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^{*} To whom correspondence should be addressed.

Received September 13, 2013; Revision received September 20, 2013

This paper presents a new experimental approach for determining the individual optical characteristics of reduced hemeain bovine heart cytochromecoxidase starting from a small selective shift of the hemeaabsorption spectrum induced by calcium ions. The difference spectrum induced by Ca^{2+}corresponds actually to a first derivative (differential) of the hemea^{2+}absolute absorption spectrum. Such an absolute spectrum was obtained for the mixed-valence cyanide complex of cytochrome oxidase (a^{2+}a_{3}^{3+}-CN) and was subsequently used as a basis spectrum for further procession and modeling. The individual absorption spectrum of the reduced hemeain the Soret region was reconstructed as the integral of the difference spectrum induced by addition of Ca^{2+}. The spectrum of hemea^{2+}in the Soret region obtained in this way is characterized by a peak with a maximum at 447 nm and half-width of 17 nm and can be decomposed into two Gaussians with maxima at 442 and 451 nm and half-widths of ~10 nm (589 cm^{–1}) corresponding to the perpendicularly oriented electronic π→π* transitionsB_{0x}andB_{0y}in the porphyrin ring. The reconstructed spectrum in the Soret band differs significantly from the “classical” absorption spectrum of hemea^{2+}originally described by Vanneste (Vanneste, W. H. (1966)Biochemistry,65, 838-848). The differences indicate that the overall γ-band of hemea^{2+}in cytochrome oxidase contains in addition to theB_{0x}andB_{0y}transitions extra components that are not sensitive to calcium ions, or, alternatively, that the Vanneste’s spectrum of hemea^{2+ }contains significant contribution from hemea_{3}^{2+}. The reconstructed absorption band of hemea^{2+}in the α-band with maximum at 605 nm and half-width of 18 nm (850 cm^{–1}) corresponds most likely to the individualQ_{0y}transition of hemea, whereas theQ_{0x}transition contributes only weakly to the spectrum.

KEY WORDS: cytochromecoxidase, Ca^{2+}, hemea, absorption spectrum, spectral shift

DOI: 10.1134/S0006297913120067

Abbreviations: CBS, cation-binding site; COX, cytochromecoxidase; EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid; TMPD,N,N,N′,N′-tetramethyl-p-phenylenediamine.

Cytochrome *c *oxidase (COX) is a terminal enzyme of the
mitochondrial and many bacterial respiratory chains that transfers
electrons to oxygen coupled to membrane potential generation and
translocation of H^{+} across the coupling membrane (reviewed
in [1-3]). Electron transfer is
mediated by four redox centers containing transition metal ions. There
are two hemes (low-spin heme *a* and high-spin heme
*a*_{3}) and two copper centers: binuclear
(Cu_{A}) and mononuclear (Cu_{B}) [4] (Fig. 1). Also, X-ray analysis
revealed an additional cation-binding site (CBS) in COX from
mitochondria and several bacterial species [5-7]. The CBS is situated at the periphery of the main
catalytic subunit (subunit I), rather close to heme *a* and within
just a few angstroms from the enzyme surface protruding from the
membrane into the external aqueous phase (Fig. 1).
In the bacterial oxidases the CBS is occupied by a tightly bound
calcium ion, whereas in the mitochondrial enzyme calcium binds
reversibly and can be easily removed by chelators such as EGTA [8, 9].

Fig. 1.Subunit I of cytochromecoxidase from bovine heart with redox centers and calcium-binding site shown ([4], pdbID: 1V55).

Optical absorption spectroscopy is one of the main methods in studies of
COX, but despite many years of research the individual spectra of the
two hemes have not yet been fully resolved. In particular, the
absorption spectra of the hemes *a* and *a*_{3}
overlap significantly in the Soret band (420-460 nm) determined
mainly by the electronic π→π* transitions in the heme
porphyrin rings (*B*_{0}-transitions). In the reduced
enzyme, the hemes give rise to a common band with a maximum at
~444 nm [10, 11], which
is somewhat unusual considering the different spin states of the two
hemes. In the past, several attempts have been made to reveal the
individual absorption spectra of hemes *a* and
*a*_{3} in the Soret band of reduced COX by binding
various ligands to heme *a*_{3} followed by algebraic
manipulation of the spectra obtained [10, 12-16], as well as by redox
titrations [17] and higher derivative analysis of
the spectra [18]. Of these approaches, it is only
the “ligand” method that provides the individual absolute
spectra of the hemes. However, the method has several disadvantages of
which the most significant is that the dependence of the spectral
characteristics of one heme on the state of the other heme is
neglected.

In the present work, an independent experimental approach is proposed
that allows reconstructing the line shape of the absolute absorption
spectrum of the reduced heme *a* on the basis of the difference
spectrum induced by binding of calcium ions. As first found by Wikstrom
and collaborators [19, 20],
calcium binding to mitochondrial COX induces a small red shift of the
absorption spectrum of the reduced heme *a*. The shift titrates
with calcium with *K*_{d} of ~1 µM [21]. As the shift is very small compared to peak
bandwidth, it provides a unique opportunity for modeling: the
difference spectrum induced by calcium binding can be treated as the
first derivative of the absorption spectrum of the reduced heme
*a*, integration of which provides the line shape of the parent
absolute spectrum.

**MATERIALS AND METHODS**

**Reagents and preparations.** The pH buffers MES and Tris were
purchased from Amresco (USA), sodium dithionite, calcium chloride,
EGTA, ascorbate, and potassium cyanide were from Sigma-Aldrich (USA),
and TMPD was from Fluka (Germany).

The *aa*_{3}-type cytochrome *c *oxidase was isolated
from bovine heart mitochondria according to a modified protocol of
Fowler et al. [22]. Dodecyl maltoside of SOL-GRADE
(Anatrace, USA) was used for enzyme solubilization.

**Measurements. **Steady-state absorption spectra were recorded in a
Cary-300 Bio spectrophotometer (Varian, USA) in 1 ml semi-micro
cuvettes with blackened walls (Hellma, Germany) and optical path of 1
cm at 26°C. The measurements were carried out in medium containing
100 mM Tris/MES buffer, pH 8.0, with 0.1% dodecyl maltoside
to solubilize COX. The use of NaOH or KOH for adjustment of pH was
avoided because Na^{+} ions expel Ca^{2+} from the CBS
of COX [23] and potassium salts are often
contaminated with Na^{+}. The cyanide complex of the oxidized
COX was obtained by incubation of the enzyme with excess cyanide
(4 mM) for 15 min. Then 100 µM TMPD and 2 mM
Tris-ascorbate were added to obtain the mixed-valence cyanide complex
[24]. Difference spectra of the
Ca^{2+}-induced spectral shift of the reduced heme *a*
were obtained by subtraction of the corresponding absolute spectra
(spectrum of the sample in the Ca^{2+}-containing buffer
*minus *spectrum of the Ca^{2+}-depleted enzyme treated
with 1 mM EGTA, to extract Ca^{2+} from the CBS of COX).
All the absorption values were normalized to COX concentration in the
sample. The concentration of the enzyme was determined from the
“dithionite-reduced *minus *oxidized” difference
absorption spectra using molar extinction coefficient
Δε_{605-630} =
27 mM^{–1}·cm^{–1}.

Algebraic transformation, integration, and deconvolution of the spectral curves into Gaussians were made with the aid of custom programs written in the Python 2.7.3 computer language with the extensions Numpy, SciPy, and Matplotlib. The commercial program Origin 7 (Microcal, USA) was used for presentation of the results.

**RESULTS**

The base experimental spectra used for subsequent modeling are shown in
Figs. 2a and 2b. Figure 2a
shows the absolute absorption spectrum of mixed-valence cyanide complex
of COX *a*^{2+}*a*_{3}^{3+}-CN (cf.
“Materials and Methods”) which is in agreement with data in
the literature [10, 13, 24]. There are two prominent absorption bands: a band
in the visible region (α-band) with a maximum at 605 nm that
belongs largely to the reduced heme *a*^{2+}, and a
stronger near-UV band at 420-460 nm called the Soret**- **or
γ*-*band with the maxima at ~429 and ~442 nm
corresponding to the cyanide-complexed oxidized heme
*a*_{3} and reduced heme *a*, respectively. Minor
absorption maxima at ~520 and ~550 nm may correspond to a weakly
expressed vibronic β-band.

Fig. 2.Base experimental spectra used for modeling. a) Absolute absorption spectrum of the mixed-valence cyanide complex of COXa^{2+}a_{3}^{3+}-CN. COX (1.7 μM) in the basic medium was incubated with 4 mM KCN as described in “Materials and Methods” and then reduced by 5 mM Tris-ascorbate in the presence of 100 μM TMPD. b) Difference spectrum of the calcium-induced spectral shift of hemea. The spectrum shows the difference between the absolute spectra of the COX mixed-valence cyanide complex in the presence of calcium and after addition of 1 mM EGTA. The concentration of adventitious calcium in the buffer in the absence of EGTA is ca. 30 μM, i.e. ~30-fold theK_{d}value, which is sufficient to saturate the calcium binding site even in the absence of added calcium.

A typical difference spectrum of the absorption shift of the reduced
heme *a* induced by addition of Ca^{2+} to the
mixed-valence cyanide complex of COX
*a*^{2+}*a*_{3}^{3+}-CN (spectrum
obtained in the Ca^{2+}-containing buffer minus spectrum after
addition of 1 mM EGTA) is shown in Figs. 2b and 3a
(spectrum *1*). The spectrum has a nearly symmetric line shape and
is characterized in the Soret band by a minimum at 439 nm, maximum
at 455 nm, and clear inflection point at 447 nm (Fig. 3a, spectrum *1*). The magnitude of the spectral
changes in the Soret normalized to concentration of COX is
11 mM^{–1}·cm^{–1}. These
characteristics are in good agreement with data in the literature [19-21]. The spectral response of
COX induced by Ca^{2+} is selective: it is not the entire Soret
band that shifts to the red, but only part of it. This can be
demonstrated by constructing the differential of the absolute spectrum
of the COX cyanide complex (spectrum *2* in Fig. 3a). Comparison of the line shape of the differential
(spectrum *2*) with that of the experimental difference spectrum
of the spectral shift induced by calcium (spectrum *1*) shows
similarity between the spectra at longer wavelengths, while at shorter
wavelengths the curves differ in position and even sign of the extrema.
This difference agrees with the fact that it is the reduced heme
*a* that responds to Ca^{2+} binding with COX and
contributes more to the long wavelength part of the overall Soret band
in the mixed-valence cyanide complex of COX. Accordingly, the
difference spectrum of the Ca^{2+}-induced spectral shift is
centered at 447 nm, which corresponds to the absorption maximum of
the reduced heme *a* but not of the cyanide complex of the
oxidized heme *a*_{3}.

Fig. 3.Reconstruction of the absorption spectrum of reduced hemeaof cytochrome oxidase from bovine heart. a) Difference spectrum of the Ca^{2+}-induced red shift of hemea(spectrum1, solid line) is compared to the first derivative (differential) of the overall spectrum of the mixed-valence cyanide complex of COX (a^{2+}a_{3}^{3+}-CN) shown in Fig. 2a (spectrum2, dotted line). b) Reconstructed line shape of the Soret absorption band of the reduced hemeaobtained as the antiderivative (the indefinite integral curve) of the difference spectrum characterizing the calcium-induced spectral shift of hemea(cf. Fig. 2b). The absorption is normalized to its maximal value. The zero line was corrected by adding a constant 0.0244. c) Deconvolution of the reconstructed Soret band of hemeainto two Gaussians. Spectrum1(solid line), the reconstructed spectrum; spectrum2(dotted line), sum of the two Gaussians. d) Comparison of the experimental and simulated difference spectra of the calcium-induced red shift of hemea. Solid line, the experimental spectrum (Fig. 2b). Dotted line, the simulated spectrum corresponding to a shift of the reconstructed absorption band of reduced hemeaby Δλ = 0.54 nm.

In the visible region, the difference spectrum induced by addition of
Ca^{2+} to COX shows a symmetric line shape without an
inflection point (Fig. 2b), i.e. it corresponds to
the first derivative of a single absorption band. The inflection point
observed in the γ**-**band difference spectrum indicates that
the spectrum represents the superposition of the first derivatives of
at least two absorption bands. Such line shape is anticipated for
energy splitting of the transitions *B _{x}* and

The absolute spectrum of the reduced heme *a* (the antiderivative
curve) in the 400-500 nm range normalized to its maximal value is
shown in Fig. 3b. The spectrum is characterized by
a maximum at 447 nm and half*-*width (full width at
half-height) of 17.3 nm, while its full width at
*e*^{–1/8} height is equal to 9.3 nm. Let us
denote the last two parameters as *w* and *l*,
correspondingly. For a single Gaussian, their ratio should be
*w*_{gauss}/*l*_{gauss} = 22
ln2 = 2.35, while for the spectrum in Fig. 3b the
ratio is *w*/*l* = 17.3/9.3 = 1.86, which indicates
that it is composed of at least two Gaussian components. Spectrum
*2* in Fig. 3c has then been modeled by
superposition of two Gaussians. It can be seen that within the
430-470 nm region, spectrum *2* is close enough to the
antiderivative curve (spectrum *1*, solid line) obtained by
integration of the Ca^{2+}-induced difference spectrum as
described above. Thus, we can conclude that two Gaussian components are
sufficient to describe the line shape of the anti-derivative curve in
the 430-470 nm range. Decomposition of the spectrum into Gaussian
components was performed in two steps: in the first step, the
parameters of the components (magnitudes, half-widths, positions) were
fitted manually; in the second step, the manually selected parameters
were refined with the TNC algorithm (Truncated Newton Constrained) that
minimizes mean-square difference between the sum of the Gaussian
components and the approximated curve; in this step, the half-widths of
the Gaussian components were set equal. The Gaussian components thus
obtained are characterized by peaks at 442 and 451 nm, half-widths
of 588.7 cm^{–1} (10.1 nm), and relative
magnitudes of 0.68 and 0.77, respectively. According to this result,
the heme *a*^{2+} Soret absorption band appears to be
strongly split in the energies of the electronic transitions
*B*_{0x} and *B*_{0y}.

The sum of the two Gaussian components approximates well enough the line
shape of the absorption spectrum of heme *a*^{2+}. To
reconstruct the absolute spectrum of heme *a*, the normalized
curve has been multiplied by the molar absorption coefficient of
95 mM^{–1}·cm^{–1} [10] corresponding to the molar extinction of heme
*a* absolute spectrum assumed in the literature. Then the
Ca^{2+}-induced spectral shift of the heme
*a*^{2+} absorption band was simulated, and the results
are demonstrated in Fig. 3d. For the simulation,
the reconstructed spectrum of heme *a* was slightly displaced to
higher wavelengths and the magnitude of the shift was varied. The shift
of Δλ = 0.54 nm proved to be optimal, and the
difference between the initial and displaced spectra approximates
satisfactorily the line shape of the experimental difference spectrum
of the Ca^{2+}-induced effect.

The spectral shift induced by calcium in the visible region corresponds
to ca. 1 nm (~27 cm^{–1}) [21], which can be seen directly in the absolute
spectra. However, in the Soret region a shift of
27 cm^{–1} will correspond to only ~0.5 nm
which is hard to observe in the absolute spectra taking into account
the broadness of the γ-band. For precise estimation of the
magnitude of the shift in the γ-band, we took advantage of the
fact that in the case of small spectral shifts, the line shape of the
difference spectra (i.e. of differential of the absolute spectrum) does
not depend significantly on the shift magnitude, Δλ, but
the amplitude of the difference spectrum depends on Δλ
linearly (Fig. 4, inset). Conceivably, the
amplitude of the difference spectrum depends also on molar extinction
of the absolute spectrum, ε_{max}. According to data in
the literature, the molar extinction of heme *a* varies within the
ε_{max} =
95-115 mM^{–1}·cm^{–1} range
(e.g. [10]). Figure 4
represents differentials for heme *a*^{2+} absorption band
at increasing shift values, assuming ε_{max} =
95 mM^{–1}·cm^{–1}. It can be
seen that the positions of the extrema are almost constant in the 0
< Δλ < 2.3 nm range, while the magnitude of the
simulated difference spectrum increases linearly with Δλ.
On a graph of the magnitude of the spectral response plotted versus the
absorption shift, the amplitude of the difference spectrum obtained
experimentally
(11 mM^{–1}·cm^{–1})
corresponds to a shift Δλ = 0.59 nm.
Accordingly, the extrema of the theoretical and experimental spectra
coincide in the region 0.33-0.66 nm. These estimates are in
agreement with the results of the optimization with the TNC algorithm,
according to which the best agreement between the experimental and
model curves is achieved at Δλ = 0.54 nm.

Fig. 4.Modeled difference spectra of the calcium-induced spectral shift of hemeaat different Δλ values. The figure shows difference spectra simulating red shift of the Soret absorption band of hemea, reconstructed as a sum of two Gaussian bands (cf. spectrum2in Fig. 3c) at different increments of Δλ (0.33, 0.66, 1.00, 1.33, 1.66, 2.00, and 2.33 nm). The arrows indicate the direction of Δλ increase. The inset shows the dependence of the difference spectrum amplitude (left axis) and positions of the extrema (right axis) on Δλ.

**DISCUSSION**

According to our results, the absolute Soret spectrum of the reduced
heme *a* reconstructed from the difference spectrum of the
calcium-induced red shift is characterized by an absorption band with
maximum at 447 nm and full width at half-maximal height
(half-width) of 17 nm; the latter value is somewhat larger than
typical of the absorption bands of reduced low-spin cytochromes:
10 nm for reduced cytochrome *c* [25],
14 nm for the heme-binding domain of reduced cytochrome
*b*_{5} [26]. The spectrum obtained
can be fitted by a sum of two transitions with centers at 442 and
451 nm and half-widths of ~589 cm^{–1}
(~10 nm).

The presence of the two bands with the indicated maxima is in good
agreement with the results obtained by second-derivative spectroscopy
of different oxidases [18]. Splitting of the
*B*_{0}-band of heme *a* into two sub-bands with
centers at 437 and 451 nm and half-width of
452 cm^{–1} but with a somewhat different intensity
ratio was obtained by our group earlier with the aid of modeling the
absorption and circular dichroism spectra of COX based on
dipole–dipole interaction theory [11].

At the same time, the reconstructed Soret band of the reduced heme
*a* differs significantly from Vanneste’s
“classical” spectrum of the reduced cytochrome *a*
(taken from Fig. 5 of [10])
obtained by comparison of the spectra of COX in the presence of various
ligands of heme *a*_{3} (solid line in Figs. 5a and 5b). In the table, the spectral characteristics
obtained by the “ligand method” and by the calcium-induced
shift approach are compared. According to our results, the maximum of
the γ-band of heme *a *is at 447 nm, i.e. shifted
slightly to the red relative to the data in [10].
More importantly, the half-width of the γ-band as given in [10] is by about 20 nm larger than the half-width
of the antiderivative for the calcium shift obtained in the present
work. To explain this discrepancy, it may be proposed that in addition
to the Ca^{2+}-sensitive *B*_{0x} and
*B*_{0y} transitions in the porphyrin ring, the
Soret band spectrum of heme *a*^{2+} contains additional
bands of other origin that are not affected by calcium; also, they do
not contribute significantly to the circular dichroism of COX [11]. Particularly notable is the presence in spectrum
*1* of a well-resolved “shoulder” at ~428 nm,
which is often observed in the absorption spectra of reduced cytochrome
oxidase. The nature of the additional band(s) is not clear. Apparently,
it cannot be a vibronic satellite of the
*B*_{0}-transitions of heme *a*, because in this case
it is expected to undergo the same red shift induced by calcium
binding. It cannot be excluded that in the Vanneste’s spectrum
the band is associated with contribution of the ligand-bound low-spin
form of heme *a*_{3 }or with a vibronic satellite of the
heme *a*_{3}^{2+ }*B*_{0} band.
Presumably, the spectrum obtained in the present work by integration of
the difference spectrum of the calcium-induced shift represents only
the sum of the perpendicularly polarized *x*- and
*y*-transitions of the *B*-band, while contribution of the
additional absorption bands becomes significant when the spectrum is
reconstructed by the “ligand” method [10].

Fig. 5.Comparison of the reconstructed spectrum of reduced hemeain the Soret region with the conventional spectrum published by Vanneste [10]. a) Soret band of reduced hemeaas reported by Vanneste (spectrum1, solid line) and that reconstructed in this work as a sum of two Gaussian bands (spectrum2, dotted line). b) Difference between the spectra shown in panel (a) (spectrum1minusspectrum2).

Parameters of the absolute absorption spectrum of the reduced heme
*a*

Neither experimental data nor theoretical considerations can provide
evidence for splitting of the Soret absorption band of reduced heme
*a*_{3} [11, 27]. In all probability, the splitting of the band in
heme *a* is caused by interaction of the heme with the protein
environment. It was shown [18] that
second-derivatives of the absorption spectra of several heme *a*
model compounds do not reveal the splitting inherent in the spectra of
heme *a* in reduced heme *a-*containing oxidases (but cf. [27]). The splitting could originate from strong
hydrogen bonding of the formyl group of heme *a* with the
conserved Arg38 (Arg54 in *Paracoccus denitrificans* enzyme [28, 29]) or could be induced by
dipole–dipole interactions. The latter possibility is favored by
the calculations [11] showing that the
*B*_{0x}**-** and
*B*_{0y}-transitions of heme *a* are virtually
degenerate if the interactions with heme *a*_{3} and the
nearby aromatic amino acid residues are not taken into account, whereas
the splitting appears if the dipole–dipole interactions are
considered.

One of the drawbacks of the “ligand method” in [10] is that possible influence of the state of one
heme on the optical properties of the other is neglected. At the same
time, it has been shown [11, 30, 31] that the hemes interact
strongly with each other, and optical properties of one heme depend on
the state of the neighboring heme. The algebraic procedure used by
Vanneste [10] operates with the experimental
spectra of the enzyme forms with different states of heme
*a*_{3}, and it is not possible to say to which state of
heme *a*_{3} the spectrum of the reduced heme *a*
obtained in [10] corresponds.

On the contrary, calcium ions induce only a small perturbation of the
heme *a* spectrum and therefore do not affect any significantly
interaction between the hemes. Therefore, the calcium shift-based
method of spectrum reconstruction represents an independent
experimental approach that allows direct determination of the optical
properties of heme *a* without perturbing its interaction with
heme *a*_{3}. Applying the calcium shift approach to
different forms of the enzyme, e.g. to the free reduced form,
*a*^{2+}*a*_{3}^{3+}-CN and
*a*^{2+}*a*_{3}^{2+}-CO, it may be
possible in the future to obtain the line shape of the heme *a*
spectrum at different states of heme *a*_{3} and to
evaluate directly to which extent the redox and spectral
characteristics of heme *a*_{3} affect the absolute
spectrum of heme *a*.

Finally, it is interesting to point out a discrepancy between the line
shapes of the γ**- **and α**-**bands of heme
*a*. With the Soret band half-width of 17 nm, one could
expect half-width of the α**-**band to be around 30 nm.
However, antiderivative of the calcium-induced spectral shift in the
visible region has half-width of only 18 nm and can be
approximated well by a single Gaussian (data not shown). This means
that either there is no splitting of the visible *Q*_{0
}band, or that the α-band of COX at ~605 nm represents
only one of the two widely split
*Q*_{0x,y}-transitions, while the other transition
is but weakly expressed in the absorption spectrum. Accordingly,
magnetic circular dichroism (MCD) data show [27]
that the α**-**band of free reduced heme *a* coordinated
by two 1-methyl-imidazoles is split, so that the centers of the
*Q*_{0x}- and *Q*_{0y}-bands
are at 570 and 595 nm, respectively. The MCD spectrum of the
reduced cytochrome *c* oxidase has the same shape but is shifted
to the red by ~10 nm, from which it can be inferred that the
absorption band observed at ~605 nm corresponds most likely to the
*Q*_{0y}-transition, while the
*Q*_{0x}-transition is not well pronounced in the
absorption spectrum.

The authors express their sincere gratitude to Dr. A. M. Arutyunyan for seminal discussions.

This work was supported in part by a grant from the Russian Foundation for Basic Research (No. 11-04-01330-a).

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