrared (FT-IR) spectroscopic
approach In this oxidase the electron donor, cytochrome c,
is covalently bound to subunit II of the cytochrome c
oxidase Oxidative electrochemical redox titrations in the
visible spectral range yielded a midpoint potential of
)0.01 ± 0.01 V (vs Ag/AgCl/3MKCl, 0.218 V vs SHE¢)
for the heme c This potential differs for about 50 mV from
the midpoint potential of isolated cytochrome c, indicating
the possible shifts of the cytochrome c potential when bound
to cytochrome c oxidase For the signals where the hemes a
and a3contribute, three potentials,¼)0.075 V ± 0.01 V,
Em2¼ 0.04 V ± 0.01 V and Em3¼ 0.17 V ± 0.02 V
(0.133, 0.248 and 0.378 V vs SHE¢, respectively) could be
obtained Potential titrations after addition of the inhibitor
cyanide yielded a midpoint potential of)0.22 V ± 0.01 V
for heme a3-CN– and of Em2¼ 0.00 V ± 0.02 V and
Em3¼ 0.17 V ± 0.02 V for heme a ()0.012 V, 0.208 V
and 0.378 V vs SHE¢, respectively) The three phases of the
potential-dependent development of the difference signals
can be attributed to the cooperativity between the hemes a,
a3and the CuBcenter, showing typical behavior for
cyto-chrome c oxidases A stronger cooperativity of CuBis dis-cussed to reflect the modulation of the enzyme to the different key residues involved in proton pumping We thus studied the FT-IRspectroscopic properties of this enzyme to identify alternative protonatable sites The vibrational modes of a protonated aspartic or glutamic acid at
1714 cm)1concomitant with the reduced form of the protein can be identified, a mode which is not present for other cytochrome c oxidases Furthermore modes at positions characteristic for tyrosine vibrations have been identified Electrochemically induced FT-IRdifference spectra after inhibition of the sample with cyanide allows assigning the formyl signals upon characteristic shifts of the m(C¼O) modes, which reflect the high degree of similarity of heme a3
to other typical heme copper oxidases A comparison with previously studied cytochrome c oxidases is presented and
on this basis the contributions of the reorganization of the polypeptide backbone, of individual amino acids and of the hemes c, a and a3upon electron transfer to/from the redox active centers discussed
Keywords: caa3 oxidase; cytochrome c oxidase; UV/VIS-spectroscopy; FT-IR-UV/VIS-spectroscopy; Thermus thermophilus
Cytochrome c oxidase is the terminal enzyme of the
respiratory chain in mitochondria and many prokaryotes
As an integral membrane protein it catalyzes the reduction
of dioxygen to water using electrons from cytochrome c
Four redox-active sites are involved in the electron transfer
Electrons from cytochrome c are first transferred to a
homobinuclear copper A site (CuA) and then subsequently
to heme a, and to heme a3, which is located close to copper
B (CuB), forming a heterobinuclear metal center where
oxygen is reduced to water Protons needed for water
formation are taken up from the cytosolic side in bacterial
membranes or from the matrix side in mitochondria The proton consumption and the coupled translocation of
n H+/e– across the membrane contribute to the proton gradient needed to synthesize ATP
Two pathways have been proposed to serve for consumed and pumped protons on the basis of site-directed mutagen-esis [1,2] and later using the crystal structures [3–5] These pathways are highly conserved among most studied cyto-chrome oxidases [2,6] However, cytocyto-chrome oxidases have been reported that lack amino acids disputed to be essential
in proton translocation In the case of caa3-oxidases from
T thermophilus, for example, as well as from Rhodothermus marinus, the amino acid Glu278 (numbering for Paracoccus denitrificans), which is proposed to pass protons in the D-pathway to the binuclear center, is missing, but proton-pumping activity is observed [3,7–9] A highly conserved Tyr–Ser couple was suggested to replace Glu278 [9] In the current understanding, two pathways are necessary for the catalytic activity, but different residues may be involved In
an important step for the understanding of the essentials for cytochrome c oxidase activity and coupled proton pump-ing, the crystal structure of the aberrant ba3-oxidase from
T thermophiluswas determined [10] and alternative path-ways discussed
Correspondance to P Hellwig, Institut fu¨r Biophysik der
Johann-Wolfgang-Goethe-Universita¨t, Theodor-Stern-Kai 7 Haus 74,
60590 Frankfurt/M., Germany.
Fax: + 49 69 6301 5838, Tel.: + 49 69 6301 4227,
E-mail: hellwig@biophysik.uni-frankfurt.de
Abbreviations: FT-IR, Fourier-transform infrared; SHE¢, standard
hydrogen electrode; TMPD,
N,N,N¢,N¢-tetramethyl-p-phenylenedi-amine dihydrochloride
*Present address: Paul Scherrer Institut, Structural Biology Group,
5232-CH, Villigen PSI, Switzerland.
(Received 13 March 2002, revised 6 August 2002, accepted 14 August 2002)
Trang 2Under restricted O2 supply, the thermophilic Gram
negative bacterium T thermophilus expresses two different
cytochrome c oxidases The heme types incorporated
belong to the caa3- and ba3-type cytochrome c oxidases,
respectively The caa3-oxidase contains analogous central
subunits and catalytic entity to the mitochondrial aa3
-oxidases, however, including a covalently bound type-c
heme [11] This is currently only found in a few bacteria
[9,12] Recent results showed that the enzyme is made of two
fusion proteins The smaller protein consists of a typical
oxidase subunit II sequence, which provides the
homonu-clear CuA binding site and is fused to a cytochrome c
domain [11,13] The larger protein is a fusion product of
subunit I, that has the hemes a, a3and the CuBsites, and
subunit III [8,12,13] The heme c center in the caa3-oxidase is
proposed to serve as the first electron acceptor from a bc1
complex [14] We note, however, that no bc1complex has
yet been described for T thermophilus No activity was
detected for a reaction with soluble horse heart
cyto-chrome c, c552 from T thermophilus and yeast iso1
cyto-chrome c, which serve as natural reductands for
cytochrome c oxidases, but a reduction can be noted for
nonphysiological reducing agents such as
N,N,N¢,N¢-tetra-methyl-p-phenylenediamine dihydrochloride (TMPD) [15]
The caa3-oxidase may be regarded as an integrated version
of the noncovalent redox complex between cytochrome c
and cytochrome c oxidase
Previous reports on the caa3-oxidase from T
thermophi-lusconcluded that this enzyme is a typical member of the
heme copper oxidase family [12], with the exception of a
different titrimetric behavior of the redox centers in the
electron transfer [16] and the lack of some key residues as
mentioned above In this work we study the electrochemical,
UV-VIS and FT-IRspectroscopic properties of the caa3
-oxidase from T thermophilus in the presence and absence of
cyanide, and compare the observed properties to previous
reports on members of the heme copper oxidase family such
as cytochrome c oxidase from bovine heart and P
denitri-ficans, and the aberrant ba3-oxidase from T thermophilus
M A T E R I A L S A N D M E T H O D S
Sample preparation
The caa3-type cytochrome c oxidase from T thermophilus
was prepared as described previously in Gerscher et al [17]
For electrochemistry the sample was solubilized in
n-decyl-b-D-maltopyranoside, 100 mM phosphate buffer
(pH 7) containing 100 mM KCl and concentrated to
approximately 0.5 mMusing Microcon ultrafiltration cells
(Millipore) Exchange of H2O against D2O was performed
by repeatedly concentrating the enzyme and rediluting it in a
D2O phosphate-buffer H/D exchange was better than 80%
as judged from the shift of the amide-II mode at 1550 cm)1
in the FT-IRabsorbance spectra (data not shown) For
inhibition with cyanide, samples were diluted with 500 lL
of 100 mM phosphate buffer containing 20 mM KCN
(pH 7), incubated overnight and reconcentrated to 0.5 mM
Electrochemistry
The ultra-thin layer spectroelectrochemical cell for the UV/
VIS and IRwas used as described previously [18] Sufficient
transmission in the 1800–1000 cm)1 range, even in the region of strong water absorbance around 1645 cm)1, was achieved with the cell pathlength set to 6–8 lm The gold grid working electrode was chemically modified by a 2-mM
cysteamine solution as reported previously [19] In order to accelerate the redox reaction, 15 different mediators were added as reported by Hellwig et al [19], with the exception
of K4[Fe(CN)6], to a total concentration of 40 lMeach At this concentration, and with the pathlength below 10 lm,
no spectral contributions from the mediators in the VIS and IRrange could be detected in control experiments with samples lacking the protein, except for the PO modes of the phosphate buffer between 1200 cm)1and 1000 cm)1 As a supporting electrolyte, 100 mM KCl was added Approxi-mately 5–6 lL of the protein solution were sufficient to fill the spectroelectrochemical cell Potentials quoted with the data refer to the Ag/AgCl/3M KCl reference electrode, adding + 208 mV for SHE¢ (pH 7) potentials Midpoint potentials are described for both electrode types
Spectroscopy FT-IRand UV/VIS difference spectra as a function of the applied potential were obtained simultaneously from the same sample with a setup combining an IRbeam from the interferometer (modified IFS 25, Bruker, Germany) for the 4000–1000 cm)1 range and a dispersive spectrometer for the 400–900 nm range as reported previously [18] First, the protein was equilibrated with an initial potential at the electrode, and single beam spectra in the VIS and IRrange were recorded A potential step to the final potential was then applied, and single beam spectra of this state were again recorded after equilibration Difference spectra as presented here were then calculated from the two single beam spectra with the initial single beam spectrum taken as
a reference No smoothing or deconvolution procedures were applied The equilibration process for each potential applied was followed by monitoring the electrode current and by successively recording spectra in the visible range until no further changes were observed The equilibration generally took less than 8 min under the conditions reported (protein concentration, electrode modification, mediators) for the full potential step from )0.5 V to 0.5 V and to selected potentials Typically, 128 interferograms at 4 cm)1 resolution were coadded for each single beam IRspectrum and Fourier-transformed using triangular apodization Differences in sample concentration and pathlength were taken into account by normalizing the FT-IRdifference spectra on the difference signal of the sample in the UV/VIS
at 602 nm
Redox titrations The redox-dependent absorbance changes of the caa3 -oxidase from T thermophilus were studied performing electrochemical redox titrations in the UV/VIS spectral range The redox titrations were performed by stepwise setting the potential and recording the spectrum after sufficient equilibration Typically data were recorded at steps of 30–50 mV All measurements were performed at
5C The midpoint potentials Em and the number n of transferred electrons were obtained by adjusting a calcula-ted Nernst curve to the measured absorbance change at a
Trang 3single wavelength by an interactive fit All parameters have
to be adjusted manually until the theoretical Nernst curve
and the measured data match well (fit by eye)
R E S U L T S A N D D I S C U S S I O N
UV/VIS difference spectra
Figure 1A shows the oxidized-minus-reduced UV/VIS
difference spectra of the caa3-oxidase from T thermophilus
obtained for a potential step from)0.5 V to 0.5 V (vs Ag/
AgCl/3MKCl) In the oxidized-minus-reduced spectra the
positive signals correlate with the oxidized and the negative
signals with the reduced form of the enzyme For the
reduced form, the Soret band can be observed at 415 and
442 nm, and for the oxidized form at 403 and 422 nm The
b–band can be seen at 517 nm and the a–band at 547 nm
and 603 nm
The difference signals that can be observed between 400
and 700 nm include the contributions of the hemes c, a and
a3 The difference signals observed at 403, 415, 517 and
547 nm are characteristic for heme c In electrochemically
induced difference spectra of horse heart cytochrome c the
Soret band was reported to absorb at 418 nm, the b-band at
520 nm and the a–band at 550 nm [18] The deviations of
approximately 3 nm between the signals of horse heart
cytochrome c and the heme c in the caa3-oxidase from
T thermophiluscan be attributed to the different environ-ment of the heme centers The difference signals observed at
442 nm and 603 nm can be assigned to the contributions of the hemes a and a3, with the position of the a-band showing
a downshift in relation to bovine heart oxidase
In Fig 1B the oxidized-minus-reduced UV/VIS differ-ence spectra of the caa3-oxidase poisoned with cyanide obtained for a potential step from)0.5 V to 0.5 V (solid line) and from)0.5–0.05 V (dotted line) can be seen The a-band shifts to 599 nm upon binding of cyanide to heme a3 This shift is reflected clearly in the critical potential step from)0.5 to )0.05 V, where mainly heme a3and heme
ccontribute Addition of cyanide was used in the electro-chemically induced FT-IRdifference spectra to separate contributions of heme a3
UV/VIS redox titrations
In Fig 2A the potential dependent development of the a–band from heme c at 548 nm in an oxidative titration can
be seen (filled circles) The theoretical Nernst fit described
in Materials and methods yields a midpoint potential of
Em¼)0.01 ± 0.01 V (vs Ag/AgCl, and 0.218 V vs SHE¢) for heme c This value was also reported by Yoshida and Fee [16] The midpoint potential of soluble horse heart cytochrome c is 0.048 V (0.256 V vs SHE¢, as obtained with the same method as described here and in [18]); other cytochrome c types show a close midpoint potential The midpoint potential of the cytochrome c in the cyto-chrome c–cytocyto-chrome c oxidase complex is unknown and may be the origin for this downshift Alternatively, the electron transfer directly from the bc1complex, as suggested for a possible mechanism, could require a lower potential Figure 2B (filled circles) shows the potential-dependent development of the difference signal at 443 nm from)0.4 V
to + 0.6 V Three phases can be clearly discriminated The theoretical Nernst fit yields midpoint potentials
Em1¼)0.075 ± 0.01 V, Em2¼ 0.04 ± 0.01 V and
Em3¼ 0.17 ± 0.02 V for an n value of 0.9–1 (these values correspond to 0.133, 0.248 and 0.378 V vs SHE¢, respect-ively) As reported previously, cytochrome c oxidase from bovine heart shows a complex titration curve reflecting the cooperative interactions between the hemes a and a3, and the CuB center [20–22] For cytochrome c oxidase from bovine heart Emvalues near 200, 260 and 340 mV have been reported [23–25], and analyzed in detail by several groups [20–22] For the caa3-oxidase we observe a noteworthy pronounced phase at 40 mV (248 mV vs SHE¢) but essentially a similar titrimetric behavior In a previously reported potential titration of the caa3-oxidase from
T thermophilus, Yoshida and Fee [16] describe a compar-able potential and n-value for the first step, but report a second step with an n-value of two at approximately
160 mV for CuB and heme a3 On this basis, CuB and heme a3 have been suggested to act as a two-electron acceptor [12] in contrast to bovine heart oxidase, where subsequent one-electron transfer is reported The three phasic curve, with a step of n¼ 1 for each step as found here, shows a significantly more comparable titrimetric behavior in comparison to other typical oxidases, but in contrast to the work by Yoshida and Fee [16] The small difference to other oxidases found here, reflected in the stronger second step at 40 mV, may be attributed to the
Fig 1 Oxidized-minus-reduced UV/VIS difference spectra of the caa 3
-oxidase from T thermophilus Results obtained for a potential step
from )0.5 V to 0.5 V (vs Ag/AgCl/3 M KCl) in the absence (A) or the
presence (B, solid line) of cyanide, and for a potential step from )0.5 to
0.05 V in the presence of cyanide (B, dotted line).
Trang 4covalently attached heme c or to a generally changed cooperativity of the other redox centers
In order to discriminate the contributions of the cofac-tors, inhibitors uncoupling or changing the cooperativity can be used Addition of cyanide strongly shifts the heme a3 potential and thus uncouples or changes cooperativity in the binuclear center [20,22,28] The shifts of the titration curve upon addition of cyanide can be seen in Fig 2B (open circles) The theoretical Nernst fit yielded midpoint poten-tials Em1¼)0.22 ± 0.01 V, Em2¼ 0.00 ± 0.01 V and
Em3¼ 0.17 ± 0.02 V (the values correspond to )0.012, 0.208 and 0.378 V vs SHE¢, respectively) The potential at )220 mV can be attributed to the heme a3–CN–signal, this shift reflecting the characteristic behavior of cytochrome c oxidases heme a is expected to contribute with two steps, reflecting the remaining interactions with CuB As seen in Fig 2B (open circles), a further interaction is observed, presenting additional evidence for a different cooperativity
of the redox centers
Whereas in the Soret Band heme a and heme a3 contribute almost equally, the heme a contribution domin-ates the a-band Figure 2C shows a comparison of the potential-dependent development of the modes at 599 nm (triangles) and 442 nm (open circles) in the presence of cyanide As seen for the curve that represents the titration curve at 602 nm (triangles) a smaller ratio is present for the potential at)220 mV than for the titration curve measured
at 442 nm in the same conditions, supporting the assign-ment to heme a3
Heme c, however, shows a relatively small difference in midpoint potential of 15 mV in the presence of cyanide (Fig 2A, empty circles) and thus does not indicate a noteworthy cooperativity between heme a3and the heme c centers Heme c can be ruled out as origin for the distinct second phase at 40 mV in the titration curve for the hemes a and a3 It may be suggested that the different cooperativity,
as well as the lower heme a3 potential, is necessary to compensate the differences caused by the presence of different key residues in the D-pathway, since the potentials are assumed to be crucial for the coupling of electron and proton transfer Interestingly, for the caa3-oxidase from
R marinuswhich also lacks the above-mentioned Glu278 side chain, downshifted potentials for the hemes a and a3
have been described, although the cooperativity is not discussed [9] In the case of the aberrant ba3-cytochrome c oxidase from T thermophilus, a completely different titri-metric behavior was observed [26], also indicating that the midpoint potentials and cooperativity are adapted to the varying proton path residues To emphasize this suggestion further, future comparative studies on the varying oxidases could be performed
FT-IR difference spectra Figure 3 shows the oxidized-minus-reduced FT-IRdiffer-ence spectra of the caa3-oxidase from T thermophilus for a potential step from)0.5 V to 0.5 V equilibrated in H2O (A) and D2O buffer (B) Numerous distinct sharp bands appear throughout the spectrum, with half-widths typically below 5–10 cm)1 The noise level in these difference spectra can be estimated at approximately 25–50· 10)6absorbance units
at frequencies above 1750 cm)1, where no signals appear Only in regions of strong absorbance of the sample, such as
Fig 2 Potential dependent development of the hemes in the caa 3
-oxid-ase from T thermophilus Heme c was monitored at 548 nm in the
absence (filled circles) and presence (open circles) of cyanide (A) and a
midpoint potential of )0.01 V ± 0.01 V (vs Ag/AgCl/3 M KCl or
0.218 V vs SHE¢) was obtained by a theoretical Nernst fit (solid line).
The hemes a and a 3 were monitored at 442 nm in the absence
(filled circles) and presence (open circles) of cyanide Midpoint
poten-tials of Em 1 ¼ )0.075 V ± 0.01 V, Em 2 ¼ 0.04 V ± 0.01 V and
Em 3 ¼ 0.17 V ± 0.02 V were determined (these values correspond to
0.133 V, 0.248 V and 0.378 V vs SHE¢, respectively) After addition of
the inhibitor cyanide (open circles) a midpoint potential of
)0.22 V ± 0.01 V for heme a 3 -CN – and of Em 2 ¼ 0.00 V ± 0.02 V
and Em 3 ¼ 0.17 V ± 0.02 V for heme a can be seen (the values
correspond to )0.012 V, 0.208 V and 0.378 V vs SHE¢, respectively).
(B) Comparison of the potential dependent development of the modes
at 599 nm (triangles) and 442 nm (open circles) in the presence of
cyanide The theoretical Nernst fit is shown as a solid line (C).
Trang 5around 1650 cm)1(water OH-bending mode and amide-I
C¼O mode), was the noise level slightly higher, though
never exceeding 10)4absorbance units
The entirety of difference signals represent the total
molecular changes concomitant with the redox reactions
In the electrochemically induced FT-IRdifference spectra,
contributions from the porphyrin ring, the heme
propio-nates and the vinyl substituent can be expected, originating
from heme c, with contributions from the formyl groups
and from the geranyl side chain expected from heme a and
a3 In addition to the signals of the hemes, the
reorgan-ization of the polypeptide backbone and amino acid side
chains occurring upon electron transfer of the five redox
active centers heme c, a, a3, CuA and CuB, and coupled
processes such as proton transfer can be expected to
manifest themselves in the spectra In the following
paragraph the difference spectra will be described and
discussed Tentative assignments will be presented on the
basis of the comparison to IRand Raman spectra of heme
model compounds, other oxidases, spectra of isolated
amino acids as model compounds and information on
contributions from the secondary structure from infrared
absorbance spectra and the deconvolution of the amide-I
region
A particular problem of the assignment in the difference
spectra is the superposition of signals from different
constituents of the oxidase, which can lead to the possibility
of multi component bands and may present ambiguities in the assignment A spectral region particularly susceptible for overlapping bands is the amide-I range Although in this range (approx 1690–1610 cm)1) typical contributions from secondary structure elements are expected, and signals may point to the alterations of local protein conformation in the course of the redox reaction, we keep in mind that the heme formyl mode also contributes here, as well as specific modes from amino acid side chains For a clearer discrimination of these overlapped bands, we used deuteration of the sample and FT-IRspectra studies in the presence of the inhibitor cyanide support our tentative assignments
Tentative assignments of difference signals
to polypeptide backbone modes Amide-I signals are predominantly caused by the C¼O stretching vibration of the polypeptide backbone For different secondary structure elements, characteristic absorptions can be distinguished In the electrochemically induced FT-IRdifference spectra, contributions from the reorganization of the polypeptide backbone upon electron transfer to and from the cofactors can be expected, and a partial attribution of the signals observed in the amide-I region (1690–1610 cm)1) to amide-I modes is conceivable The different secondary structure elements show a different sensitivity to H/D exchange In Fig 3A strong positive signals can be observed at 1694 cm)1, 1684 cm)1,
1674 cm)1and 1646 cm)1, and prominent negative differ-ence modes are present at 1660 cm)1, 1626 cm)1 and
1614 cm)1 After H/D exchange (Fig 3B) the increase of the signal at 1696 cm)1and 1626 cm)1can be observed A clear shift from 1634 cm)1to 1658 cm)1and to 1650 cm)1
is visible The modes involved in the signals at 1660 cm)1 contribute in the range characteristic for the absorbance from a–helical secondary structure elements However, absorbance changes induced by the reorganization of a-helical secondary structure elements are expected to show very small shifts after H/D exchange at most (2–10 cm)1) and an assignment is unlikely Unordered secondary structure elements show a higher sensitivity to H/D exchange and also contribute in this spectral range An involvement of reorganizations of b–sheet secondary struc-ture elements is possible for the difference in the signals at 1696–1674 cm)1, and at 1646–1620 cm)1 However, con-clusive assignments are difficult in this spectral range where difference bands strongly overlap
In the amide-II region (1575 cm)1)1480 cm)1), strong negative signals at 1546 cm)1 and 1516 cm)1 as well as positive signals at 1562 cm)1 and 1498 cm)1 can be observed An assignment of the signals in the amide-II region in the electrochemically induced FT-IRdifference spectrum of the caa3-oxidase from T thermophilus to amide-II modes, however, appears less probable since little
or no shift for H/D exchange is observed
Assignment of heme vibrational modes Formyl substituent.The C¼O bond of the formyl group at the porphyrin ring of hemes a and a3 can be expected to contribute between 1680 cm)1and 1606 cm)1, depending
on hydrogen bonding with neighboring amino acids The formyl substituent of heme a is predicted to form a
Fig 3 Oxidized-minus-reduced FT-IR difference spectra of the caa 3
-oxidase from T thermophilus Results obtained for a potential step
from )0.5 V to 0.5 V (vs Ag/AgCl/3 M KCl) equilibrated in H 2 O (A)
and D 2 O buffer (B).
Trang 6hydrogen bond with an arginine side chain, while the same
substituent for heme a3appears to be free from H-bonding
to nearby amino acid residues Different frequencies for the
m(C¼O) stretching mode of the formyl group can thus be
expected In resonance Raman spectra of caa3-oxidase from
T thermophilusa signal at 1611 cm)1could be assigned to
the m(C¼O) CHO from reduced heme a and at 1649 cm)1
to the oxidized form [17] Comparable signals can be
observed here at 1650 cm)1for the oxidized form and at
1608 cm)1for the reduced form
The resonance Raman spectroscopic characterization of
the m(C¼O) CHO vibrational modes for heme a3 showed
the presence of a mode for the reduced form at 1664 cm)1
and for the oxidized form at 1673 cm)1 [17] In the
electrochemically induced FT-IRdifference spectra in
Fig 3A corresponding bands can be seen at 1678 cm)1
(oxidized form) and 1660 cm)1 (reduced form) These
modes have been previously attributed to the formyl side
chain from cytochrome c oxidase from bovine heart [27–29]
reported to be sensitive to CN–binding in a characteristic
way [27] In Fig 4A the spectra in the presence of cyanide
clearly reflect a shift of the mode at 1678–1668 cm)1and of
the mode at 1660–1652 cm)1, supporting the assignment to
the heme a3formyl mode In a direct comparison of these
vibrational modes to those observed for the cytochrome c
oxidase from P denitrificans, an analog environment of the
protein site of the heme a3formyl group in the absence and
presence of cyanide can be concluded
Porphyrin ring vibrations Porphyrin ring vibrations of
the heme centers, for example the CaCm vibration (m37) or
the CbCb vibration (m38) can be expected between
1620 cm)1 and 1500 cm)1 and are involved in the
electrochemically induced FT-IRdifference spectra shown
in Figs 3 and 4 On the basis of recent resonance Raman work on the caa3-oxidase from T thermophilus [17] and a direct comparison to resonance Raman and FT-IR investigations on other oxidases [17,23,30] tentative as-signments of porphyrin ring vibrations have been made and summarized in Table 1 As described previously by Gerscher et al [17], the spectroscopic properties of the hemes a and a3 sites are comparable to other typical aa3 oxidases Additionally contributions of the heme c center can be expected
It is clear that, in addition to the modes assigned here, further C–C or C–N vibrations of the porphyrin ring (m4,m39) will contribute to the electrochemically induced FT-IRdifference spectra However, we refrain from dis-cussing and assigning these modes on the basis of the data presented here, in spite of the fact that bands in the difference spectra are observed in the region where the modes were attributed
The vibrational modes of bound cyanide Electrochemically induced FT-IRdifference spectra of cyanide bound to heme a3 were characterized to specify possible variations of the binuclear center in direct compari-son to other oxidases, as for example the cytochrome c oxidase from P denitrificans In the spectral range from 2200–2000 cm)1contributions from cyanide ligand bound
to heme a3can be expected In the inset in Fig 4 a strong positive mode can be seen at 2148 cm)1 and a negative signal at 2040 cm)1for a potential step from)0.5 to 0.5 V (unbroken line) and for)0.5 to 0.05 V (dotted line) A small mode at 2092 cm)1 can be seen in the reduced state, indicating the presence of free cyanide upon reduction The band at 2148 cm)1may be assigned to the C–N stretching of the Fe3+–C¼N–CuB2+entity (also a Fe3+–C¼N–CuB2+– C¼N structure was discussed) based on the spectral shifts observed for isotopically labeled cyanide complexes [31–33] Correspondingly, the band at around 2040 cm)1 of the reduced form could be attributed tentatively to the C–N stretching of a nonbridging cyanide ligand of heme a3 In the spectra observed for the critical potential step from)0.5
to 0.05 V, where the reorganization upon oxidation of the inhibited heme a3center is induced, the cyanide modes are completely developed To allow the above-mentioned bridged structure to be present, CuB must be oxidized at this potential in the presence of cyanide, since the contri-bution of the unbridged Fea33+–CN–structure was repor-ted to be observable at 2132 cm)1
The position of the cynide vibrational modes are essentially identical to the ones observed for bovine heart oxidase [31] and from P denitrificans (Hellwig et al unpublished results) reflecting a close environment and ligand binding properties of the binuclear heme a3–CuB center
Identification of protonable sites Aspartic and Glutamic acid side chains The m(C¼O) mode of protonated aspartic and glutamic side chains absorb typically above 1710 cm)1, the exact absorption depending on the hydrogen bonding A negative mode is present at 1714 cm)1, which shifts to 1716 cm)1upon H/D exchange This indicates the presence of a protonated
Fig 4 Oxidized-minus-reduced FT-IR difference spectra of the caa 3
-oxidase from T thermophilus Results obtained for a potential step
from )0.5 V to 0.5 V (vs Ag/AgCl/3 M KCl) in the absence (dotted
line) and presence of cyanide (solid line) The inset shows an enlarged
view of the spectral region characteristic for the CN modes from 2200
to 2000 cm)1.
Trang 7aspartic or glutamic acid side chain in the reduced state of
the enzyme, which is coordinated with a strong hydrogen
bond A small very broad positive mode is found at
1744 cm)1, indicating the possible contribution of a
surface group in the oxidized form of the protein This
mode shifts to 1740 cm)1upon H/D exchange The signal
may originate from heme c reduction, but also reflect a
distinct protonable site in subunit I, involved in a different
proton pathway
For the cytochrome c oxidase from P denitrificans
difference modes at 1746 cm)1and 1734 cm)1have been
observed and attributed to Glu278 [19,34], and to the
equivalent residues in the cytochrome bo3 quinol oxidase
from E coli [35,36], a residue which lacks the caa3-oxidase
as mentioned above Correspondingly no analogous
con-tribution can be seen here
Tyrosines Pereira et al [9] recently suggested a Tyr–Ser
motif, conserved in several of the cytochrome c oxidases
which lack the above-mentioned Glu278 residue, to be involved in proton pumping For tyrosine side chains, the
m19(CC) ring mode for the protonated form of tyrosines is proposed to absorb with an strong signal at 1518 cm)1and for the deprotonated form at 1496–1486 cm)1 [37,38] Clearly a difference mode in the spectral region character-istic for the protonated form can be seen concomitant with the reduced state at 1515 cm)1and the mode typical for the deprotonated form at 1498 cm)1, indicating the protonation
of a tyrosine residue with the reduction of the protein Also the m7¢a(CO) and d(COH) of tyrosine side chains expected at approximately 1265 cm)1and 1245 cm)1respective to the protonation state, can be seen We note that these assignments are highly tentative until this data can be supported by combining the technique with site-directed mutants or labeled compounds
Heme propionates.The heme propionates at the hemes a and a are discussed to be involved in proton translocation
Table 1 Summary of tentative assignments of the vibrational modes involved in the electrochemically induced FT-IR difference spectra of the caa 3 -oxidase from T thermophilus.
caa 3
FT-IR
caa 3
RR [17] Redox state Tentative assignments
Comparable modes for
aa 3 P denitrificans [30]
1744 ox m(C¼O) Glu278 for P denitrificans 1746
– red m(C¼O) Glu278 for P denitrificans 1734
1678 1674 ox m(C¼O) CHO heme a 3
m(C¼O) heme propionates m(C¼O) Asn/Gln m(CN 3 H 5 ) as Arg
1676
1660 1665 red m(C¼O) CHO heme a 3
amide-I (a-helical) m(CN 3 H 5 ) as Arg
1662
1650 1650 ox amide-I (a-helical)
m(C¼O) CHO heme a
1656/1644
m(CN 3 H 5 )sArg amide-I (b-sheet)
1632
1602 1604 ox m 37 heme a
(m 8a / 8b (CC) Tyr-OH)
1562 1567/1558 ox m 38x heme a/a 3
m(COO–)asheme propionate m(COO–)asAsp/Glu m(CC) ring Tyr-O –
1564
1530 1532 red m 38y heme a 3
m(COO – ) as heme a 3 propionate
1528
1498 – ox m 19 (CC) Tyr-O–
1265 – ox m 7¢a (CO) Tyr-O–
1245 – red m7¢a(CO) and d(COH) Tyr-OH
Trang 8during catalytic cycle [39] The contributions of protonated
and ionized carboxylic groups of the heme propionates
for the cytochrome c oxidase from P denitrificans were
assigned by specific13C-labelling of the carboxylic groups
of the four heme propionates and site-directed mutagenesis
in the vicinity of its site [40,41] A signal at 1676 cm)1
was attributed to contributions of protonated carboxylic
groups Difference bands at 1570 cm)1and 1538 cm)1were
assigned to the m(COO–)asvibration and at 1380 cm)1to the
m(COO–)s vibration of deprotonated heme propionates
[40,41] Signals at comparable positions can be seen in the
spectra shown for the caa3-oxidase In addition the
contributions of the heme propionates of the heme c can
be expected in a comparable spectral region
C O N C L U S I O N S
The superfamily of heme copper oxidases includes a
number of enzymes, which show deviations to the centrally
discussed oxidases The study of these aberrant systems is
important to understand the principles of these enzymes
The cytochrome c oxidase from T thermophilus studied in
this work lacks a key residue in the so-called D-pathway,
although it does show proton pumping activity A Tyr–Ser
motif was previously suggested to replace the absent acidic
group in several oxidases [9] In this work we could
observe modes at characteristic positions for the
protona-tion of a tyrosine side chain concomitant with the
reduction of the enzyme A further alternative protonable
site could be seen at 1714 cm)1 This mode is observable in
the spectral range characteristic for protonated aspartic or
glutamic acid side chains and reflects its protonation with
the reduction of the protein We note that these
assign-ments are tentative and can be supported by the
combi-nation with site-directed mutagenesis or labeling
experiments
Interestingly potential titrations of the enzyme show a
slightly different redox-dependent behavior It may be
suggested that the stronger cooperativity displays the
modulation of the enzyme to the different residues involved
This is in line with the observation reported previously for
the caa3-oxidase from R marinus and ba3-oxidase from
T thermophilus[9,26] An influence of the attached heme c
center is less likely on the basis of titrations in the presence
of cyanide
The electrochemically induced FT-IRdifference spectra
also include the contributions of the heme centers c, a and
a3 Together with the spectra in the presence of cyanide and
in direct comparison to previous resonance Raman data it
can be concluded that the hemes a and a3 have a similar
structural environment comparised with bovine heart and
P denitrificansoxidases [17]
In summary, the caa3-cytochrome c oxidase shows the
characteristic complex redox behavior and shows several
structural properties of a typical cytochrome c oxidase
The presence of the two proton pathways is discussed as
one essential of the cytochrome c oxidase The so-called
D-pathway seems to involve different residues here, most
likely a tyrosine and an aspartic or glutamic acid It may
also be suggested that the complex redox behavior is crucial
for the cytochrome c oxidase mechanism, with some
variations, as observed here
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