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Structure of cytochrome c oxidase: a comparison of the bacterial and mitochondrial enzymes
Biochimica et Biophysica Acta 1544 (2001) 1^9 www.elsevier.com/locate/bba
Review
Structure of cytochrome c oxidase: a comparison of the bacterial and mitochondrial enzymes Je¡ Abramson a , Margareta Svensson-Ek a , Bernadette Byrne
a;b
, So Iwata
a;b;
*
a
b
Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, SE-751 23 Uppsala, Sweden Department of Biochemistry and Division of Biomedical Sciences, Imperial College of Science, Technology and Medicine, London SW7 2AY, UK Received 8 June 2000; received in revised form 28 September 2000; accepted 3 October 2000
Abstract It has been almost 5 years since the first structures of cytochrome c oxidase, from Paracoccus denitrificans and bovine heart mitochondria, were revealed. Since then many different proton pumping mechanisms have been proposed for the enzyme; however, no definitive conclusion has been achieved. In this article, we revisit the original structures of bacterial and mitochondrial oxidases and try to clarify similarities as well as differences between the two structures. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Cytochrome c oxidase; X-ray crystallography; Proton pump; Membrane protein; Proton pathway
1. Introduction Cytochrome c oxidase belongs to the superfamily of terminal oxidases known as the heme-copper oxidases. Members of this superfamily are redox driven proton pumps that couple the reduction of molecular oxygen to vectorial translocation of protons across the membrane [1,2]. The superfamily is de¢ned by a high sequence similarity within the largest subunit (subunit I) and a binuclear active site consisting of a high-spin heme (heme a3 ) and a closely associated copper ion (CuB ). In addition to the binuclear center, cytochrome c oxidase has a low-spin heme (heme a) within subunit I and another copper center (CuA ), Abbreviations: BOX, cytochrome c oxidase from bovine heart mitochondria; POX, cytochrome c oxidase from Paracoccus denitri¢cans * Corresponding author, at address a. Fax: +46-18-511-755.
which is located in the extrinsic domain of subunit II. Electrons from cytochrome c enter the protein at the binuclear CuA center where they are transferred to heme a and then further channeled to the binuclear oxygen binding site. Fig. 1 summarizes the metal centers and the electron transfer pathway in cytochrome c oxidase. During the reduction of molecular oxygen to water four protons are consumed from the `IN' side of the membrane [2]. Four additional protons are translocated from the `IN' side to the `OUT' side utilizing the free energy available from the exergonic reaction of the reduction of oxygen to water (Fig. 1) [1,3]. The active transport of protons generates a proton and voltage gradient that is converted to more useful energy forms via energy conserving systems such as ATP synthase. A major advance in cytochrome c oxidase research was achieved with the X-ray structures of Paracoccus
0167-4838 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 0 0 ) 0 0 2 4 1 - 7
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Fig. 1. Schematic drawing of subunits I and II of cytochrome c oxidase. In the ¢gure, a and a3 represent heme a and heme a3 , respectively. The electron transfer and the proton translocation pathways are shown by arrows.
denitri¢cans (POX) and mitochondrial (BOX) enzymes in 1995. The POX structure (four-subunit enî [4]; this zyme) was initially determined to a of 2.8 A was followed by the structure of the two-subunit î [5] and then an oxidase (subunits I and II) at 2.7 A improved structure of the four-subunit enzyme [6]. The BOX structure was also initially reported to a î resolution [7] but has been largely improved 2.8 A î ), fully with the structures of fully oxidized (2.3 A î î reduced (2.35 A), azide bound (2.9 A) and carbon î ) forms published [8]. monoxide-bound (2.8 A In spite of rapid progress in structural studies, the proton pumping mechanism of cytochrome c oxidase remains largely unsolved. Utilizing the POX structure and the histidine cycle mechanism from Wikstro«m [9], Michel and his colleagues originally proposed the `Histidine shuttle' mechanism [4]. In recent publications [6,10,11] Michel has discarded the `Histidine shuttle' mechanism in favor of a new model based on electron-proton transfer coupling mainly deduced from pKa calculations on the POX structure. In the model of Wikstro«m, the proton pumping is directly coupled to the oxygen reduction reaction rather than the electron transfer by itself. One common feature of their respective mechanisms is that the energy from oxygen reduction is directly used for proton translocation within close proximity to the binuclear center.
The proton pumping model proposed by Yoshikawa and his colleagues for the bovine enzyme [8] is substantially di¡erent from the models presented by Michel and Wikstro«m. Both Michel and Wikstro«m's models attempt to be consistent with the structural data for the POX as well as the BOX enzyme; however, Yoshikawa's model is speci¢cally tailored for the BOX enzyme. Yoshikawa's model was inspired by observable conformational changes at the outlet of the H-channel between the reduced and oxidized structure of BOX. Using these conformational changes, they explain proton pumping in BOX as an indirect conformational coupling mechanism. There are two main arguments that contradict the model proposed by Yoshikawa as a general mechanism for terminal oxidases: (1) the H-channel is not present in the bacterial oxidases; (2) a computational simulation has shown that the H-channel is not likely to be a proton channel [12]. In this article, we revisit the original structures of bacterial and mitochondrial oxidases and try to clarify similarities and di¡erences between the POX and BOX structures. We also highlight results obtained with mutant enzymes from other sources, especially from Escherichia coli and Rhodobacter sphaeroides. We will focus on the structure of subunit I, the heart of the proton pump. 2. Overall structure of subunit I In the following discussion, we refer to the two subunit oxidase structure of POX (PDB entry, 1AR1) and the fully oxidized structure of BOX (PDB entry, 2OCC), unless otherwise speci¢ed. The numbering for BOX is used for all residues with the POX residue number in brackets. BOX and POX have 13 and four subunits, respectively. Amino acid sequences of the three core subunits (subunits I, II and III) are well conserved for both enzymes and the structures of these subunits are, indeed, very similar. In Fig. 2A,B structural overviews of subunit I (POX) containing 12 transmembrane helices are shown. Fig. 2B (viewed from the `OUT' side of the membrane) depicts three arcs, which are related by a pseudo-threefold symmetry [4]. Each arc is shaped by four transmembrane helices and together with the last segment of the previous semicircle a pore-like
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Fig. 2. Structure of subunit I of cytochrome c oxidase from P. denitri¢cans (POX). (A) View parallel to the membrane. The subunit contains 12 transmembrane helices. Heme a (red), heme a3 (cyan), CuB (blue sphere), magnesium ion (magenta sphere) and calcium ion (green sphere) are also shown. (B) View along the membrane normal from `OUT' side of the membrane. Only transmembrane helices are shown with hemes and metal centers. The coloring of the hemes and metal centers are the same as in panel A. This ¢gure was drawn with a modi¢ed version of MOLSCRIPT [41].
appearance is formed. The three pores (pores A, B and C) vary in function. Pores B and C hold the binuclear center (heme a3 and CuB ) and heme a, respectively. The `IN' side half of pores A and B house the D- and K-proton pathways, respectively (see below); and the `OUT' side half of pore A is a possible oxygen pathway [13].
(OH) group of the hydroxylethylfarnesyl side chain because the OH group is pointing in the opposite direction (Fig. 3A). In the BOX structure, the hydrogen bond network around the OH group is part of the H-channel, the third proton pathway. Lack of this network suggests that there is no H-channel in POX (see below).
2.1. Metal centers
2.1.2. Binuclear center The binuclear center, where molecular oxygen is reduced to water, is composed of heme a3 and the CuB ion (Fig. 3B). The axial ligand of heme a3 is His 376 (411) and the CuB ligands are His 240 (276), His 290 (325) and His 291 (326). The iron of heme a3 is î out of the heme plane in POX but almost 0.36 A within the plane in BOX. In POX, the heme a3 î and 5.2 A î in the two and CuB distance is 4.5 A four subunit enzymes, respectively, the distance is independent of the redox state [6]. In the bovine enzyme, the distance varies depending on the redox î , 5.2 A î state and the presence of a bound ligand; 4.9 A î and 5.3 A have been reported for the fully oxidized enzyme (no ligand), the fully reduced enzyme (no
2.1.1. Heme a Heme a is a low-spin heme maintaining two axial histidine ligands, His 61 (94) and His 378 (413) (Fig. 3A). The heme environments in POX and BOX are very similar. The conformation of the hydroxylethylfarnesyl chain of heme a is slightly, but signi¢cantly di¡erent between the two enzymes (Fig. 3A). The hydroxyl group of the farnesyl chain in BOX has hydrogen bonds to Ser 382 (417) and a water molecule. This water molecule has additional hydrogen bonds to Ser 461 (496) and Thr 424 (Ile 459). Although Ser 382 (417) is conserved in the POX enzyme, there is no hydrogen bond to the hydroxyl
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ligand) and the fully reduced enzyme with bound CO, respectively. According to Yoshikawa and his colleagues, the position of heme a3 is always ¢xed but the position of CuB di¡ers depending on either the oxidation state of the metal sites or the binding of a ligand [8]. Even without adding any inhibitor or substrate, some electron density is always observed between heme a3 and CuB for both POX and BOX. In the fully oxidized BOX structure a peroxide ligand has been modeled into the density [8]. However, in the POX structure, a hydroxide and a water molecule have been proposed as the bridging ligands [5] and this model is further supported by spectroscopic data [14]. A covalent bond between the NO2 of His 240 (276) and CO2 of Tyr 244 (280) has been reported for both BOX [8] and POX [5]. In the BOX structure, it was reported that this covalent bond exists regardless of the redox state or the presence of a bound ligand. It has been suggested that this covalent linkage could be caused by a radical reaction as reported for catalase HPII from E. coli where a similar covalent bond is formed between the CL atom of Tyr 415 and the
NN1 atom of His 392 [15]. It has further been suggested that the pK of Tyr 244 (280) could be considerably reduced by the formation of this bond [8] but no experimental data that support this have been reported. On the other hand, a related terminal oxidase, the cbb3 -type cytochrome c oxidase lacks a homologue to this Tyr, as well as several other key residues, but still functions as a proton pump albeit at reduced levels [16,17] leaving the role of this HisTyr bond still unclear. 2.1.3. Non-redox metal centers In addition to the redox-active centers mentioned above, the enzyme also contains tightly bound nonredox-active metal centers. These are Mg2 and Ca2 in cytochrome c oxidase from POX and Mg2 , Na and Zn2 in cytochrome c oxidase from BOX. The zinc ion in BOX is bound by a nuclear encoded subunit (subunit Vb) on the matrix side of the membrane (`IN'). All of the other non-redox-active metals are bound within subunit I. The Mg2 binding site is located at the interface between subunits I and II and is in close proximity to the heme a3 propionates (Fig.
Fig. 3. Structures of heme a and heme a3 and surrounding residues in cytochrome c oxidase. (A) Structure of heme a and surrounding residues of cytochrome c oxidase from bovine heart mitochondria (BOX) and P. denitri¢cans (POX). The BOX structure is colored based on the atom type (C: yellow, N: blue, O: red, Fe: red). The POX structure is shown in cyan. A water molecule and the hydroxyl group of the hydroxylethylfarnesyl groups are labeled by w and OH, respectively. The water molecule was not in the PDB ¢le (2OCC) but added based on the description of [8]. (B) Structure of heme a3 and surrounding residues of cytochrome c oxidase from bovine heart mitochondria (BOX) and P. denitri¢cans (POX). The coloring scheme is the same as in panel A. In the BOX structure, Y244 (280)-H240 (276) is covalently bonded. For the POX structure, a similar structure was observed but these are not connected in the PDB entry 1AR1. In the BOX structure, a bridge ligand between heme a and CuB has been putatively assigned as a peroxide molecule. This ¢gure was generated in program O [42].
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interface, which could be important for proton pumping.
Fig. 4. The proton pathways and the Mg2 binding site. The residues in bovine cytochrome c oxidase (BOX, red) and P. denitri¢cans oxidase (POX, blue) are superimposed. Structures of helices, heme a3 and metal centers are based on the POX structure. This ¢gure was drawn with a modi¢ed version of MOLSCRIPT [41].
2.1.3.2. Ca2 +/Na + binding site. The other non-redox metal binding site is formed by a loop between the transmembrane helices I and II on the `OUT' side surface of subunit I (Fig. 5). In the crystal structures, calcium and sodium ions are assigned for POX and BOX, respectively, based on the ligand coordination patterns. It has been suggested, however, that the metal in the POX could also be a sodium ion [21]. The function of this metal binding site is also unknown. In the quinol oxidase from E. coli, no metal binding site has been found at this position [22]. It has been suggested that the Ca2 /Na site could also be involved in the exit pathway of the pumped protons [21]. The ligands of the POX Ca2 site are the backbone carbonyl oxygen atoms from residues Glu 40
2). The Ca2 (POX)/Na (BOX) binding site is formed by a loop between the transmembrane helices I and II at the `OUT' side surface of the membrane. 2.1.3.1. Mg2 + site. In both POX and BOX structures, the non-redox-active metal that was observed in the interface between subunits I and II (Fig. 4) was assigned as a magnesium ion, although it can be replaced by a manganese ion in POX [5]. The ligands of the magnesium ion are His 368 (403), Asp 369 (404), Glu 198 (218) of subunit II and three water molecules (not shown in Fig. 4). The function of the Mg2 site is unknown. Since the site is in the vicinity of a possible proton exit pathway, it was suggested that it could be involved in proton transfer; however, mutant enzymes lacking this magnesium ion maintain an activity level greater than 50% and show no change in proton pumping e¤ciency [18,19]. Moreover, quinol oxidases do not have the Mg2 site but still pump protons [20]. The site could have a structural role for stabilization of the interface between subunits I and II since both subunits have a number of negatively charged residues at the
Fig. 5. Structure of Ca2 /Na binding site. The structure of bovine cytochrome c oxidase (BOX) is colored based on the atom type (C: yellow, N: blue, O: red, Na: yellow). The structure of P. denitri¢cans oxidase (POX) is shown in cyan. BOX and POX bind Ca2 and Na , respectively. The water molecules mentioned in the main text are not shown since the PDB ¢les (1AR1 and 2OCC) do not contain the coordinates. This ¢gure was generated in program O [42].
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(56), His 43 (Phe 59), and Gly 45 (61), the side chain oxygen atoms from Glu 40 (56) and Gln 63 (POX) and one water molecule (not shown in Fig. 5). In BOX, the backbone carbonyl oxygen atoms of Glu 40 (56), Gly 45 (61) and Ser 441 (Ile 476), the side chain oxygen atom of Glu 40 (56) and a water molecule (not shown in the ¢gure) are reported as metal ligands to the sodium ion. The di¡erence between the metal binding sites of the two reported structures may be due to the lack of an equivalent side chain for Gln 63 (POX) in BOX. This metal binding site is within close proximity to î ). Many years ago a spectral heme a (approx. 17 A perturbation of heme a upon the binding of the Ca2 was observed in mitochondrial cytochrome c oxidase [23]. This spectral change was not observed in wild type POX [24] but it has been shown that the same perturbation can be reproduced in POX when mutations have been made around the Ca2 site [21]. The physiological relevance of this interaction is not clear; however, since the metal binding site is close to the exit of the H-channel in the bovine enzyme, it could be related to the function of this pathway. 2.2. Proton pathways The oxygen binding site is located in the membrane spanning hydrophobic region of the protein, î from the `IN' side surface of the memabout 35 A brane. Thus, the enzyme needs a proton translocating pathway for substrate protons as well as for protons to be pumped across the membrane. Extensive mutational studies were performed and key residues involved in proton uptake and translocation have been identi¢ed [18,25,26]. When the ¢rst structures of cytochrome c oxidase were unveiled, two polar cavities were revealed as possible proton pathways (Fig. 4). This ¢nding was supported by many of the previous as well as proceeding mutational studies [27^30]. These two pathways, the D- and K-channels named after residue Asp 91 (124) and Lys 319 (354), are essentially the same for both structures. The Kchannel leads into pore B from Ser 255 (291) to the binuclear center through Lys 319 (354), Thr 316 (351), the OH group of the hydroxylethylfarnesyl side chain of heme a3 , Tyr 244 (280) and His 240 (276). A tightly bound water molecule is observed between Thr 316 (351) and the hydroxyl group.
There is a large hydrophobic gap between Lys 319 (354) and Thr 316 (351) where no water molecules have been found. Therefore, Lys 319 (354) is assumed to shuttle between Ser 255 (291) and Thr 316 (351) during proton translocation. Studies on the related ubiquinol oxidase from E. coli show that within close proximity to Lys 319 (354), there is a conserved glutamate, Glu 62 (78) of subunit II which could be an alternative entrance of the K-channel since mutations of this residue reduce the activity of the enzyme [31]. The second pathway, the D-channel, starts with Asp 91 (124) and leads to Glu 242 (278) through a large polar cavity ¢lled with water molecules in pore A. These polar residues along with water molecules form a hydrogen bond network, which connects Asp 91 (124) to Glu 242 (278). Thus, protons can be translocated through the hydrogen bonds in this pathway. For many years Glu 242 (278) has been shown to be a key component in the proton pumping mechanism of terminal oxidases [27,28,30,32]. Surprisingly, the hydrogen bonding environment of Glu 242 (278) between POX and BOX is di¡erent. The Glu 242 (278) of POX is hydrogen bonded to the main chain carbonyl oxygen of Ile 66 (Met 99) and seems to always be protonated at neutral pH. While in the BOX structure, Glu 242 (278) is hydrogen bonded to the SN atom of the Met 71 (Ile 104) side chain. Recent reports have shown that some terminal oxidases are lacking this key glutamate in the D-channel but yet still pump protons [33,34]. However, in these structurally distant members of the heme-copper oxidase, the fourth residue upstream from the Glu 242 (278) is often a Tyr residue. In a recent publication by Backgren et al. [35] a Tyr residue was shown to be able to facilitate enzyme activity as well as proton pumping in a triple mutant of POX. After this glutamic acid the connection of the D-channel is not clear. The pathway could be connected to the binuclear center or a heme a3 propionate through a rearrangement in the water chain depending on the charge distribution in the enzyme. In this way, the area could form a proton gate that is essential for the proton pumping. Many details of the two pathways have been studied over the years using mutational and spectroscopic studies in conjunction [36] but the precise
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mechanistic approach for delivering and pumping of protons has yet to be con¢rmed. Mutational studies of Lys 319 (354) concluded that the K-channel is only used during the reduction step of the enzyme and takes up one or two chemical protons [29]. The results of mutational studies on Asp 91 (124) and Glu 242 (278) [28,37] strongly support that all other protons are taken up through the D-channel. For a full understanding of the roles of these two pathways, the exact timing of proton pumping in relation to the oxygen reduction cycle must be worked out and correlated with structures of oxygen intermediates. The proton exit pathway is less clear. In both enzymes, many tightly bound water molecules are observed in the space between subunits I and II. The heme a3 propionate, mentioned above, also exists within this cavity (pore B) and is connected to the outside of the protein through a hydrogen bond network of the water molecules. In addition, there seems to be more than one possible proton exit pathway. In the BOX structure, a third proton pathway, the H-channel, has been suggested. The H-channel leads into pore C from Asp 407 (Glu 442) to Asp 51 (Gly 84) through seven polar side chains and two water molecules and one main chain peptide bond. In the oxidized enzyme structure, Asp 51 (Gly 84) has hydrogen bonds to Ser 205 (Ala 225) and Ser 441 (Ile 476) which are located in the interior of the molecule and renders the pathway closed. In the fully reduced enzyme, however, this residue is exposed to the solvent. Based on this conformational change, Yoshikawa and his colleagues propose a conformationally coupled proton pumping mechanism in BOX. However, there are several di¤culties applying this model for all terminal oxidases. To begin with, the hydrogen bond network is not entirely connected through the H-channel. In their model, protons need to be transferred through the protein polypeptide chain, which is highly unlikely. In addition, the conformation of the hydroxylethylfarnesyl chain of heme a is di¡erent in the POX structure which prevents the formation of the hydrogen bond network seen in the H-channel in the BOX structure. Furthermore, the residues in the channel are not fully conserved in bacterial enzymes, i.e. POX lacks the key residue Asp 51 (Gly 84). Mutations of residues around this area have little to no e¡ect on the activity of the
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bacterial oxidase [38]. However, it is still possible that BOX has a di¡erent proton pumping mechanism from the bacterial enzymes, but there have been no experimental data that support the H-channel model. 3. Other terminal oxidase structures Recently two additional structures of heme copper oxidases have been published and a third is soon on the way. The hope is by combining information available from all structures, a general mechanistic approach for proton consumption and translocation can be achieved. Here we will shortly summarize the new structures. The structure of the ba3 -cytochrome c oxidase from Thermus thermophilus has been reported to a î [34]. The amino acid sequence resolution of 2.4 A of this enzyme shows a clear but distant homology to other members of the super family. In brief, this structure identi¢es both the D- and K-pathways although many key residues are not conserved. In addition, a third proton pathway, termed the `Qpathway' has been identi¢ed and is not equivalent to any other reported pathway. Other key observations are a distinct covalent bond between Tyr 244 (280) and His 240 (276) (Tyr 237 and His 233 ba3 numbering) and the lack of a Glu 242 (278) homologue. Recently the structure of cytochrome bo3 oxidase î resolution [22]. Unlike all prewas reported at 3.5 A vious terminal oxidase structures, this is a quinol oxidase that utilizes ubiquinol as a substrate instead of cytochrome c. The structure has con¢rmed both the D- and K-pathways in ubiquinol oxidase as well as the newly found ubiquinol binding site. Resolution limits of cytochrome bo3 prevent the assigning of a covalent bond between Tyr 244 (280) and His 240 (276). Finally, the structure of another aa3 cytochrome c oxidase from R. sphaeroides is soon to be published î (M. Svensson-Ek, L. Rodto a resolution of 2.3 A gers, J. Abramson, P. Brzezinski, S. Iwata, unpublished data). The work of cytochrome aa3 from R. sphaeroides compares the structures of wild type and mutant enzymes showing, for the ¢rst time, a rearrangement of the water pathway around the binu-
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clear center. Again both the D- and K-pathways are seen but the question of a covalent bond between Tyr 244 (280) and His 240 (276) is uncertain. The electron density indicates that the bond could be only partially formed in this enzyme. 4. Summary On the whole, protein crystallography, mutational studies and spectroscopy have revealed the structural and functional framework of a fascinating enzyme, cytochrome c oxidase. Despite signi¢cant results within these areas, the proton pumping mechanism of the enzyme remains largely unresolved. This is mainly due to the di¤culties to resolve the timing of the proton pumping during the oxygen reduction and the structural rearrangements in the reaction cycle. Invisibility of the protons by X-ray crystallography and the rather small conformational changes during proton pumping are additional di¤culties. Upcoming structures at higher resolution and from other species will certainly help the understanding of the mechanism. In bacteriorhodopsin several intermediate structures have been obtained, intermediate structures that are slightly di¡erent from one another but together reveal the pumping mechanism in this enzyme [39,40]. For this to occur for heme copper oxidases, a similar approach of combining structural studies with sophisticated biochemical and spectroscopic analysis must be implemented. Acknowledgements The Swedish Research Council, NFR and the EC biotechnology program, support this work. We would also like to thank the reviewer whose comments made this a more complete study.
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J. Abramson et al. / Biochimica et Biophysica Acta 1544 (2001) 1^9 î delroth, M.S. Ek, D.M. Mitchell, R.B. Gennis, P. Brze[28] P. A zinski, Biochemistry 36 (1997) 13824^13829. î delroth, R.B. Gennis, P. Brzezinski, Biochemistry 37 [29] P. A (1998) 2470^2476. [30] A.A. Konstantinov, S. Siletsky, D. Mitchell, A. Kaulen, R.B. Gennis, Proc. Natl. Acad. Sci. USA 94 (1997) 9085^ 9090. [31] J. Ma, P.H. Tsatsos, D. Zaslavsky, B. Barquera, J.W. Thomas, A. Katsonouri, A. Puustinen, M. Wikstro«m, P. Brzezinski, J.O. Alben, R.B. Gennis, Biochemistry 38 (1999) 15150^15156. [32] M.L. Verkhovskaya, A. Garcia-Horsman, A. Puustinen, J.L. Rigaud, J.E. Morgan, M.I. Verkhovsky, M. Wikstro«m, Proc. Natl. Acad. Sci. USA 94 (1997) 10128^10131. [33] M.M. Pereira, M.L. Verkhovskaya, M. Teixeira, M.I. Verkhovsky, Biochemistry 39 (2000) 6336^6340. [34] T. Soulimane, G. Buse, G.P. Bourenkov, H.D. Bartunik, R. Huber, M.E. Than, EMBO J. 19 (2000) 1766^1776.
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[35] C. Backgren, G. Hummer, M. Wikstro«m, A. Puustinen, Biochemistry 39 (2000) 7863^7867. [36] R.B. Gennis, Science 280 (1998) 1712^1713. î delroth, R.B. Gennis, P. Brzezinski, Bio[37] I.A. Smirnova, P. A chemistry 38 (1999) 6826^6833. [38] H.M. Lee, T.K. Das, D.L. Rousseau, D. Mills, S. FergusonMiller, R.B. Gennis, Biochemistry 39 (2000) 2989^2996. [39] K. Edman, P. Nollert, A. Royant, H. Belrhali, E. PebayPeyroula, J. Hajdu, R. Neutze, E.M. Landau, Nature 401 (1999) 822^826. [40] A. Royant, K. Edman, T. Ursby, E. Pebay-Peyroula, E.M. Landau, R. Neutze, Nature 406 (2000) 645^648. [41] R.M. Esnouf, J. Mol. Graph. Model. 15 (1997) 132^134, 112^133. [42] T.A. Jones, J.-Y. Zou, S.W. Cowan, M. Kjeldgaard, Acta Crystallogr. A 47 (1991) 110^119.
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Review
Structure of cytochrome c oxidase: a comparison of the bacterial and mitochondrial enzymes Je¡ Abramson a , Margareta Svensson-Ek a , Bernadette Byrne
a;b
, So Iwata
a;b;
*
a
b
Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, SE-751 23 Uppsala, Sweden Department of Biochemistry and Division of Biomedical Sciences, Imperial College of Science, Technology and Medicine, London SW7 2AY, UK Received 8 June 2000; received in revised form 28 September 2000; accepted 3 October 2000
Abstract It has been almost 5 years since the first structures of cytochrome c oxidase, from Paracoccus denitrificans and bovine heart mitochondria, were revealed. Since then many different proton pumping mechanisms have been proposed for the enzyme; however, no definitive conclusion has been achieved. In this article, we revisit the original structures of bacterial and mitochondrial oxidases and try to clarify similarities as well as differences between the two structures. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Cytochrome c oxidase; X-ray crystallography; Proton pump; Membrane protein; Proton pathway
1. Introduction Cytochrome c oxidase belongs to the superfamily of terminal oxidases known as the heme-copper oxidases. Members of this superfamily are redox driven proton pumps that couple the reduction of molecular oxygen to vectorial translocation of protons across the membrane [1,2]. The superfamily is de¢ned by a high sequence similarity within the largest subunit (subunit I) and a binuclear active site consisting of a high-spin heme (heme a3 ) and a closely associated copper ion (CuB ). In addition to the binuclear center, cytochrome c oxidase has a low-spin heme (heme a) within subunit I and another copper center (CuA ), Abbreviations: BOX, cytochrome c oxidase from bovine heart mitochondria; POX, cytochrome c oxidase from Paracoccus denitri¢cans * Corresponding author, at address a. Fax: +46-18-511-755.
which is located in the extrinsic domain of subunit II. Electrons from cytochrome c enter the protein at the binuclear CuA center where they are transferred to heme a and then further channeled to the binuclear oxygen binding site. Fig. 1 summarizes the metal centers and the electron transfer pathway in cytochrome c oxidase. During the reduction of molecular oxygen to water four protons are consumed from the `IN' side of the membrane [2]. Four additional protons are translocated from the `IN' side to the `OUT' side utilizing the free energy available from the exergonic reaction of the reduction of oxygen to water (Fig. 1) [1,3]. The active transport of protons generates a proton and voltage gradient that is converted to more useful energy forms via energy conserving systems such as ATP synthase. A major advance in cytochrome c oxidase research was achieved with the X-ray structures of Paracoccus
0167-4838 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 0 0 ) 0 0 2 4 1 - 7
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Fig. 1. Schematic drawing of subunits I and II of cytochrome c oxidase. In the ¢gure, a and a3 represent heme a and heme a3 , respectively. The electron transfer and the proton translocation pathways are shown by arrows.
denitri¢cans (POX) and mitochondrial (BOX) enzymes in 1995. The POX structure (four-subunit enî [4]; this zyme) was initially determined to a of 2.8 A was followed by the structure of the two-subunit î [5] and then an oxidase (subunits I and II) at 2.7 A improved structure of the four-subunit enzyme [6]. The BOX structure was also initially reported to a î resolution [7] but has been largely improved 2.8 A î ), fully with the structures of fully oxidized (2.3 A î î reduced (2.35 A), azide bound (2.9 A) and carbon î ) forms published [8]. monoxide-bound (2.8 A In spite of rapid progress in structural studies, the proton pumping mechanism of cytochrome c oxidase remains largely unsolved. Utilizing the POX structure and the histidine cycle mechanism from Wikstro«m [9], Michel and his colleagues originally proposed the `Histidine shuttle' mechanism [4]. In recent publications [6,10,11] Michel has discarded the `Histidine shuttle' mechanism in favor of a new model based on electron-proton transfer coupling mainly deduced from pKa calculations on the POX structure. In the model of Wikstro«m, the proton pumping is directly coupled to the oxygen reduction reaction rather than the electron transfer by itself. One common feature of their respective mechanisms is that the energy from oxygen reduction is directly used for proton translocation within close proximity to the binuclear center.
The proton pumping model proposed by Yoshikawa and his colleagues for the bovine enzyme [8] is substantially di¡erent from the models presented by Michel and Wikstro«m. Both Michel and Wikstro«m's models attempt to be consistent with the structural data for the POX as well as the BOX enzyme; however, Yoshikawa's model is speci¢cally tailored for the BOX enzyme. Yoshikawa's model was inspired by observable conformational changes at the outlet of the H-channel between the reduced and oxidized structure of BOX. Using these conformational changes, they explain proton pumping in BOX as an indirect conformational coupling mechanism. There are two main arguments that contradict the model proposed by Yoshikawa as a general mechanism for terminal oxidases: (1) the H-channel is not present in the bacterial oxidases; (2) a computational simulation has shown that the H-channel is not likely to be a proton channel [12]. In this article, we revisit the original structures of bacterial and mitochondrial oxidases and try to clarify similarities and di¡erences between the POX and BOX structures. We also highlight results obtained with mutant enzymes from other sources, especially from Escherichia coli and Rhodobacter sphaeroides. We will focus on the structure of subunit I, the heart of the proton pump. 2. Overall structure of subunit I In the following discussion, we refer to the two subunit oxidase structure of POX (PDB entry, 1AR1) and the fully oxidized structure of BOX (PDB entry, 2OCC), unless otherwise speci¢ed. The numbering for BOX is used for all residues with the POX residue number in brackets. BOX and POX have 13 and four subunits, respectively. Amino acid sequences of the three core subunits (subunits I, II and III) are well conserved for both enzymes and the structures of these subunits are, indeed, very similar. In Fig. 2A,B structural overviews of subunit I (POX) containing 12 transmembrane helices are shown. Fig. 2B (viewed from the `OUT' side of the membrane) depicts three arcs, which are related by a pseudo-threefold symmetry [4]. Each arc is shaped by four transmembrane helices and together with the last segment of the previous semicircle a pore-like
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Fig. 2. Structure of subunit I of cytochrome c oxidase from P. denitri¢cans (POX). (A) View parallel to the membrane. The subunit contains 12 transmembrane helices. Heme a (red), heme a3 (cyan), CuB (blue sphere), magnesium ion (magenta sphere) and calcium ion (green sphere) are also shown. (B) View along the membrane normal from `OUT' side of the membrane. Only transmembrane helices are shown with hemes and metal centers. The coloring of the hemes and metal centers are the same as in panel A. This ¢gure was drawn with a modi¢ed version of MOLSCRIPT [41].
appearance is formed. The three pores (pores A, B and C) vary in function. Pores B and C hold the binuclear center (heme a3 and CuB ) and heme a, respectively. The `IN' side half of pores A and B house the D- and K-proton pathways, respectively (see below); and the `OUT' side half of pore A is a possible oxygen pathway [13].
(OH) group of the hydroxylethylfarnesyl side chain because the OH group is pointing in the opposite direction (Fig. 3A). In the BOX structure, the hydrogen bond network around the OH group is part of the H-channel, the third proton pathway. Lack of this network suggests that there is no H-channel in POX (see below).
2.1. Metal centers
2.1.2. Binuclear center The binuclear center, where molecular oxygen is reduced to water, is composed of heme a3 and the CuB ion (Fig. 3B). The axial ligand of heme a3 is His 376 (411) and the CuB ligands are His 240 (276), His 290 (325) and His 291 (326). The iron of heme a3 is î out of the heme plane in POX but almost 0.36 A within the plane in BOX. In POX, the heme a3 î and 5.2 A î in the two and CuB distance is 4.5 A four subunit enzymes, respectively, the distance is independent of the redox state [6]. In the bovine enzyme, the distance varies depending on the redox î , 5.2 A î state and the presence of a bound ligand; 4.9 A î and 5.3 A have been reported for the fully oxidized enzyme (no ligand), the fully reduced enzyme (no
2.1.1. Heme a Heme a is a low-spin heme maintaining two axial histidine ligands, His 61 (94) and His 378 (413) (Fig. 3A). The heme environments in POX and BOX are very similar. The conformation of the hydroxylethylfarnesyl chain of heme a is slightly, but signi¢cantly di¡erent between the two enzymes (Fig. 3A). The hydroxyl group of the farnesyl chain in BOX has hydrogen bonds to Ser 382 (417) and a water molecule. This water molecule has additional hydrogen bonds to Ser 461 (496) and Thr 424 (Ile 459). Although Ser 382 (417) is conserved in the POX enzyme, there is no hydrogen bond to the hydroxyl
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ligand) and the fully reduced enzyme with bound CO, respectively. According to Yoshikawa and his colleagues, the position of heme a3 is always ¢xed but the position of CuB di¡ers depending on either the oxidation state of the metal sites or the binding of a ligand [8]. Even without adding any inhibitor or substrate, some electron density is always observed between heme a3 and CuB for both POX and BOX. In the fully oxidized BOX structure a peroxide ligand has been modeled into the density [8]. However, in the POX structure, a hydroxide and a water molecule have been proposed as the bridging ligands [5] and this model is further supported by spectroscopic data [14]. A covalent bond between the NO2 of His 240 (276) and CO2 of Tyr 244 (280) has been reported for both BOX [8] and POX [5]. In the BOX structure, it was reported that this covalent bond exists regardless of the redox state or the presence of a bound ligand. It has been suggested that this covalent linkage could be caused by a radical reaction as reported for catalase HPII from E. coli where a similar covalent bond is formed between the CL atom of Tyr 415 and the
NN1 atom of His 392 [15]. It has further been suggested that the pK of Tyr 244 (280) could be considerably reduced by the formation of this bond [8] but no experimental data that support this have been reported. On the other hand, a related terminal oxidase, the cbb3 -type cytochrome c oxidase lacks a homologue to this Tyr, as well as several other key residues, but still functions as a proton pump albeit at reduced levels [16,17] leaving the role of this HisTyr bond still unclear. 2.1.3. Non-redox metal centers In addition to the redox-active centers mentioned above, the enzyme also contains tightly bound nonredox-active metal centers. These are Mg2 and Ca2 in cytochrome c oxidase from POX and Mg2 , Na and Zn2 in cytochrome c oxidase from BOX. The zinc ion in BOX is bound by a nuclear encoded subunit (subunit Vb) on the matrix side of the membrane (`IN'). All of the other non-redox-active metals are bound within subunit I. The Mg2 binding site is located at the interface between subunits I and II and is in close proximity to the heme a3 propionates (Fig.
Fig. 3. Structures of heme a and heme a3 and surrounding residues in cytochrome c oxidase. (A) Structure of heme a and surrounding residues of cytochrome c oxidase from bovine heart mitochondria (BOX) and P. denitri¢cans (POX). The BOX structure is colored based on the atom type (C: yellow, N: blue, O: red, Fe: red). The POX structure is shown in cyan. A water molecule and the hydroxyl group of the hydroxylethylfarnesyl groups are labeled by w and OH, respectively. The water molecule was not in the PDB ¢le (2OCC) but added based on the description of [8]. (B) Structure of heme a3 and surrounding residues of cytochrome c oxidase from bovine heart mitochondria (BOX) and P. denitri¢cans (POX). The coloring scheme is the same as in panel A. In the BOX structure, Y244 (280)-H240 (276) is covalently bonded. For the POX structure, a similar structure was observed but these are not connected in the PDB entry 1AR1. In the BOX structure, a bridge ligand between heme a and CuB has been putatively assigned as a peroxide molecule. This ¢gure was generated in program O [42].
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interface, which could be important for proton pumping.
Fig. 4. The proton pathways and the Mg2 binding site. The residues in bovine cytochrome c oxidase (BOX, red) and P. denitri¢cans oxidase (POX, blue) are superimposed. Structures of helices, heme a3 and metal centers are based on the POX structure. This ¢gure was drawn with a modi¢ed version of MOLSCRIPT [41].
2.1.3.2. Ca2 +/Na + binding site. The other non-redox metal binding site is formed by a loop between the transmembrane helices I and II on the `OUT' side surface of subunit I (Fig. 5). In the crystal structures, calcium and sodium ions are assigned for POX and BOX, respectively, based on the ligand coordination patterns. It has been suggested, however, that the metal in the POX could also be a sodium ion [21]. The function of this metal binding site is also unknown. In the quinol oxidase from E. coli, no metal binding site has been found at this position [22]. It has been suggested that the Ca2 /Na site could also be involved in the exit pathway of the pumped protons [21]. The ligands of the POX Ca2 site are the backbone carbonyl oxygen atoms from residues Glu 40
2). The Ca2 (POX)/Na (BOX) binding site is formed by a loop between the transmembrane helices I and II at the `OUT' side surface of the membrane. 2.1.3.1. Mg2 + site. In both POX and BOX structures, the non-redox-active metal that was observed in the interface between subunits I and II (Fig. 4) was assigned as a magnesium ion, although it can be replaced by a manganese ion in POX [5]. The ligands of the magnesium ion are His 368 (403), Asp 369 (404), Glu 198 (218) of subunit II and three water molecules (not shown in Fig. 4). The function of the Mg2 site is unknown. Since the site is in the vicinity of a possible proton exit pathway, it was suggested that it could be involved in proton transfer; however, mutant enzymes lacking this magnesium ion maintain an activity level greater than 50% and show no change in proton pumping e¤ciency [18,19]. Moreover, quinol oxidases do not have the Mg2 site but still pump protons [20]. The site could have a structural role for stabilization of the interface between subunits I and II since both subunits have a number of negatively charged residues at the
Fig. 5. Structure of Ca2 /Na binding site. The structure of bovine cytochrome c oxidase (BOX) is colored based on the atom type (C: yellow, N: blue, O: red, Na: yellow). The structure of P. denitri¢cans oxidase (POX) is shown in cyan. BOX and POX bind Ca2 and Na , respectively. The water molecules mentioned in the main text are not shown since the PDB ¢les (1AR1 and 2OCC) do not contain the coordinates. This ¢gure was generated in program O [42].
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(56), His 43 (Phe 59), and Gly 45 (61), the side chain oxygen atoms from Glu 40 (56) and Gln 63 (POX) and one water molecule (not shown in Fig. 5). In BOX, the backbone carbonyl oxygen atoms of Glu 40 (56), Gly 45 (61) and Ser 441 (Ile 476), the side chain oxygen atom of Glu 40 (56) and a water molecule (not shown in the ¢gure) are reported as metal ligands to the sodium ion. The di¡erence between the metal binding sites of the two reported structures may be due to the lack of an equivalent side chain for Gln 63 (POX) in BOX. This metal binding site is within close proximity to î ). Many years ago a spectral heme a (approx. 17 A perturbation of heme a upon the binding of the Ca2 was observed in mitochondrial cytochrome c oxidase [23]. This spectral change was not observed in wild type POX [24] but it has been shown that the same perturbation can be reproduced in POX when mutations have been made around the Ca2 site [21]. The physiological relevance of this interaction is not clear; however, since the metal binding site is close to the exit of the H-channel in the bovine enzyme, it could be related to the function of this pathway. 2.2. Proton pathways The oxygen binding site is located in the membrane spanning hydrophobic region of the protein, î from the `IN' side surface of the memabout 35 A brane. Thus, the enzyme needs a proton translocating pathway for substrate protons as well as for protons to be pumped across the membrane. Extensive mutational studies were performed and key residues involved in proton uptake and translocation have been identi¢ed [18,25,26]. When the ¢rst structures of cytochrome c oxidase were unveiled, two polar cavities were revealed as possible proton pathways (Fig. 4). This ¢nding was supported by many of the previous as well as proceeding mutational studies [27^30]. These two pathways, the D- and K-channels named after residue Asp 91 (124) and Lys 319 (354), are essentially the same for both structures. The Kchannel leads into pore B from Ser 255 (291) to the binuclear center through Lys 319 (354), Thr 316 (351), the OH group of the hydroxylethylfarnesyl side chain of heme a3 , Tyr 244 (280) and His 240 (276). A tightly bound water molecule is observed between Thr 316 (351) and the hydroxyl group.
There is a large hydrophobic gap between Lys 319 (354) and Thr 316 (351) where no water molecules have been found. Therefore, Lys 319 (354) is assumed to shuttle between Ser 255 (291) and Thr 316 (351) during proton translocation. Studies on the related ubiquinol oxidase from E. coli show that within close proximity to Lys 319 (354), there is a conserved glutamate, Glu 62 (78) of subunit II which could be an alternative entrance of the K-channel since mutations of this residue reduce the activity of the enzyme [31]. The second pathway, the D-channel, starts with Asp 91 (124) and leads to Glu 242 (278) through a large polar cavity ¢lled with water molecules in pore A. These polar residues along with water molecules form a hydrogen bond network, which connects Asp 91 (124) to Glu 242 (278). Thus, protons can be translocated through the hydrogen bonds in this pathway. For many years Glu 242 (278) has been shown to be a key component in the proton pumping mechanism of terminal oxidases [27,28,30,32]. Surprisingly, the hydrogen bonding environment of Glu 242 (278) between POX and BOX is di¡erent. The Glu 242 (278) of POX is hydrogen bonded to the main chain carbonyl oxygen of Ile 66 (Met 99) and seems to always be protonated at neutral pH. While in the BOX structure, Glu 242 (278) is hydrogen bonded to the SN atom of the Met 71 (Ile 104) side chain. Recent reports have shown that some terminal oxidases are lacking this key glutamate in the D-channel but yet still pump protons [33,34]. However, in these structurally distant members of the heme-copper oxidase, the fourth residue upstream from the Glu 242 (278) is often a Tyr residue. In a recent publication by Backgren et al. [35] a Tyr residue was shown to be able to facilitate enzyme activity as well as proton pumping in a triple mutant of POX. After this glutamic acid the connection of the D-channel is not clear. The pathway could be connected to the binuclear center or a heme a3 propionate through a rearrangement in the water chain depending on the charge distribution in the enzyme. In this way, the area could form a proton gate that is essential for the proton pumping. Many details of the two pathways have been studied over the years using mutational and spectroscopic studies in conjunction [36] but the precise
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mechanistic approach for delivering and pumping of protons has yet to be con¢rmed. Mutational studies of Lys 319 (354) concluded that the K-channel is only used during the reduction step of the enzyme and takes up one or two chemical protons [29]. The results of mutational studies on Asp 91 (124) and Glu 242 (278) [28,37] strongly support that all other protons are taken up through the D-channel. For a full understanding of the roles of these two pathways, the exact timing of proton pumping in relation to the oxygen reduction cycle must be worked out and correlated with structures of oxygen intermediates. The proton exit pathway is less clear. In both enzymes, many tightly bound water molecules are observed in the space between subunits I and II. The heme a3 propionate, mentioned above, also exists within this cavity (pore B) and is connected to the outside of the protein through a hydrogen bond network of the water molecules. In addition, there seems to be more than one possible proton exit pathway. In the BOX structure, a third proton pathway, the H-channel, has been suggested. The H-channel leads into pore C from Asp 407 (Glu 442) to Asp 51 (Gly 84) through seven polar side chains and two water molecules and one main chain peptide bond. In the oxidized enzyme structure, Asp 51 (Gly 84) has hydrogen bonds to Ser 205 (Ala 225) and Ser 441 (Ile 476) which are located in the interior of the molecule and renders the pathway closed. In the fully reduced enzyme, however, this residue is exposed to the solvent. Based on this conformational change, Yoshikawa and his colleagues propose a conformationally coupled proton pumping mechanism in BOX. However, there are several di¤culties applying this model for all terminal oxidases. To begin with, the hydrogen bond network is not entirely connected through the H-channel. In their model, protons need to be transferred through the protein polypeptide chain, which is highly unlikely. In addition, the conformation of the hydroxylethylfarnesyl chain of heme a is di¡erent in the POX structure which prevents the formation of the hydrogen bond network seen in the H-channel in the BOX structure. Furthermore, the residues in the channel are not fully conserved in bacterial enzymes, i.e. POX lacks the key residue Asp 51 (Gly 84). Mutations of residues around this area have little to no e¡ect on the activity of the
7
bacterial oxidase [38]. However, it is still possible that BOX has a di¡erent proton pumping mechanism from the bacterial enzymes, but there have been no experimental data that support the H-channel model. 3. Other terminal oxidase structures Recently two additional structures of heme copper oxidases have been published and a third is soon on the way. The hope is by combining information available from all structures, a general mechanistic approach for proton consumption and translocation can be achieved. Here we will shortly summarize the new structures. The structure of the ba3 -cytochrome c oxidase from Thermus thermophilus has been reported to a î [34]. The amino acid sequence resolution of 2.4 A of this enzyme shows a clear but distant homology to other members of the super family. In brief, this structure identi¢es both the D- and K-pathways although many key residues are not conserved. In addition, a third proton pathway, termed the `Qpathway' has been identi¢ed and is not equivalent to any other reported pathway. Other key observations are a distinct covalent bond between Tyr 244 (280) and His 240 (276) (Tyr 237 and His 233 ba3 numbering) and the lack of a Glu 242 (278) homologue. Recently the structure of cytochrome bo3 oxidase î resolution [22]. Unlike all prewas reported at 3.5 A vious terminal oxidase structures, this is a quinol oxidase that utilizes ubiquinol as a substrate instead of cytochrome c. The structure has con¢rmed both the D- and K-pathways in ubiquinol oxidase as well as the newly found ubiquinol binding site. Resolution limits of cytochrome bo3 prevent the assigning of a covalent bond between Tyr 244 (280) and His 240 (276). Finally, the structure of another aa3 cytochrome c oxidase from R. sphaeroides is soon to be published î (M. Svensson-Ek, L. Rodto a resolution of 2.3 A gers, J. Abramson, P. Brzezinski, S. Iwata, unpublished data). The work of cytochrome aa3 from R. sphaeroides compares the structures of wild type and mutant enzymes showing, for the ¢rst time, a rearrangement of the water pathway around the binu-
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clear center. Again both the D- and K-pathways are seen but the question of a covalent bond between Tyr 244 (280) and His 240 (276) is uncertain. The electron density indicates that the bond could be only partially formed in this enzyme. 4. Summary On the whole, protein crystallography, mutational studies and spectroscopy have revealed the structural and functional framework of a fascinating enzyme, cytochrome c oxidase. Despite signi¢cant results within these areas, the proton pumping mechanism of the enzyme remains largely unresolved. This is mainly due to the di¤culties to resolve the timing of the proton pumping during the oxygen reduction and the structural rearrangements in the reaction cycle. Invisibility of the protons by X-ray crystallography and the rather small conformational changes during proton pumping are additional di¤culties. Upcoming structures at higher resolution and from other species will certainly help the understanding of the mechanism. In bacteriorhodopsin several intermediate structures have been obtained, intermediate structures that are slightly di¡erent from one another but together reveal the pumping mechanism in this enzyme [39,40]. For this to occur for heme copper oxidases, a similar approach of combining structural studies with sophisticated biochemical and spectroscopic analysis must be implemented. Acknowledgements The Swedish Research Council, NFR and the EC biotechnology program, support this work. We would also like to thank the reviewer whose comments made this a more complete study.
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