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

Characterization of mutations in crucial residues around the Q o binding site of the cytochrome bc 1 complex from Paracoccus denitrificans Thomas Kleinschroth 1 , Oliver Anderka 1 , Michaela Ritter 2 , Andreas Stocker 1,2 , Thomas A. Link 2 , Bernd Ludwig 1 and Petra Hellwig 3 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 bc 1 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 bc 1 com- plexes contain three catalytic subunits: cytochrome c 1 Keywords bc 1 complex; 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 Q o binding site of the cyto- chrome bc 1 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 b L 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 Q o site 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; Q i, ubiquinone reduction site; Q o, ubiquinol oxidation site. FEBS Journal 275 (2008) 4773–4785 ª 2008 The Authors Journal compilation ª 2008 FEBS 4773 with covalently bound c-type heme, cytochrome b with two b-type hemes (b L and b H ), 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 bc 1 complexes follow the same catalytic mechanism, the so-called Q-cycle [7–9], which relies on two separate binding sites for quinones, Q o and Q i . The Q o site is located close to heme b L and the [2Fe– 2S] cluster, and the Q i site is close to heme b H on 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 Q o site have been discussed [10–15]. Inhibitors are an important tool for analysis of the molecular mechanism of the bc 1 complex, and have been extensively used to characterize the various qui- none binding sites [16]. Depending on their binding properties, Q o and Q i site-specific inhibitors may be distinguished. Stigmatellin binds at the Q o site and interacts with the Rieske protein and cytochrome b, and also influences the heme b L spectral properties. The crystal structure of the bc 1 complex with stigma- tellin bound at the Q o site [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 bc 1 complex). On the heme b L 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 bc 1 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 Q o site of the bc 1 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 bc 1 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 bc 1 complexes from other organisms. The mutated residues are highlighted in Fig. 1. Results Site-directed mutations in the Q o binding site Mutations in conserved positions of cytochrome b at the Q o site were constructed (Fig. 1). The three subun- its of the P. denitrificans bc 1 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 Q o site 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. Infrared spectroscopic characterization of mutations in the Q o site T. Kleinschroth et al. 4774 FEBS Journal 275 (2008) 4773–4785 ª 2008 The Authors Journal compilation ª 2008 FEBS solubilization, the complex was purified using a DEAE–Sepharose column, and the cytochrome bc 1 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. IC 50 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 IC 50 value. A distinct increase of the IC 50 value was observed for the E295Q and Y147F mutant enzymes. FTIR difference spectra of mutations in the Q o 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 b L , b H and c 1 , 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 bc 1 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 bc 1 monomer 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 IC 50 fold increase over wild-type Wild-type 100 a 1 b E81Q 120 3.5 D86N 66 1.4 D278N 105 1.3 E295Q 10 4.6 Y147F 55 5.2 Y297F 90 2.0 Y302F 95 2.1 a 100% indicates a turnover number of 327 s )1 based on one cyto- chrome b (per monomer). b 1 indicates an IC 50 value for the wild- type of 131 ± 7 n M under our experimental conditions. 1800 1700 1600 1500 1400 1300 1200 1455 1559 1474 1560 Δ Abs 0.001 1656 1654 1746 1746 1726 1724 1656 1656 1570 1724 1694 1628 1612 1561 1540 1289 1432 1496 1470 1264 1658 1746 WT E295Q D278N E81Q D86N Wavenumber (cm –1 ) 1800 1750 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 bc 1 complexes from P. denitrificans obtained for a change in potential from )0.292 to +0.708 V. The inset shows double difference spec- tra obtained by subtracting the wild-type red-ox difference spec- trum from that of each mutant. T. Kleinschroth et al. Infrared spectroscopic characterization of mutations in the Q o site FEBS Journal 275 (2008) 4773–4785 ª 2008 The Authors Journal compilation ª 2008 FEBS 4775 acidic residues were perturbed. The decrease is shown in the inset to Fig. 2, showing double difference spec- tra obtained by subtracting 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 )1 that 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 m 19 (CC) ring mode. At 1249 cm )1 , a signal com- posed of the m 7’a (CO) vibration and the d(COH) vibra- tion is expected, and the position is sensitive to the hydrogen-bonding environment [23,25,28,29]. For deprotonated tyrosine in solution, the m 8a ⁄ 8b (CC) ring mode was identified at 1560 cm )1 and the m 19 (CC) ring mode at 1499 cm )1 , thus reflecting the sensitivity of the ring modes to the protonation state of the phenyl group. The m 7’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 )1 for m(C = N), 1633 cm )1 for d(NH 3 + ) as and 1522 cm )1 for d(NH 3 + ) 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. 1522 1626 1666 1264 1516 1575 1561 1561 1520 1516 1507 1507 Δ Abs 1540 1507 1540 1540 1800 1700 1600 1500 1400 1300 1200 1658 1644 1644 1644 1658 1656 1746 1746 1746 1644 1550 1508 1498 1520 1630 1658 1746 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. Infrared spectroscopic characterization of mutations in the Q o site T. Kleinschroth et al. 4776 FEBS Journal 275 (2008) 4773–4785 ª 2008 The Authors Journal compilation ª 2008 FEBS 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 )1 is 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 )1 both 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 bc 1 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 bc 1 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 1750 16501700 1600 D278N E295Q WT 1724 1726 1751 1746 1724 1693 1612 1644 1658 1746 Wavenumber (cm –1 ) 1800 1700 1600 1500 1400 1300 1200 0.001 Δ Abs D86N E81Q D278N E295Q WT 1448 1639 1560 1540 1266 1692 1448 1655 1657 1655 1635 1452 1448 1639 1639 1743 Wavenumber (cm –1 ) Fig. 4. Overview of the oxidized-minus-reduced FTIR difference spectra of wild-type and mutant cytochrome bc 1 complexes 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 H 2 O buffer. T. Kleinschroth et al. Infrared spectroscopic characterization of mutations in the Q o site FEBS Journal 275 (2008) 4773–4785 ª 2008 The Authors Journal compilation ª 2008 FEBS 4777 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 )1 is seen. This is in line with a previous study on the yeast bc 1 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 )1 indicate 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 bc 1 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 subtracting 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 )1 in 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 Δ s b A A B 8 8 5 1 7 4 4 1 2 4 2 1 8 2 4 1 0 4 5 1 8 1 6 1 5 3 6 1 6 4 6 1 0 7 6 1 8 9 6 1 4 4 7 1 3 2 7 1 0.002 Δ s b A 4 0 7 1 C 3 1 5 1 4 9 2 1 6 4 3 1 3 8 3 1 4 4 4 1 7 6 4 1 5 3 5 1 3 6 5 1 0 0 6 1 2 2 6 1 3 9 6 1 4 4 6 1 0 7 6 1 2 5 2 1 Wavenumber (cm –1 ) Wavenumber (cm –1 ) 1775 3 2 7 1 (cm –1 ) 4 4 7 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. Infrared spectroscopic characterization of mutations in the Q o site T. Kleinschroth et al. 4778 FEBS Journal 275 (2008) 4773–4785 ª 2008 The Authors Journal compilation ª 2008 FEBS In the case of the D278N mutant enzyme, a differen- tial signal was observed at 1750 ⁄ 1728 cm )1 upon 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 Q o site, 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 )1 cannot 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 bc 1 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 Q o binding site, and residues E81, D86 and D278, located close to the surface. These structural regions are analyzed and 1700 1600 1500 1400 0.0005 ΔΔ sbA 6461 88 5 1 2051 20 5 1 21 5 1 5361 3371 4471 0 571 0 6 5 1 8241 644 1 4351 7441 8 2 41 5 651 0451 2 4 51 2561 056 1 8661 0761 0761 89 6 1 2071 7071 3271 8271 ddwt ddD278N ddE295Q Wavenumber (cm –1 ) 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. 1800 1700 1600 1500 1400 1300 1200 0.001 Δ s bA 1651 6461 4471 6 471 2471 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 bc 1 complex from P. denitrificans obtained for a change in potential from )0.292 to +0.708 V in the presence of stigmatellin. T. Kleinschroth et al. Infrared spectroscopic characterization of mutations in the Q o site FEBS Journal 275 (2008) 4773–4785 ª 2008 The Authors Journal compilation ª 2008 FEBS 4779 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 Q o 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 b L edge, 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 bc 1 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 Q o binding site. The observed shifts may be a sec- ondary-order effect induced by perturbation of the water chain that leads to the heme b L 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 AssignmentBefore After 1746 (+) m(C = O) D278, E295 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 b L , b H 1680 (+) m(C = O) heme propionates b L , b H , c 1 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 1644 (+) m(C = O) quinone m 37 heme c 1 1628 ()) Amide I (Rieske b-sheet) m(CN 3 H 5 ) Arg (cytochrome b H ) 1612 (+) m(C = C) quinone 1592 (+) 1570 (+) Amide II m 37 heme b L m 38 heme c 1 1561 ()) m(COO ) ) as D278, E295 1565 ⁄ 1540 ()) m(COO ) ) as heme propionates b L , b H , c 1 m(COO ) ) as Asp ⁄ Glu (cytochrome b H ) D278, E295 1550 (+) Amide II m 38 heme b H 1540 ()) Amide II m(COO ) ) as Asp ⁄ Glu (cytochrome b H ) m(COO ) ) as heme propionates b L , b H 1520 (+) Y297, Y302 m 19 (CC) ring mode, protonated Tyr 1516 ()) Y297, Y302 m 19 (CC) ring mode, protonated Tyr 1508 (+) Amide II (Rieske) 1496 ()) Quinone ring 1470 ()) Quinone ring 1447 (+) 1447 (+) m(COO ) ) s D278 1432 ()) Quinone ring 1428 ()) m(COO ) ) s D278 1408 ()) Quinone ring 1388 ()) Quinone ring 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 Before After 1289 (+) m(C–O) methoxy group, quinone 1264 (+) m(C–O) methoxy group, quinone m 42 heme c 1 1240 ()) m 42 heme b H 1204 (+) Quinone Infrared spectroscopic characterization of mutations in the Q o site T. Kleinschroth et al. 4780 FEBS Journal 275 (2008) 4773–4785 ª 2008 The Authors Journal compilation ª 2008 FEBS 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 bc 1 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 b L reduction only. Obviously, the heme b L redox 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 Q o 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 previous 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 bc 1 complexes were also found to be important for the bc 1 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 )1 appears 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 T. Kleinschroth et al. Infrared spectroscopic characterization of mutations in the Q o site FEBS Journal 275 (2008) 4773–4785 ª 2008 The Authors Journal compilation ª 2008 FEBS 4781 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 Q o bind- ing site of the bc 1 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 StuI ⁄ XhoI cassette excised from the wild-type fbc operon [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 and Y147F, mutagenesis was performed using an NcoI ⁄ 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 TCATGCGCAACGTGAACGGCGGCTAC-3¢; bY147F, 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. fbc operons encoding the wild-type or mutated P. deni- trificans bc 1 complex were cloned into the HindIII ⁄ SacI 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 purification were performed essentially as described previously [57], with the following modifications: membranes were solubilized with n-dodecyl b-d-maltoside (DDM; 1.2 gÆg )1 protein), subsequently diluted to a salt concentration of 350 mm NaCl using 50 mm Mes ⁄ NaOH (pH 6.0), 0.02% w ⁄ v DDM before anion-exchange chromatography, and eluted using a salt gradient between 350 and 600 mm NaCl in the above mentioned detergent buffer (50mm Mes ⁄ NaOH, pH 6.0, 0.02% v DDM). Pooled fractions were concentrated by ultrafiltration (Amicon Centriprep ⁄ Centricon, Milipore, 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 again to a final bc 1 enzyme concentration of approxi- mately 0.5–2 mm. For H ⁄ D exchange, samples were equili- brated in a 100-fold excess of the corresponding D 2 O buffer, re-concentrated using ultrafiltration devices (Ami- con Microcon, exclusion limit 100 kDa), and washed twice with the same buffer for 30 min. H ⁄ D exchange was found to be better than 80% as determined from the shift of the amide II mode (data not shown). For inhibition of the Q o site, the concentrated samples were incubated for 1 h on ice in the presence of a 2-fold molar excess of stigmatellin. Activity assay Ubihydroquinone–cytochrome c oxidoreductase activities 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 contain- ing 50 mm Mops ⁄ NaOH pH 7.5, 1 mm EDTA, 1 mm KCN 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 50 mm Mops ⁄ NaOH pH 7.5, 100 mm NaCl, 0.04% DDM, 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. The IC 50 value was determined under activity test 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 added before the addition of the enzyme. V max was plotted against the common logarithm (log 10) of the stigmatellin concentration and fitted non-linearly. The IC 50 value is defined as the inflection point of the curve. Infrared spectroscopic characterization of mutations in the Q o site T. Kleinschroth et al. 4782 FEBS Journal 275 (2008) 4773–4785 ª 2008 The Authors Journal compilation ª 2008 FEBS [...]... 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Characterization of mutations in crucial residues around the Q o binding site of the cytochrome bc 1 complex from Paracoccus denitrificans Thomas Kleinschroth 1 ,. 2008) doi:10.1111/j.1742-4658.2008.06611.x The protonation state of residues around the Q o binding site of the cyto- chrome bc 1 complex from Paracoccus denitrificans and their interaction with