Báo cáo khoa học: Characterization of mutations in crucial residues around the Qo binding site of the cytochrome bc1 complex from Paracoccus denitrificans pdf

By monitoring the interaction with the inhibitor stigmatellin for the wild-type enzyme at various redox states, interactions of the bound stigmatellin with amino acid side chains such as

the Qo binding site of the cytochrome bc1 complex from Paracoccus denitrificans

Thomas Kleinschroth1, Oliver Anderka1, Michaela Ritter2, Andreas Stocker1,2, Thomas A Link2, Bernd Ludwig1 and Petra Hellwig3

1 Institut fu¨r Biochemie der Johann Wolfgang Goethe Universita¨t, Molekulare Genetik, Biozentrum, Frankfurt am Main, Germany

2 Institut fu¨r Biophysik der Johann Wolfgang Goethe Universita¨t, Frankfurt am Main, Germany

3 Institut de Chimie, UMR 7177 CNRS, Laboratoire de Spectroscopie Vibrationnelle et Electrochimie des Biomole´cules, Universite´ Louis Pasteur, Strasbourg, France

Ubiquinol–cytochrome c oxidoreductase (cytochrome

bc1 complex; complex III) [1] is a fundamental

compo-nent of the respiratory electron transfer chains located

in the inner mitochondrial or bacterial cytoplasmic membrane As a minimum requirement, all bc1 com-plexes contain three catalytic subunits: cytochrome c1

Keywords

bc1complex; FTIR spectroscopy;

Paracoccus denitrificans; proton and

electron transfer; quinones

Correspondence

P Hellwig, Institut de Chimie, UMR 7177

CNRS, Laboratoire de Spectroscopie

Vibrationnelle et Electrochimie des

Biomole´cules, Universite´ Louis Pasteur 4,

rue Blaise Pascal, 67000 Strasbourg, France

Fax: +33 390 241431

Tel: +33 390 241273

E-mail: hellwig@chimie.u-strasbg.fr

(Received 31 March 2008, revised 14 June

2008, accepted 28 July 2008)

doi:10.1111/j.1742-4658.2008.06611.x

The protonation state of residues around the Qo binding site of the cyto-chrome bc1 complex from Paracoccus denitrificans and their interaction with bound quinone(s) was studied by a combined electrochemical and FTIR difference spectroscopic approach Site-directed mutations of two groups of conserved residues were investigated: (a) acidic side chains located close to the surface and thought to participate in a water chain leading up to the heme bL edge, and (b) residues located in the vicinity of this site Interestingly, most of the mutants retain a high degree of catalytic activity E295Q, E81Q and Y297F showed reduced stigmatellin affinity On the basis of electrochemically induced FTIR difference spectra, we suggest that E295 and D278 are protonated in the oxidized form or that their mutation perturbs protonated residues Mutations Y302, Y297, E81 and E295, directly perturb signals from the oxidized quinone and of the protein backbone By monitoring the interaction with the inhibitor stigmatellin for the wild-type enzyme at various redox states, interactions of the bound stigmatellin with amino acid side chains such as protonated acidic residues and the backbone were observed, as well as difference signals arising from the redox active inhibitor itself and the replaced quinone The infrared difference spectra of the above Qosite mutations in the presence of stigma-tellin confirm the previously established role of E295 as a direct interaction partner in the enzyme from P denitrificans as well The protonated residue E295 is proposed to change the hydrogen-bonding environment upon stigmatellin binding in the oxidized form, and is deprotonated in the reduced form Of the residues located close to the surface, D278 remains protonated and unperturbed in the oxidized form but its frequency shifts in the reduced form The mechanistic implications of our observations are discussed, together with previous inhibitor binding data, and referred to the published X-ray structures

Abbreviations

bc 1 complex, ubihydroquinone–cytochrome c oxidoreductase; b H, high-potential b-type heme; b L, low-potential b-type heme; DDM, n-dodecyl b- D -maltoside; Qi,ubiquinone reduction site; Qo,ubiquinol oxidation site.

with covalently bound c-type heme, cytochrome b with

two b-type hemes (bLand bH), and the Rieske iron

sul-fur protein with a [2Fe–2S] cluster Crystal structures

of several mitochondrial complexes that contain

addi-tional subunits have been reported [2–5] Recently, a

new crystal structure for a bacterial complex has been

solved [6]

The enzyme couples the electron transfer from

ubiquinol to cytochrome c to the translocation of

pro-tons across the membrane Both bacterial and

mito-chondrial bc1 complexes follow the same catalytic

mechanism, the so-called Q-cycle [7–9], which relies on

two separate binding sites for quinones, Qo and Qi

The Qo site is located close to heme bL and the [2Fe–

2S] cluster, and the Qisite is close to heme bHon the

opposite side of the membrane Although this

mech-anism is generally accepted, not all aspects of the

quinol⁄ quinone binding and redox reaction are yet

fully understood at the molecular level, and various

models for the quinol oxidation mechanism at the Qo

site have been discussed [10–15]

Inhibitors are an important tool for analysis of the

molecular mechanism of the bc1 complex, and have

been extensively used to characterize the various

qui-none binding sites [16] Depending on their binding

properties, Qo and Qi site-specific inhibitors may be

distinguished Stigmatellin binds at the Qo site and

interacts with the Rieske protein and cytochrome b,

and also influences the heme bL spectral properties

The crystal structure of the bc1 complex with

stigma-tellin bound at the Qosite [2] shows tight and specific

binding of the inhibitor The position of the

conju-gated trienes is stabilized by several van der Waals

interactions with cytochrome b residues The chromone

headgroup is oriented by numerous nonpolar and a

few polar interactions, including a hydrogen bond

from the carbonyl group (4-C = O) to His155 (His188

in yeast), one of the [2Fe–2S] cluster ligands of the

Rieske protein, which is thereby fixed in a

cyto-chrome b docking position [2] (unless otherwise

indi-cated, numbering of the amino acids corresponds to

the Paracoccus denitrificans bc1 complex) On the heme

bL facing side of the inhibitor, the 8-hydroxy group is

within hydrogen-bonding distance of the side chain of

cytochrome b residue Glu295 (272 in yeast) Bound

stigmatellin is thought to mimic an intermediate of

ubiquinol oxidation [2] Based on published structures

and biochemical characterization of variants, Glu295

has been proposed to be part of the proton exit

path-way for ubiquinol oxidation [2,16]

The cytochrome bc1 complex of P denitrificans

represents a small bacterial version of the

mitochon-drial enzyme, lacking any additional subunits Its 3D

structure is not yet known; however, due to extensive sequence identity, mostly in the cytochrome b and Rieske subunits, a similar architecture for the three catalytic subunits between the mitochondrial and the bacterial complex is assumed In order to probe poten-tial similarities and dissimilarities, we have investigated the Qosite of the bc1 complex from P denitrificans by

a combination of site-directed mutagenesis, protein electrochemistry and FTIR difference spectroscopy Reaction-induced FTIR spectroscopy is a method that

is suitable for the study of the protonation state of acidic residues or quinone binding as described previ-ously for several membrane proteins including bc1 com-plexes [17–22] Identification of interaction partners for stigmatellin binding in the oxidized and reduced forms

as well as the protonation state of the residues involved

in proton transfer are described and discussed in the light of studies on bc1complexes from other organisms The mutated residues are highlighted in Fig 1

Results

Site-directed mutations in the Qobinding site Mutations in conserved positions of cytochrome b at the Qosite were constructed (Fig 1) The three subun-its of the P denitrificans bc1 complex are expressed in all mutants and assembled into a stable complex that corresponds to the wild-type enzyme as determined

by SDS–PAGE and Western blot analysis After

Fig 1 3D representation of the Qosite environment of the cyto-chrome bc 1 complex based on the structure obtained from Rhodob-acter sphaeroides [46] Cytochrome c 1 is shown in blue, cytochrome b in red, and the Rieske protein in green The iron–sul-fur cluster is shown in purple and yellow, and the bound inhibitor stigmatellin is shown in turquoise Heme is shown in light purple, and the heme iron is shown in purple Mutations of conserved amino acids introduced in seven positions of the P denitrificans enzyme are indicated as follows: 1, D71 ⁄ 86 (mitochondrial ⁄ bacte-rial complex); 2, E66 ⁄ 81; 3, D255 ⁄ 278; 4, Y132 ⁄ 147; 5, E272 ⁄ 295;

6, Y274 ⁄ 297; 7, Y279 ⁄ 302.

solubilization, the complex was purified using a

DEAE–Sepharose column, and the cytochrome bc1

eluted as a single peak Samples were > 95% pure as

determined by silver staining The

ubiquinol–cyto-chrome c oxidoreductase activities of the purified

com-plexes were measured in buffer containing n-dodecyl

b-d-maltoside and compared to that of the wild-type

enzyme (Table 1)

The activities of the E81Q, D278N, Y297F and

Y302F mutant enzymes ranged from 90% to 120% of

that of the wild-type enzyme Significantly reduced

activity (66 and 55%) was observed for the D86N and

Y147F mutant enzymes A drastic reduction in

turn-over was seen for the E295Q mutation, with only 10%

residual activity The activity of the wild-type and all

mutant complexes is strongly inhibited to < 1% of

wild-type activity by the addition of 2 lm of the

inhib-itor stigmatellin IC50 values, defined as the inflection

point of the curve, are listed in Table 1 Interestingly

the E81Q mutant enzyme showed both a slightly

increased turnover and also an increased IC50value A

distinct increase of the IC50value was observed for the

E295Q and Y147F mutant enzymes

FTIR difference spectra of mutations in the Qo

binding site

Figure 2 shows an overview of the

oxidized-minus-reduced FTIR difference spectra of the E295Q,

D278N, E81Q and D86N mutant enzymes in

compari-son with wild-type The redox-induced FTIR difference

spectra include contributions from reorganization of

the cofactors, heme bL, bHand c1, the bound quinones,

individual amino acids, the backbone and coupled

pro-tonation reactions All purified mutants retained their

bound quinones, as their spectra include the character-istic contributions that dominate the overall spectrum

of the P denitrificans bc1 complex, such as the typical contribution of the methoxy side chain at 1264 cm)1,

as detailed below The number of molecules of quinone per bc1monomer has been reported as 2.6–3.3 for this type of preparation [19]

Contribution of acidic side chains For the redox-induced FTIR difference spectra of the E295Q, D278N and D86N mutant enzymes, the signals

in the spectral region characteristic for protonated

Table 1 Enzymatic activities and IC 50 values for stigmatellin of

purified cytochrome bc 1 mutants at the Q o quinone binding site.

Values are the means of triplicate measurements.

Enzyme ⁄ mutant

Percentage

of the activity

in wild-type

IC50fold increase over wild-type

a 100% indicates a turnover number of 327 s)1based on one

cyto-chrome b (per monomer).b1 indicates an IC 50 value for the

wild-type of 131 ± 7 n M under our experimental conditions.

Abs 0.001

WT

E295Q

D278N

E81Q

D86N

D278N

(cm –1 )

E295Q

E81Q

1743

Fig 2 Overview of the oxidized-minus-reduced FTIR difference spectra of wild-type and acidic side-chain mutant cytochrome bc1 complexes from P denitrificans obtained for a change in potential from )0.292 to +0.708 V The inset shows double difference tra obtained by subtracting the wild-type red-ox difference spec-trum from that of each mutant.

acidic residues were perturbed The decrease is shown

in the inset to Fig 2, showing double difference

spec-tra obtained by subspec-tracting the spectrum of the

E295Q, D278N and E81Q variants from that of the

wild-type Both D278N and E295Q show a decrease in

the mode at 1746 cm)1 associated with the oxidized

form, without a complete loss of the signal (see Fig 4

below), so both residues may contribute to this signal

or indirectly influence the contributing C = O group

In the case of the D86N mutant enzyme, the negative

mode at 1724 cm)1 is decreased In contrast, the E81Q

mutation does not induce changes in this region In

the spectral range that includes the signals for

deproto-nated acidic side chains [23–27], clear variations occur

at 1560 cm)1 for E295Q, at 1563 cm)1 for D86N and

at 1559 cm)1 for D278N, at positions typical for the

d(COO))as vibrational mode The d(COO))s

vibra-tional mode can be tentatively assigned to the shifts

observed between 1455 and 1423 cm)1 These shifts

may be attributed to the acidic residues that are

per-turbed due to the mutations or alternatively loss of

interaction with the heme propionates from the nearby

heme b

Contributions from tyrosine side chains

Figure 3 gives an overview on the

oxidized-minus-reduced FTIR difference spectra of the Y147F, Y297F

and Y302F mutant enzymes in comparison with

wild-type The wild-type spectrum shows contributions in

the spectral range around 1516 and 1500 cm)1that are

characteristic of tyrosine side chains In previously

reported model spectra of the protonated tyrosine, the

signal at approximately 1518 cm)1 was attributed to

the m19(CC) ring mode At 1249 cm)1, a signal

com-posed of the m7’a(CO) vibration and the d(COH)

vibra-tion is expected, and the posivibra-tion is sensitive to the

hydrogen-bonding environment [23,25,28,29] For

deprotonated tyrosine in solution, the m8a⁄8b(CC) ring

mode was identified at 1560 cm)1and the m19(CC) ring

mode at 1499 cm)1, thus reflecting the sensitivity of

the ring modes to the protonation state of the phenyl

group The m7’a(CO) mode was present at 1269 cm)1

In the difference spectra shown in Fig 3, changes

were only observed for the Y302F and the Y297F

mutant enzymes These shifts are rather small as

com-pared to previously published absorption coefficients

for these modes [23,25,28,29] In the spectra of the

Y302F mutation, the signal at 1666 cm)1 is absent

This spectral range typically includes contributions

from the m(C = O) mode of the backbone or

proton-ated heme propionates Additionally, we suggest the

perturbation of arginine side chains This is supported

by model compound studies that indicated that vibra-tional modes are expected at 1673 cm)1for m(C = N),

1633 cm)1 for d(NH3+)asand 1522 cm)1for d(NH3+)s [23,25] For the Y302 mutant, perturbations were seen

at 1666, 1626 and 1522 cm)1

Contributions of the quinones and the protein backbone

In redox-induced FTIR difference spectra of quinones

in solution, the positive signals between 1670 and

1540 cm)1, as well as at 1610, 1288, 1264 and

1204 cm)1, correlate with the neutral quinone, while the negative signals at 1490, 1470, 1432 and 1388 cm)1 represent the reduced and protonated quinol form The mode between 1670 and 1640 cm)1 was previously assigned to the C = O vibration of the quinone, and the mode at 1610 cm)1 was attributed to the C = C vibration [32–34] The C–O modes of the methoxy groups contribute to the signals at 1288 and 1264 cm)1 Figure 4 shows the spectra after hydrogen⁄ deuterium (H⁄ D) exchange, and an enlarged view for the wild-type, E295Q and D278N mutant enzymes before the exchange

0.001

wt

Y297F

Y302F

Y147F

Wavenumber (cm–1) Fig 3 Overview of the oxidized-minus-reduced FTIR difference spectra of wild-type and tyrosine side-chain mutant cytochrome bc 1 complexes from P denitrificans obtained for a change in potential from )0.292 to +0.708 V.

The effect of H⁄ D exchange has been described

pre-viously [19] Interestingly, the strong positive feature

around 1655 cm)1, previously tentatively assigned to

the m(C = O) mode of neutral, fully oxidized quinones,

is perturbed in most of the mutants The position of

this vibration is dependent on hydrogen bonding to the

C = O group, as previously found in quinone spectra

of other enzymes [32–34] The most prominent shift

occurs for the E295Q mutation, for which an increase

of the shoulder at 1646 cm)1is observed (Figs 2 and 4,

insets), indicating that at least one of the involved

quinones experiences weaker hydrogen bonding

Simi-larly, the signal at 1639 cm)1 is significantly increased

in the H⁄ D-exchanged sample We note, however, that these changes may also originate from contributions of the protein backbone, varied due to the mutations Another potential explanation for the variation in signal intensity seen for the various mutants might be the differences in quinone content; however, other characteristic signals of the quinone, such as the mode at 1264 cm)1 (1266 cm)1 in the H⁄ D-exchanged sample) remain unperturbed (Fig 3) As an alternative explanation for the loss of signal intensity, e.g for the E81Q mutation, the dependence of the m(C = O) signal for up to 50% of its intensity on the orientation of the methoxy side chains in relation to the position of the quinone ring should be noted, as previously reported [36] The change in intensity was confirmed in the

H⁄ D-exchanged sample, for which the signals at 1655 and 1639 cm)1both strongly decrease due to the muta-tion This may indicate a change of the quinone envi-ronment in some of the mutants In addition, we note some broadening of the m(C = O) signals, for example

in the case of the E295Q mutation This may be due to the loss of a hydrogen-bonding partner, allowing greater rotational freedom of the C = O groups In order to differentiate between the effects on the protein backbone and on the quinones, further experiments on isotopically labeled quinones are necessary

Wild-type FTIR difference spectra in the presence

of stigmatellin Figure 5 shows the oxidized-minus-reduced FTIR difference spectra of the wild-type cytochrome bc1 complex from P denitrificans obtained for a potential step from )0.292 to +0.708 V, in comparison with spectra obtained in the presence of a 2- or 10-fold molar excess of stigmatellin

Upon binding of stigmatellin, shifts reflecting the changes within the binding site and the immediate envi-ronment are expected, together with signals for the inhibitor itself, which undergoes a redox reaction [18,37] The spectra obtained with a 10-fold excess of stigmatellin help to identify the signals originating from the oxidized and reduced inhibitor; signals for the inhib-itor were observed at 1704, 1670 and 1252 cm)1, for the oxidized form and several features between 1598 and

1346 cm)1 were observed for the reduced form These signals are in line with the spectra identified using iso-tope-labeled derivatives characterized in the presence of the bc1 complex from yeast [18] For interpretation of the effects of inhibitor binding, the oxidized-minus-reduced FTIR difference spectra in the presence of a 2-fold excess of stigmatellin are discussed below, enabling us to focus solely on contributions from the

D278N E295Q WT

1746 1724

0.001

D86N

E81Q

D278N

E295Q

WT

Wavenumber (cm–1) Fig 4 Overview of the oxidized-minus-reduced FTIR difference

spectra of wild-type and mutant cytochrome bc1complexes from

P denitrificans, with samples equilibrated in D 2 O buffer The inset

shows an enlarged view of the spectral region characteristic of

pro-tonated acidic residues as well as perturbations on the m(C = O)

vibrational mode of ubiquinone and the protein backbone for

wild-type and the D278N and E295A mutant enzymes equilibrated in

H2O buffer.

inhibited protein and the bound inhibitor, but not from

the unbound inhibitor Double difference spectra were

obtained by subtracting wild-type spectra from those

obtained in the presence of a 2-fold excess of

stigmatel-lin to further elucidate the observed shifts (Fig 5)

Large variations were seen over the full spectral

range The spectral region between 1760 and

1710 cm)1 is characteristic of variations in the

m(C = O) mode for protonated acidic residues

[26,27,37] A new positive feature appears at 1723 cm)1, and a small decrease of the signal at 1744 cm)1is seen This is in line with a previous study on the yeast bc1 complex [18] These difference signals include contribu-tions from several acidic residues (Fig 5) Shifts at approximately 1540 cm)1 as well as at 1447 and

1428 cm)1indicate possible variations of a

deprotonat-ed acidic residue, like, for example, amino acid side chains and heme propionates [39] Further significant shifts, not arising from contributions of the inhibitor itself, are seen in the amide I range, i.e at 1635, 1646 and 1670 cm)1, as observed previously for inhibitor binding to the yeast bc1 complex [18] These may reflect changes in the backbone that occur upon inhibi-tor binding, such as reorientation of the Rieske domain upon stigmatellin binding as reported previ-ously [2,4,40–42] In addition, the variation of the sig-nal at 1646 and 1635 cm)1 upon addition of stigmatellin may at least be partially attributed to

C = O modes of the displaced quinone loosely bound

to the protein Stigmatellin is added to the sample without any further separation, and the displaced qui-none should be observable in the difference spectra

FTIR difference spectra of the mutants in the presence of stigmatellin

Figure 6 shows the redox-induced FTIR difference spectra of the E295Q, D278N and Y302F mutant enzymes in the presence of a 2-fold excess of stigmatel-lin in comparison to that of wild-type In the amide I range, all mutants showed a typical shift at 1646 cm)1 upon binding of stigmatellin, as also observed for wild-type This shift is thought to indicate the quinone displacement or a variation in backbone due to the bound inhibitor Nevertheless, major differences among the mutants with respect to the spectroscopic binding characteristics were seen in the double difference spec-tra obtained by subspec-tracting the oxidized-minus-reduced FTIR difference spectra of the mutants recorded in the presence and absence of stigmatellin (Fig 7)

The redox-induced FTIR difference spectrum of the E295 mutant in the presence of stigmatellin displays most of the typical signals of the inhibitor binding, except for the spectral range specific for protonated acidic residues around 1744 cm)1 No obvious varia-tion was seen here Interestingly, a new signal arose at

1560 cm)1, reflecting changes in the binding pocket Additional variations were seen around 1637 cm)1 in the amide I region, possibly due to displacement of the differently bound quinone The signal seen at

1744⁄ 1723 cm)1in the wild-type spectrum can thus be attributed to the E295 side chain

1800 1700 1600 1500 1400 1300 1200

1800 1700 1600 1500 1400 1300 1200

0.002

A

B

0.002

C

Wavenumber (cm –1 )

Wavenumber (cm–1)

1775

(cm –1 )

1750 1725

Fig 5 (A) Oxidized-minus-reduced FTIR difference spectra of the

cytochrome bc 1 complex from P denitrificans obtained for a

change in potential from )0.292 to +0.708 V with a 2-fold excess

of stigmatellin (black line) in comparison with wild-type (gray line).

(B) Double difference spectrum (wild-type inhibited with 2-fold

excess of stigmatellin minus its inhibitor-free counterpart) The

spectral region characteristic for protonated residues is enlarged in

the inset above (A) (C) Effect of addition of a 10-fold excess of

stigmatellin (dotted line) in comparison with the spectrum obtained

for a 2-fold excess (black line), highlighting the contributions of

stigmatellin The spectra are normalized to the a-band

(553 ⁄ 559 nm) in the visible spectrum.

In the case of the D278N mutant enzyme, a

differen-tial signal was observed at 1750⁄ 1728 cm)1upon

bind-ing of the inhibitor On the basis of the up-shift of the

differential signals by about 6–4 cm)1 in comparison

with wild-type, weaker hydrogen bonding or a more

hydrophobic environment of the C = O group of the

E295 side chain can be deduced Differential features

in the spectral range for deprotonated acidic residues

at 1588⁄ 1565 cm)1 and 1446⁄ 1428 cm)1 were lost in

the double difference spectra of the D278N mutant as

highlighted by arrows The signals in the amide I range

are clearly shifted in comparison to wild-type D278

appears to be deprotonated in the stigmatellin-bound

form, and this residue obviously influences the

stigma-tellin binding site

In the redox-induced FTIR difference spectra of the

Y302F variant in the presence of stigmatellin (Fig 6),

only a small amount of inhibitor is observed, but most

of the typical shifts are observed Interestingly, the

negative signals at 1668 and 1702 cm)1 are not

decreased as seen for wild-type and the D278N and

E295Q mutant enzymes, and instead only a broad shift

at 1707 cm)1 was noted in the double difference

spec-tra This indicates that, after mutation at residue Y302, an alternative residue is involved in the proton displacement that takes place around the Qosite, pos-sibly accompanied by a small change in the backbone This ‘rescue’ would also explain why mutation of this crucial residue does not lead to any significant loss in activity The typical shifts at approximately 1670 and

1646 cm)1cannot be seen in the same intensity ratio

Discussion and Conclusions

In this study, the effects of mutations in conserved residues of cytochrome b from the cytochrome bc1 complex of P denitrificans were studied A detailed redox-induced FTIR difference spectroscopic study of the variants was performed in the presence and absence of stigmatellin, and band assignments are summarized in Table 2 Two regions were addressed: residues in the immediate vicinity of the Qo binding site, and residues E81, D86 and D278, located close to the surface These structural regions are analyzed and

0.0005

ddwt

ddD278N

ddE295Q

Fig 7 Double difference spectra obtained by subtracting the oxi-dized-minus-reduced FTIR difference spectra of the D278N and E295Q mutations of cytochrome bc 1 in the presence of stigmatellin from those of their inhibitor-free counterparts.

0.001

Y302F

E295Q

D278N

WT

Wavenumber (cm–1) Fig 6 Oxidized-minus-reduced FTIR difference spectra for the

D278N, E295Q and Y302F mutants of the cytochrome bc1complex

from P denitrificans obtained for a change in potential from )0.292

to +0.708 V in the presence of stigmatellin.

discussed below in the light of current views on the role of the so-called PEWY loop

Residues E81 and D86 are positioned close to the surface of cytochrome b at a distance of approximately

29 and 20 A˚, respectively, from the Qo binding site (see Fig 1), as measured from the chromone head-group of the inhibitor [2] Their involvement in a water chain leading up to the heme bLedge, and their parti-cipation in proton exit from the quinol site has been suggested previously on the basis of molecular dynam-ics modeling [43] of the bc1 structure from chicken [4] This water chain was later experimentally visualized in the structure for the complex from Saccharomyces cerevisiae[2] Interactions with the binding site may be based on hydrogen bonding and include lipids (as sug-gested in [2,4]) In the study presented here, decreased activity was found for the D86N mutant enzyme The E81Q mutant enzyme showed a lower affinity towards stigmatellin The redox-induced FTIR difference spec-tra were perturbed with respect to signals for an acidic residue that is protonated in the reduced form, and, interestingly, the quinone and backbone contributions were also shifted For both the D86N and E81Q mutant enzymes, changes in quinone contributions were observed in the respective difference spectra, indi-cating an interaction between these acidic residues and the Qobinding site The observed shifts may be a sec-ondary-order effect induced by perturbation of the water chain that leads to the heme bL edge and resi-dues of the PEWY loop, including the E295 and Y297 residues studied here

E295 is a heavily discussed position in close proxim-ity to the quinone binding site, as suggested by site-directed mutagenesis [10,13,16,41–45] and X-ray crystallography [1–3,46] All crystallographic data were obtained in the presence of stigmatellin under the assumption that the inhibitor remains oxidized In the FTIR spectroscopic analysis of the E295 mutant in the absence of inhibitor, signals characteristic of pro-tonated acidic residues in the fully oxidized form are partially lost in direct comparison to the wild-type

Table 2 Summary of tentative assignments for the

oxidized-minus-reduced FTIR difference spectra of the P denitrificans bc 1

complex based on recent data from potential titrations [18] and

site-directed mutants in this study A positive symbol (+) indicates

the oxidized state, a negative symbol ( )) indicates the reduced

state In case of a composite signal, the main peak is given.

Band position (cm)1)

before and after

stigmatellin addition

Assignment Before After

1724 ( )) m(C = O) D86 and further Asp ⁄ Glu

1723 (+) m(C = O) E295

1710 (+) m(C = O) Asp ⁄ Glu (cytochrome b H )

1693 (+) 1698 (+) Amide I (Rieske b-sheet)

m(C = O) heme propionates bL, bH

1680 (+) m(C = O) heme propionates bL, bH, c1

m(C = O) Gln ⁄ Asn (cytochrome b H ) Amide I (loop structures Rieske)

1670 (+) m(CN 3 H 5 ) Arg (cytochrome b H )

1670 (+) Stigmatellin when added in excess

Perturbed m(C = O) heme propionates

1658 (+) Amide I (a-helical, unordered)

m(C = O) quinone

1646 ⁄ 1635 (+) Amide I

m(C = O) quinone

m 37 heme c 1

m(CN3H5) Arg (cytochrome bH)

1592 (+)

m 37 heme b L

m38heme c1

1565 ⁄ 1540 ( )) m(COO)) as heme propionates bL, bH, c1

m(COO)) as Asp ⁄ Glu (cytochrome b H ) D278, E295

m 38 heme b H

m(COO)) as Asp ⁄ Glu (cytochrome b H ) m(COO)) as heme propionates b L , b H

m19(CC) ring mode, protonated Tyr

m19(CC) ring mode, protonated Tyr

1447 (+) 1447 (+) m(COO))sD278

1428 ( )) m(COO)) s D278

m(COO)) s Asp ⁄ Glu (cytochrome b H )

1368 ( )) m(COO)) s heme propionates

Table 2 (Continued) Band position (cm)1) before and after stigmatellin addition

Assignment

m42heme c1

On this basis, we suggest that the side chain is

proton-ated in the oxidized form (signal at 1746 cm)1) and

de-protonated in the reduced form (signal at 1561 cm)1)

In the presence of inhibitor, the residue remains

pro-tonated in the oxidized form, but exhibits stronger

hydrogen bonding (signal at 1723 cm)1) In the

reduced form, however, it is possibly deprotonated

(signal at 1565 cm)1) The redox-induced FTIR

differ-ence spectrum of the D278 mutant indicates the partial

contribution of this side chain to the signals of the

protonated acidic residues for the oxidized form in the

absence of the inhibitor The shifts of the signals

attributed to E295 indicate perturbation of the

hydro-gen-bonding network in the D278N mutant

In a recent study, the influence of the mutation

E295 in the bc1 complex from Rhodobacter capsulatus

was assessed [50] No obvious influence of this

muta-tion on the FTIR spectra in comparison with wild-type

was reported for either the spectral region of

proton-ated acidic residues or the spectral region characteristic

of contributions from quinones and the backbone

While our approach targets the fully oxidized and

reduced forms of the enzyme, the data for R

capsula-tus present the reorganizations induced by heme bL

reduction only Obviously, the heme bLredox reaction

alone does not affect this residue We suggest that this

side chain is addressed by the quinone reaction, as also

suggested by the strong perturbations of the signals

around 1660–1630 cm)1 In the P denitrificans E295Q

mutant, this residue is perturbed, and the quinones are

involved in the redox reaction The data from both

studies may therefore be considered complementary

However, this may not be the only conflicting

evi-dence regarding mutations at position 295 Recently,

the stigmatellin resistance of yeast mutations at this

position has been studied by various groups: whereas

conservative replacements lead to increased

stigmatel-lin resistance [48], more pronounced exchanges had no

noteworthy effects [6] Indeed, none of the mutations

completely abolished the prominent signals

characteris-tic for protonated acidic residues We suggest that

resi-dues D278 and E295 both contribute to the signal of

the oxidized form Contributions from other acidic

res-idues within the enzyme cannot be excluded The

observation that several acidic residues participate in

this spectral feature is in line with the elaborate pH

dependency previously described [19]

The tyrosine mutations appear rather unperturbed

in comparison with wild-type, despite the close

prox-imity of the tyrosines to the Qo binding site Most of

the mutants studied here alter the spectral features of

the quinone, indicating a variation of the

hydrogen-bonding environment and⁄ or structure within the

binding site This observation is not surprising in the light of previous data showing that mutations on the Y302 site induce noticeable conformational changes, perturb kinetics, and affect inhibitor as well as quinone binding [30]

A second quinone has been discussed to be located

at the site [19,44], probably in direct interaction with the first quinone The exact position of this second quinone is not clear, and it is not possible to distin-guish which quinone is primarily perturbed by the var-ious mutations On the basis of prevvar-ious data and the intensity of the quinone modes, the second quinone bound is clearly observed in the redox-induced spectra [19] The intensity of the typical quinone signals pre-sented above indicates that more than one quinone is also present in the mutants The broadening of the m(CO) vibration at about 1654 cm)1, however, indi-cates that one of the quinones is less tightly bound Essential features observed for specific side chains studied in other bc1 complexes were also found to be important for the bc1 complex from P denitrificans Interestingly, most of the mutants retain a high degree

of catalytic activity (see Table 1), indicating a rather flexible binding site in the bacterial enzyme In a recent FTIR spectroscopic study, the infrared spectroscopic characteristics of the E295 mutant (E272 in yeast) were studied by a parallel approach [18] Stigmatellin bind-ing was found to induce a similar effect to that shown here: a signal for a protonated acidic residue at approximately 1724 cm)1appears and the original sig-nal decreases [18] These results are not unambiguous, especially in light of currently discussed mechanisms and experimental observations suggesting that E295 is deprotonated upon inhibitor binding [2,43] Certainly, the suggested proton transfer via residue E295 within the hydrogen-bonding network of a water channel could also occur with a protonated E295 residue [2,43,51] The binding of quinol to the protonated resi-due, however, is difficult to substantiate We note that binding of stigmatellin was previously suggested to mimic the interaction with the quinone radical [52] and the stable intermediate that involves binding of the Rieske iron sulfur protein [53] According to the cur-rent view, stigmatellin displaces a quinol molecule [51], and the spectra shown here (Fig 7) reflect this interac-tion We suggest that the high pK seen here for E295

in the oxidized form (> 7) may shift during the cata-lytic cycle, allowing deprotonation and thus stabiliza-tion of the quinol

The redox activity of the stigmatellin reported previ-ously [18] poses a challenge for data interpretation, as the structure of the reduced form is not clear A recent study [54] has suggested reduction of the C = O group

in the stigmatellin ring to a hydroxyl group, with the

COH moiety no longer interacting with the His group

from the Rieske protein Based on the redox potential

of the stigmatellin (P Hellwig and C Boudon, Institute

de Chimie, Louis Pasteur University of Strasbourg;

unpublished results), we note that the Rieske center is

exclusively affected by a change in the redox state of

the stigmatellin

In conclusion, the redox-induced FTIR difference

spectra of the site-directed mutations in the Qo

bind-ing site of the bc1 complex from P denitrificans, a

small bacterial version of the mitochondrial enzyme,

allow specific monitoring of the protonation state of

several crucial residues in the presence and absence of

stigmatellin Interestingly, several residues perturb the

orientation of the quinone binding site and are

poten-tial partners in a hydrogen-bonding network D278

and E81 have been found to be critically involved in

the interaction, in addition to the highly discussed

res-idues E295 and Y302 We conclude that a strong

interaction occurs among the residues of the quinone

binding site

Experimental procedures

Sample preparation

Mutagenesis

Mutagenesis for the Y302F protein was carried out using a

[45], into which a StuI site was introduced between the fbcF

and fbcB open reading frames at residue 1024, and

subcl-oned into the vector pSL1180 For mutations E81Q, D86N

SmaI cassette from the wild-type fbc operon introduced

into the pUC18 vector

The following primers were used: bE81Q, 5¢-CGCC

TCGGTCCAGCATATCATGCG-3¢; bD86N, 5¢-GCATA

5¢-GCCTTCATGGGCTTCGTGCTGCCCTGG-3¢; bD278N,

5¢-CTCGATATAGTTGTTGGGATGGCCCAG-3¢; bD295Q,

5¢-CATATCGTGCCGCAATGGTATTTCGTG-3¢; bY297F,

5¢-GTGCCGCAATGGTTCTTCCTGCCCTTC-3¢; bY302F,

5¢-GGTATTTCCTGCCCTTCTTCGCCATCCTGCG-3¢

These were phosphorylated with T4 kinase (Fermentas, St

Leon-Rot, Germany) as specified by the manufacturer

Mutations E81Q, D86N, Y147F and Y302F were

intro-duced into the wild-type fbc operon using the ‘Quik

Change’ mutagenesis kit from Stratagene (La Jolla, CA,

USA) The mutated cassettes were reinserted into the fbc

operon Mutations E295Q, Y297F, and D278N were

introduced using the Altered Sites system (Promega,

Man-nheim, Germany) All mutations were confirmed by DNA

sequencing

sites of the vector pRI2 [55] The resulting plasmids were conjugated into MK6, a chromosomal fbc deletion mutant

of P denitrificans [56], resulting in strains overexpressing the enzyme Cell growth, membrane isolation, solubiliza-tion and subsequent protein purificasolubiliza-tion were performed essentially as described previously [57], with the following modifications: membranes were solubilized with n-dodecyl

diluted to a salt concentration of 350 mm NaCl using

anion-exchange chromatography, and eluted using a salt gradient between 350 and 600 mm NaCl in the above

0.02% v DDM) Pooled fractions were concentrated by

Schwalbach, Germany; exclusion limit 100 kDa), equili-brated with the standard buffer for the FTIR experiments (100 mm phosphate buffer pH 7, 150 mm KCl, 0.02% DDM) by gel filtration (Sephadex G25 fine; GE Health-care, Munich, Germany), and subsequently ultrafiltrated

buffer, re-concentrated using ultrafiltration devices (Ami-con Micro(Ami-con, exclusion limit 100 kDa), and washed twice

found to be better than 80% as determined from the shift

of the amide II mode (data not shown) For inhibition of

1 h on ice in the presence of a 2-fold molar excess of stigmatellin

Activity assay

for the isolated wild-type and mutant preparations were measured using decyl-ubihydroquinone (80 lm) and horse heart cytochrome c (25 lm) as substrates in a buffer

and 0.04% DDM The reduction of cytochrome c was fol-lowed at 550 nm Dilutions of the concentrated samples for the activity measurements were made in a buffer containing

5% glycerol and 0.05% BSA To inhibit enzyme activity, stigmatellin from a stock solution of 10 mm in ethanol was added to a final concentration of 2 lm

condi-tions, but stigmatellin (0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10 lm final concentration from 10 mm stock in ethanol) was

against the common logarithm (log 10) of the stigmatellin

defined as the inflection point of the curve