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Beef heart cytochrome c oxidase
574
Beef heart cytochrome c oxidase Shinya Yoshikawa During the past two years, the crystal structures of beef heart cytochrome c oxidase with 13 subunits and the bacterial enzyme with four subunits have been reported at atomic resolution, ushering in a new era for cytochrome c oxidase research. Different proton pumping mechanisms have been proposed for the two organisms.
Addresses
Department of Life Science, Himeji Instituteof Technology, Kamigohri, Akoh Hyogo 678-12, Japan; e-mail:[email protected] Current Opinion in Structural Biology 1997, 7:574-579
http:llbiomednet.comlelecreflO959440XO0700574 © Current Biology Lid ISSN 0959-440X
Introduction
Cytochrome c oxidase has been one of the most extensively investigated systems in the field of bioenergetics [I°',2] since its discovery [3], because of its physiological importance as well as its intriguing r e a c t i o n - - t h e reduction of 02 to water coupled with the active transport of protons. Time-resolved resonance Raman spectroscopy has been a powerful experimental technique for identifying transient intermediates during the 02 reduction, such as the dioxygen (Fe2+-Oz) , the ferryl oxide (Fe4+-O) and the hydroxide (Fe3+-OH) intermediates [4"*]. In addition, substantial efforts have been directed toward elucidating the mechanism of the coupling of the 02 reduction with the pumping of protons [1*',5]. T h e two papers on the crystal structure of the enzymes isolated from beef heart [6] and the bacteria Paracoccus denitrificans [7], published at atomic resolution in 1995, have heralded the beginning of the new era in the history of research on this enzyme. In this review, I will summarize the crystal structure of beef heart cytochrome c oxidase at its current level of atomic resolution [6,8 "°] and will discuss new insights gained from the structure for understanding the mechanism of this enzyme reaction.
Overview of the structure of beef heart cytochrome c oxidase Cytochrome c oxidase isolated from beef heart muscle is dimetic in the crystals prepared in the presence of decyl maltoside, as shown in Figure 1. T h e biggest subunits, I, II and III [9], are encoded by mitochondrial genes and form a core part of each monomer. T h e remaining ten nuclear-coded subunits surround the core. This enzyme has four redox-active metal sites which include hemes a and a3, Cu A and CUB, and two redox-inactive metals, magnesium and zinc [10]. CUA is a dinuclear copper site located within the extramembrane domain of subunit 11 in
the cytosolic side of the enzyme [11-13]. A nuclear-coded subunit, Vb, located on the matrix surface, holds the zinc ion [6]; the other four metals are found in subunit I. T h e planes of both hemes lie perpendicular to the membrane surface [6]. T h e distances from the center of the two copper atoms of Cu A to the irons of hemes a and a 3 are 19 ~ and 22 ~, respectively. T h e magnesium site is midway between CUA and heine a3. In addition to these metals, eight phospholipids, five phosphatidyl ethanolamines and three phosphatidyl glycerols are detectable within the crystal structure [8°°]. Two cholate molecules are located near the membrane surface levels [8°']. T h e cholate molecules are probably contaminants as the enzyme preparation used for the custallization is exposed to cholatc during purification from beef heart muscle. ADP and cholate are very similar in their size and shape, and the cholate-binding sites are therefore likely to be the sites for A D P binding; an atomic model of ADP fits quite well into electron density corresponding to either of the cholate-binding sites. Furthermore, one of the cholate-binding sites, near the N terminus of subunit Via, corresponds to an immunochemically determined ADP-binding site [14]. T h e effect of ADP/ATP ratio on the efficiency of proton pumping has also been demonstrated [15]. Cu A site
T h e oxidation state of CUA in the fully oxidized state has been proposed to be [CuI'S+'"Cul'5+], in which one electron is delocalized between the two Cu 2+ ions, on the basis of the similarity between the electron paramagnetic resonance spectra of Cu A and NeO reductase [11], a dinuclear copper protein. T h e redox equilibrium behavior of Cu A indicates that the midpoint potential of Cu A, as well as those of hemes a and a 3 and possibly Cu B, is influenced by the oxidation states of the other redox sites. Thus, the potentials of all these sites are identical in any given overall oxidation state, and the absolute potential depends on the overall oxidation state [16}. A dinuclear copper center will demonstrate a greater flexibility to influence the midpoint potential compared with a mononuclear center in the same conformation. This is supported by the existence of iron-sulfur proteins that contain a dinuclear iron site and that show high variability in their midpoint potential [17]. Furthermore, the dinuclear center has a greater number of ligands compared with the mononuclear center; these can transfer the influence of the change in the oxidation state of the metal site to another part of the protein. Thus, a dinuclear metal site can better serve to promote redox coupling between the cofactors. In order to elucidate the mechanism of the interaction, the redox-dependent changes in the conformation of the Cu A site should be examined crystallographically. Redox-coupled changes
Beef heart cytochrome c oxidase Yoshikawa
575
Figure 1
Co~ backbone trace of dimer of beef heart cytochrome c oxidase. Each monomer consists of 13 different subunits, each shown in a different color. The subunit name is given in the color of the subunit. (a) Side view against the transmembrane helices. (b) Top view from the cytosolic side.
in the reactivity to the h e m e ligands, examined using Fourier transform infrared (Fq'IR), has provided unique information on the interaction b e t w e e n heine a 3 and the other redox metal sites [18]. Heme a and the long alkyl sidechain
H e i n e a is coordinated by two histidines, His61 and His378, of subt, nit I. T h e iron atom is in the h e m e plane thus indicating a low spin ferric state [6]. T h e h e m e plane is positioned perpendicular to the m e m b r a n e surface with the hvdroxylfarnesylethyl sidechain of the h e m e directed toward the m e m b r a n e surface of the matrix side in almost the fully e x t e n d e d conformation [6,7]. On the one hand, this e x t e n d e d conformation seems to anchor the h e m e plane quite stronglx: On the other hand, the corresponding long sidechain of h e m e a 3 serves as a structural e l e m e n t of one of the possible 02 channels to the 0 2 reduction site [8"]. It is widely accepted that all the enzymes in the terminal oxidase family reduce 02 to water coupled with the p u m p i n g of protons via an identical reaction mechanism [19[. T h e insertion of the large alkyl groups of h e m e s a and a 3 into the protein moiety within the t r a n s m e m b r a n e region, however, will induce significantly large conformational changes. If the reaction mechanism of the enzymes is identical regardless of the structure of heroes, c o m p e n s a t i n g conformational changes must occur in other regions of the protein. Alternatively, the mechanism of the e n z y m e reaction may be modified without the need for large, c o m p e n s a t i n g conformational changes. Thus, it is important to soh, e the crystal structure of the terminal oxidase that does not contain the hydroxyl fa rnesylethyl group.
0 2 reduction site
T h r e e histidine imidazoles coordinate to CUB [6]. "File copper atom is at the c e n t e r of an equilateral triangle consisting of the three coordinating histidine nitrogens; the histidine triangle is parallel to the h e m e a 3 porphyrin plane. CuB is 4.7~1 away from the heine a 3 iron and 1.0.~l away from a line passing through the iron atom of h e m e a3 and normal to the plane of the porphyrin ring (Figure 2). On the one hand, electron density maps indicate the absence of any larger atom such as chloride b c t w e e n the two metals, however, smaller atoms such as oxygen and nitrogen may be present. On the other hand, a cupric copper in a trigonal planar coordination is unlikel-~; Thus, the presence of another ligand to Cul~ will probably form a tetrahedtal coordination. It should be noted that the m e t a l - m e t a l distance, 4.7 ~i, is too long for a bridging coordination by a single atom. Tyr248 is hydrogen b o n d e d to the hydroxyl group of the hydroxylfarnesylethyl group of h e m e a.~ (Figure 2). This tyrosine is likely to be a proton donor to a reaction i n t e r m e d i a t e formed during the O 2 reduction, as a hydrogen-bond network has been identified from Tyr244 to Lys265 at the matrix surface of the protein. In addition to the proton donating site, a possible water channel leads to the O 2 reduction site [8°°]. T h e channel begins at one of the propionate groups of h e m e a 3, which forms a hydrogen bond with His368 - - one of the ligands of the magnesium atom. "IM'o arrays of hydrophilic amino acids, one belonging to subunit 1 and the other belonging to subunit II, are situated at the interface b e t w e e n subunits 1 and II. This intert:ace forms a hydrophilic channel from the propionate of h e m e a 3 to the cytosolic surface. T h e channel is too narrow to accept even a single water
576
Membrane proteins
Figure 2
between Cu A and berne a compared with the one between CUA and heme a3 (19,~ versus 22.~). Furthermore, it is still not clear why the crystal structure indicates a possible pathway for the direct electron transfer from Cu A to heine a3.
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molecule, suggesting a tightly controlled mechanism for draining water from the active site and preventing the back flow of water from the cytosolic side. Such a back-flow could result in the influx of protons and a short circuit of the transmembrane proton gradient.
Electron transfer between the redox centers Hemes a and a 3 are in close proximity, which suggests t h a t extremely rapid electron transfer can occur between them. The closest approach between the two hemes is 4 ~. T h e fifth ligands of heroes a and a3 are separated in the amino acid sequence by only one residue. A hydrogen-bond network between CUA and heme a includes His204, one of the ligands to CUA, a peptide bond between Arg438 and Arg439, and a propionate group of heme a (Figure 3). T h e network seems to provide a facile electron transfer path from Cu A to heme a. T h e double bond character of the peptide bond promotes the efficiency of electron transfer. An alternate network connecting Cu A and heine a 3 via the magnesium site (Figure 3) could also be an effective electron transfer path directly from Cu A to heme a3 [7,8°°]. A direct electron transfer between CUA and heme a3, however, has never been detected in transient kinetic investigations [20]. These kinetic measurements suggest that the electron transfer rate from Cu A to heme a is at least two orders of magnitude higher than the rate from Cu A to heine a 3. Differences between the two electron transfer rates can be rationalized by the shorter distance
The hydrogen-bond network between the redox active metal sites in beef heart cytochrome c oxidase. Dotted lines denote possible hydrogen bonds and broken lines denote metal ion coordination bonds. Blue and pink balls are copper and magnesium ions, respectively. Pink structures with and without a blue ball are hemes a3 and a, respectively. Three amino acid residues, His368, Arg438, Arg439, belong to subunit I; the others belong to subunit II.
Mechanism of 0 2 reduction A one-electron reduction of 0 2, the triplet dioxygen, is well known to be energetically unfavorable, whereas a two-electron reduction is quite favorable [21]. This property of Oz provides the stability for the oxygenated forms of hemoglobin and myoglobin, in which sequestering the FeZ+-O2 inside the protein provides protection from the two-electron reduction. Thus, CUB placed near the Oz-binding site must trigger the reduction of 0 2 or Fea3 z+ via the two-electron process to the level of per~,xide. This process is likely to be limited by the election transfer across a short distance of approximately 3,~, from CUB 1÷ to 0 2 on Fea32+. T h e process is expected to be very r a p i d - - o f the order of a picosecond. Unexpectedly, however, time-resolved resonance Raman spectroscopy detects the existence of the oxygenated form of the enzyme (Fe-O z) with a fairly long life time, tl/z =0.1 msec at 4°C, during the reaction of the fully reduced enzyme with O 2 [22]. T h e isotopic shifts of the Fe-O 2 vibration determined by exchanging 1 6 0 2 with 1 8 0 2 and 1 6 0 1 8 0
Beef heart cytochrome c oxidase Yoshikawa
indicate that O 2 binds to Fe z+ in an end-on fashion similar to its binding in hemoglobin [4°',23]. The resonance Raman results suggest a structure at the active site that is consistent with a diminished electron-transfer rate from CUB to the bound O 2. Currentl3, only the crystal structure of cytochrome c oxidase in the fully oxidized state has been reported. Crystal structures of many other metalloproteins indicate that the conformational differences induced by a change in the oxidation state at the metal site are very small. Thus, the structure of the O 2 reduction site in the fully reduced state of cytochrome c oxidase is likely to closely resemble the structure in the fully oxidized state. When O z initially binds to the Fea3 z+ in an end-on fashion without any interaction occurring with CuB, coordination
577
of CUBI+ is trigonal planar, with the three histidine imidazoles as the ligands. The Cu I+ in such a coordination is very stable [24t, and this stability limits the electron transfer from CUB 1+ to the 02 bound to Fea3 2+. Thus, the second electron is likely to come from heme a. The crystal structure indicates that Tyr244 hydrogen bonds to the bound O 2 concomitantly with a small conformational change [25]. Formation of this hydrogen bond is likely to promote the reduction of the bound O2 to a hydroperoxy species. The formation can not be very rapid because a conformational change of Tyr244, albeit small, takes place. In peroxidase, the hydroperoxo intermediate very rapidly decomposes to compound I, which is an oxoferryl intermediate (Fe4+-O) with one extra oxidation equivalent, concomitantly with the release of HeO [26]. By analogy,
Figure 4
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Schematic representations of two possible channels for proton pumping. Shaded ovals, dotted lines and dotted lines with arrows denote cavities, hydrogen bonds, and possible hydrogen-bond configurations, respectively. The cavities contain no electron density but are large enough for keeping randomly oriented or mobile water molecules. A pair of amino acid sidechains in a possible hydrogen-bond configuration are placed too far apart with each other to form a hydrogen bond; however, a small conformational change in the pair without movement of peptide bond could provide a new hydrogen bond. The matrix side of each network is shown at the bottom and the cytosolic side at the top. Both (a) and (b) show only the structure connecting the matrix port to the cytosolic port. No dead-end branch is shown except for a branch leading to heme a in the channel B. Both channels occur in subunit I, therefore, all the amino acid residues shown belong to subunit I.
578
Membrane proteins
a similar process is likely to occur for the hydroperoxy intermediate of cytochrome c. Recent resonance Raman evidence suggests that the extra oxidation equivalent is on the iron atom as FeS+-O [4"]. CUB, with its extremely high tedox potential, would be able to reduce the Fea3 at this high oxidation state during enzymic turnover.
Mechanism of proton pumping The coupling of the 0 2 reduction with proton translocation has been carefully and ingeniously investigated by examining the effect of the electrochemical potential on the reverse of the Oz reduction - - t h e oxidation of water [5]. These investigations have demonstrated that only two out of four electron transfer steps during the 0 2 reduction to water are driven in the reverse direction when an electrochemical potential opposite in polarity to that of physiological conditions is applied. Papa and coworkers [27] have reported that the efficiency of proton uptake is dependent on the electron transfer rate, which suggests an indirect coupling of the Oz reduction with proton translocation. One year before the crystal structures of cytochrome c oxidase appeared, Wikstrs6m et al. [28] proposed a mechanism for redox-coupled proton pumping on the basis of the coordination structure of the 0 2 reduction site deduced from mutagenesis results [28]. In their mechanism, one of the three histidine ligands of Cu B acts as a unidirectional proton shuttle by dissociating itself from Cu B. The dissociation is dependent on protonation of the histidine imidazole as well as on the oxidation and ligand-binding states of the O 2 reduction site. The mechanism seems consistent with the crystal structure of the azide-bound fully oxidized bacterial cytochrome c oxidase [7,29°]. One of the three histidine imidazoles of this enzyme is missing in the crystal structure which does suggest the mobility of this residue. In the mechanism of Wikstr/Sm et al. [28], this histidine imidazole forms the proton pumping site in addition to being a part of the 0 2 reduction site. Thus, this proton pumping site must keep the protons completely away from the dioxygen reduction site, othei~visc the pumping protons are likely to be taken by O Z reduction intermediates that form during the O 2 reduction and that have an extremely high affinity to protons. Such proton trapping would ultimately short circuit the proton pumping mechanism [28,30]. As mentioned above, the lack of one of the histidine of Cu B ligands in the crystal structure of bacterial enzyme suggests a mobility of the imidazole; however, it would appear to be very difficult for such a mobile imidazole to keep the protons completely away from the intermediate species of O z reduction on Yea3. Two hydrogen-bond networks from the matrix side to the cytosolic side have been mapped. (Figure 4) [8°°]. These networks are probable conduits for proton pumping; both
are well separated from the redox-active metal site. No probable proton pumping pathways have been detected at the 0 2 reduction site in the beef heart crystal structure. For the elucidation of the proton pumping mechanism, the identification of redox-coupled conformational changes is of paramount importance. Redox-linked difference infrared spectra obtained from bacterial cytochrome c oxidasc suggest conformational and/or protonation changes at a carboxyl sidechain arc involved [31"]. In addition, redox-linked conformational changes have been suggested by the circular dichroism spectrum of beef heart enzyme [32].
Conclusions The crystal structure of beef heart cytochrome c oxidase in the fully oxidized form at 2.8 ~, resolution suggests that heine a, not CUB, is the donor of the second electron to reduce 0 2 bound at Fea32+. Tvr244 positioned close to the 0 2 reduction site is the proton donor for the production of water. Two possible proton pumping networks well separated from the O z reduction site have been identified within the crystal structure of beef heart enzyme. The crystal structures of the enzyme in various oxidation and ligand-binding states at the level of 2.0]~ resolution are desirable for further understanding the mechanism of the redox coupled proton pumping of this enzyme. Hopefull>, at such a high resolution, the 02 reduction intermediate species will be detectable at the Fea3-Cu B site. Beef heart cytochrome c oxidase crystals at 2.1-2.3 ~ resolution have been obtained; the crystal analysis of these as well as further improvement of the crystallization conditions are underwa.v
Acknowledgement I wish to thank Herbert [, Axclrod for his valuable discussion.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest • ,, of outstanding interest 1. Ferguson-Miller S, Babcock GT: Heme/copper terminal •. oxidases. Chem Rev 1996, 96:2889-290Z A comprehensive and well-organized review on the structure and function of cytochrome c oxidase. It is currently the best review of the present level of the mechanistic research of this enzyme. A comparison of the paragraphs in this review related to the 02 reduction with those of [4 °°] will be .'elpful for considering this intriguing enzyme reaction. 2.
Malmstr6m BG: Cytochrome c oxidase as a redox-linked pro, ,,1 pump. Chem Rev 1990, 90:1247-1260.
3.
Warburg O: Uber eisen, den sauerstoffiJbertragenden bestandteil des atmungsfermentes. Biochem Z 1924, 152:479494. [Title translation: Iron and oxygen transporters of the respiratory system.]
4. °,
Kitagawa T, Ogura T: Oxygen activation mechanism at the binuclear site of heme-copper oxidase superfamily as revealed by time-resolved resonance Raman spectroscopy, Progr/norg Chem 1997, 45:431-4'79. A good summary of resonance Raman studies on the 02 activation of cytochrome c oxidase, showing how powerful this spectroscopy is for investigating the 02 reduction mechanism. It includes a well-written introduction of
Beef heart cytochrome c oxidase Yoshikawa
the principles and practice of time-resolved resonance Raman spectroscopy, which is suitable for biochemists not familiar to this technique. 5.
18.
Yoshikawa S, Mochizuki M, Zhao X-J, Caughey WS: Effects of overall oxidation state on infrared spectra of heine a 3 cyanide in bovine heart cytochrome c oxidase. J Biol Chem 1995, 270:4270-4279.
19.
Calhoun MW, Thomas JW, Gennis RB: The cytochrome oxidase superfamily of redox-driven proton pumps. Trends Biochem Sci 1994, 19:325-330.
20.
Hill BC: Modeling the sequence of electron transfer reactions in the single turnover of reduced, mammalian cytochrome c oxidase. J Biol Chem 1994, 269:2419-2425.
21.
Caughey WS, Wallace WJ, Volpe JA, Yoshikawa S: Cytochrome c oxidase. In The Enzymes, edn 3. Edited by Boyer PD. New York: Academic Press; 1976:299-344.
22.
Ogura 1", Takahashi S, Shinzawa-ltoh K, Yoshikawa S, Kitagawa T: Time-resolved resonance Raman investigation of cytochrome oxidase catalysis: observation of a n e w oxygen-isotope sensitive Raman band. Bu//Chem Soc Jpn 1991, 64:2901-2907.
23.
Ogura T, Takahashi S, Hirota S, Shinzawa Itoh K, Yoshikawa S, Appelman EH, Kitagawa T: Time-resolved resonance Raman elucidation of the pathway for dioxygen reduction by cytochrome c oxidase. J Am Chem Soc t993, 115:8527-8536.
24.
Cotton FA, Wilkinson G: Chemistry of the transition elements, copper. In Advanced Inorganic Chemistry, edn 4. New York: John Wiley Sons; 1980:798-821.
WikstrSm M, Morgan JE: The dioxygen cycle. J Biol Chem 1992, 267:10266-10273
6.
7.
Tsukihara T, Aoyama H, Yamashita E, Tomizaki T, Yamaguchi H, Shinzawa-ltoh K, Nakashima R, Yaono R, Yoshikawa S: Structures of metal sites of oxidized bovine heart cytochrome c oxidase at 2.8 A. Science 1995, 269:1069-1074. Iwata S, Ostermeier C, Ludwig B, Michel H: Structure at 2.8 A resolution of cytochrome c oxidase from Paracoccus denitrificans. Nature 1995, 376:660-669.
8. •-
Tsukihara T, Aoyama H, Yamashita E, Tomizaki T, Yamaguchi H, Shinzawa-ltoh K, Nakashima R, Yaono R, Yoshikawa S: The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A. Science 1996, 272:1136-1144. The detailed crystal structures of 13-subunit beef heart cytochrome c oxidase are described at 2.8 ~ resolution in the fully oxidized state. Two hydrogen bond networks, which do not involve the 02 reduction site, are shown to be possible proton pumping sites. Phospholipids and ADP-binding sites in the crystal structure are also given.
579
9.
Kadenbach B, Ungibauer M, Jarausch J, Buge U, Kuhn-Nentwig L: The complexity of respiratory complexes. Trend Biochem Sci 1983, 8:398-400.
10.
Steffens GCM, Soulinane T, Wolff G, Buse G: Stoichiometry and redox behavior of metals in cytochrome-c oxidase. Eur J Biochem 1993, 213:1149-1157
25.
Verkhovsky MI, Morgan JE, Wikstr6m M: Oxygen binding and activation: early steps in the reaction of oxygen with cytochrome c oxidase. Biochemistry 1994, 33:3079-3086.
11.
Kroneck PMH, Antholine WA, Riester J, Zumft WG: The cupric site in nitrous oxide reductase contains a mixed-valence [Cu(ll), Cu(I)] binuclear center: a multifrequency electron paramagnetic resonance investigation. FEBS Let1 1988, 242:70-74.
26.
Kitagawa T, Mizutani Y: Resonance Raman spectra of highly oxidized metalloporphyrins and heine proteins. Coordination Chem Rev 1994, 135/136:685-735.
27.
Capitanio N, Capitanio G, Demarinis DA, De Nitto E, Massari S, Papa S: Factors affecting the H+/e - stoichiometry in mitochondrial cytochrome c oxidase: influence of the rate of electron flow and transmembrane ApH. Biochemistry 1996, 35:10800-10806.
28.
Wikstrom M, Bogachev A, Finel M, Morgan JE, Puustinen A, Raitio M, Verkhovskaya M, Verkhovsky MI: Mechanism of proton translocation by the respiratory oxidases. The histidine cycle. Biochim Biophys Acta 1994, 1187:106-111.
12.
Larsson S, K~llebring B, Wittung P, Malmstr6m BG: The Cu A center of cytochrome-c oxidase: electronic structure and spectra of models compared to the properties of Cu A domains. Proc Natl Acad Sci USA 1995, 92:7167-7171.
13.
Wimanns M, Lappalainen P, Kelly M, Sauer-Eriksson E, Saraste M: Crystal structure of the membrane-exposed domain from a respiratory quinol oxidase complex with an engineered dinuclear copper center. Proc Nat/Acad Sci USA 1995, 92:11955-11959.
14.
Anthony G, Reimann A, Kadenbach B: Tissue-specific regulation of bovine heart ¢ytochrome-c oxidase activity by ADP via interaction with subunit Via. Proc Nat/Acad Sci USA 1993, 90:1652-1656.
15.
Frank V, Kadenbach B: Regulation of the H+/e - stoichiometry of cytochrome c oxidase from bovine heart by intramitochondrial ATP/ADP ratios. FEBS Lett 1996, 382:121-124.
16.
Nicholls P, Wrigglesworth JM: Routes of cytochrome a 3 reduction. Ann New York Acad Sci 1988, 550:59-67.
17.
Jensen LH: The iron-sulfur proteins: an overview. In Iron-Sulfur Protein Research. Edited by Matsubara H, Katsube Y, Wade K. Berlin: Springer-Verlag; 1986:3-21.
29. Ostermeier C, Iwata S, Michel H: Cytochrome c oxidase. Curr * Opin Struct Bio11996, 6:460-466. A summary of the crystal structure of the bacterial enzyme and a discussion of the reaction mechanism. 30.
Williams RIP: Purpose of proton pathways. Nature 1995, 376:643.
31. •
Hellwig P, Rest B, Kaiser U, Ostermeier C, Michel H, M&ntele W: Carboxyl group protonation upon reduction of the Paracoccus denitrificans cytochrome c oxidase: direct evidence by FTIR spectroscopy. FEBS Lett 1996, 385:53-57. This is the first report showing redox-linked conformational and/or protonation changes of the carboxyl group of glutamate or aspartate. This finding may promote the real-time measurement of proton pump behavior. 32.
Wittung P, MalmstrSm BG: Redox-linked conformational changes in cytochrome c oxidase. FEBS Lett 1996, 388:47-49.
Beef heart cytochrome c oxidase Shinya Yoshikawa During the past two years, the crystal structures of beef heart cytochrome c oxidase with 13 subunits and the bacterial enzyme with four subunits have been reported at atomic resolution, ushering in a new era for cytochrome c oxidase research. Different proton pumping mechanisms have been proposed for the two organisms.
Addresses
Department of Life Science, Himeji Instituteof Technology, Kamigohri, Akoh Hyogo 678-12, Japan; e-mail:[email protected] Current Opinion in Structural Biology 1997, 7:574-579
http:llbiomednet.comlelecreflO959440XO0700574 © Current Biology Lid ISSN 0959-440X
Introduction
Cytochrome c oxidase has been one of the most extensively investigated systems in the field of bioenergetics [I°',2] since its discovery [3], because of its physiological importance as well as its intriguing r e a c t i o n - - t h e reduction of 02 to water coupled with the active transport of protons. Time-resolved resonance Raman spectroscopy has been a powerful experimental technique for identifying transient intermediates during the 02 reduction, such as the dioxygen (Fe2+-Oz) , the ferryl oxide (Fe4+-O) and the hydroxide (Fe3+-OH) intermediates [4"*]. In addition, substantial efforts have been directed toward elucidating the mechanism of the coupling of the 02 reduction with the pumping of protons [1*',5]. T h e two papers on the crystal structure of the enzymes isolated from beef heart [6] and the bacteria Paracoccus denitrificans [7], published at atomic resolution in 1995, have heralded the beginning of the new era in the history of research on this enzyme. In this review, I will summarize the crystal structure of beef heart cytochrome c oxidase at its current level of atomic resolution [6,8 "°] and will discuss new insights gained from the structure for understanding the mechanism of this enzyme reaction.
Overview of the structure of beef heart cytochrome c oxidase Cytochrome c oxidase isolated from beef heart muscle is dimetic in the crystals prepared in the presence of decyl maltoside, as shown in Figure 1. T h e biggest subunits, I, II and III [9], are encoded by mitochondrial genes and form a core part of each monomer. T h e remaining ten nuclear-coded subunits surround the core. This enzyme has four redox-active metal sites which include hemes a and a3, Cu A and CUB, and two redox-inactive metals, magnesium and zinc [10]. CUA is a dinuclear copper site located within the extramembrane domain of subunit 11 in
the cytosolic side of the enzyme [11-13]. A nuclear-coded subunit, Vb, located on the matrix surface, holds the zinc ion [6]; the other four metals are found in subunit I. T h e planes of both hemes lie perpendicular to the membrane surface [6]. T h e distances from the center of the two copper atoms of Cu A to the irons of hemes a and a 3 are 19 ~ and 22 ~, respectively. T h e magnesium site is midway between CUA and heine a3. In addition to these metals, eight phospholipids, five phosphatidyl ethanolamines and three phosphatidyl glycerols are detectable within the crystal structure [8°°]. Two cholate molecules are located near the membrane surface levels [8°']. T h e cholate molecules are probably contaminants as the enzyme preparation used for the custallization is exposed to cholatc during purification from beef heart muscle. ADP and cholate are very similar in their size and shape, and the cholate-binding sites are therefore likely to be the sites for A D P binding; an atomic model of ADP fits quite well into electron density corresponding to either of the cholate-binding sites. Furthermore, one of the cholate-binding sites, near the N terminus of subunit Via, corresponds to an immunochemically determined ADP-binding site [14]. T h e effect of ADP/ATP ratio on the efficiency of proton pumping has also been demonstrated [15]. Cu A site
T h e oxidation state of CUA in the fully oxidized state has been proposed to be [CuI'S+'"Cul'5+], in which one electron is delocalized between the two Cu 2+ ions, on the basis of the similarity between the electron paramagnetic resonance spectra of Cu A and NeO reductase [11], a dinuclear copper protein. T h e redox equilibrium behavior of Cu A indicates that the midpoint potential of Cu A, as well as those of hemes a and a 3 and possibly Cu B, is influenced by the oxidation states of the other redox sites. Thus, the potentials of all these sites are identical in any given overall oxidation state, and the absolute potential depends on the overall oxidation state [16}. A dinuclear copper center will demonstrate a greater flexibility to influence the midpoint potential compared with a mononuclear center in the same conformation. This is supported by the existence of iron-sulfur proteins that contain a dinuclear iron site and that show high variability in their midpoint potential [17]. Furthermore, the dinuclear center has a greater number of ligands compared with the mononuclear center; these can transfer the influence of the change in the oxidation state of the metal site to another part of the protein. Thus, a dinuclear metal site can better serve to promote redox coupling between the cofactors. In order to elucidate the mechanism of the interaction, the redox-dependent changes in the conformation of the Cu A site should be examined crystallographically. Redox-coupled changes
Beef heart cytochrome c oxidase Yoshikawa
575
Figure 1
Co~ backbone trace of dimer of beef heart cytochrome c oxidase. Each monomer consists of 13 different subunits, each shown in a different color. The subunit name is given in the color of the subunit. (a) Side view against the transmembrane helices. (b) Top view from the cytosolic side.
in the reactivity to the h e m e ligands, examined using Fourier transform infrared (Fq'IR), has provided unique information on the interaction b e t w e e n heine a 3 and the other redox metal sites [18]. Heme a and the long alkyl sidechain
H e i n e a is coordinated by two histidines, His61 and His378, of subt, nit I. T h e iron atom is in the h e m e plane thus indicating a low spin ferric state [6]. T h e h e m e plane is positioned perpendicular to the m e m b r a n e surface with the hvdroxylfarnesylethyl sidechain of the h e m e directed toward the m e m b r a n e surface of the matrix side in almost the fully e x t e n d e d conformation [6,7]. On the one hand, this e x t e n d e d conformation seems to anchor the h e m e plane quite stronglx: On the other hand, the corresponding long sidechain of h e m e a 3 serves as a structural e l e m e n t of one of the possible 02 channels to the 0 2 reduction site [8"]. It is widely accepted that all the enzymes in the terminal oxidase family reduce 02 to water coupled with the p u m p i n g of protons via an identical reaction mechanism [19[. T h e insertion of the large alkyl groups of h e m e s a and a 3 into the protein moiety within the t r a n s m e m b r a n e region, however, will induce significantly large conformational changes. If the reaction mechanism of the enzymes is identical regardless of the structure of heroes, c o m p e n s a t i n g conformational changes must occur in other regions of the protein. Alternatively, the mechanism of the e n z y m e reaction may be modified without the need for large, c o m p e n s a t i n g conformational changes. Thus, it is important to soh, e the crystal structure of the terminal oxidase that does not contain the hydroxyl fa rnesylethyl group.
0 2 reduction site
T h r e e histidine imidazoles coordinate to CUB [6]. "File copper atom is at the c e n t e r of an equilateral triangle consisting of the three coordinating histidine nitrogens; the histidine triangle is parallel to the h e m e a 3 porphyrin plane. CuB is 4.7~1 away from the heine a 3 iron and 1.0.~l away from a line passing through the iron atom of h e m e a3 and normal to the plane of the porphyrin ring (Figure 2). On the one hand, electron density maps indicate the absence of any larger atom such as chloride b c t w e e n the two metals, however, smaller atoms such as oxygen and nitrogen may be present. On the other hand, a cupric copper in a trigonal planar coordination is unlikel-~; Thus, the presence of another ligand to Cul~ will probably form a tetrahedtal coordination. It should be noted that the m e t a l - m e t a l distance, 4.7 ~i, is too long for a bridging coordination by a single atom. Tyr248 is hydrogen b o n d e d to the hydroxyl group of the hydroxylfarnesylethyl group of h e m e a.~ (Figure 2). This tyrosine is likely to be a proton donor to a reaction i n t e r m e d i a t e formed during the O 2 reduction, as a hydrogen-bond network has been identified from Tyr244 to Lys265 at the matrix surface of the protein. In addition to the proton donating site, a possible water channel leads to the O 2 reduction site [8°°]. T h e channel begins at one of the propionate groups of h e m e a 3, which forms a hydrogen bond with His368 - - one of the ligands of the magnesium atom. "IM'o arrays of hydrophilic amino acids, one belonging to subunit 1 and the other belonging to subunit II, are situated at the interface b e t w e e n subunits 1 and II. This intert:ace forms a hydrophilic channel from the propionate of h e m e a 3 to the cytosolic surface. T h e channel is too narrow to accept even a single water
576
Membrane proteins
Figure 2
between Cu A and berne a compared with the one between CUA and heme a3 (19,~ versus 22.~). Furthermore, it is still not clear why the crystal structure indicates a possible pathway for the direct electron transfer from Cu A to heine a3.
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molecule, suggesting a tightly controlled mechanism for draining water from the active site and preventing the back flow of water from the cytosolic side. Such a back-flow could result in the influx of protons and a short circuit of the transmembrane proton gradient.
Electron transfer between the redox centers Hemes a and a 3 are in close proximity, which suggests t h a t extremely rapid electron transfer can occur between them. The closest approach between the two hemes is 4 ~. T h e fifth ligands of heroes a and a3 are separated in the amino acid sequence by only one residue. A hydrogen-bond network between CUA and heme a includes His204, one of the ligands to CUA, a peptide bond between Arg438 and Arg439, and a propionate group of heme a (Figure 3). T h e network seems to provide a facile electron transfer path from Cu A to heme a. T h e double bond character of the peptide bond promotes the efficiency of electron transfer. An alternate network connecting Cu A and heine a 3 via the magnesium site (Figure 3) could also be an effective electron transfer path directly from Cu A to heme a3 [7,8°°]. A direct electron transfer between CUA and heme a3, however, has never been detected in transient kinetic investigations [20]. These kinetic measurements suggest that the electron transfer rate from Cu A to heme a is at least two orders of magnitude higher than the rate from Cu A to heine a 3. Differences between the two electron transfer rates can be rationalized by the shorter distance
The hydrogen-bond network between the redox active metal sites in beef heart cytochrome c oxidase. Dotted lines denote possible hydrogen bonds and broken lines denote metal ion coordination bonds. Blue and pink balls are copper and magnesium ions, respectively. Pink structures with and without a blue ball are hemes a3 and a, respectively. Three amino acid residues, His368, Arg438, Arg439, belong to subunit I; the others belong to subunit II.
Mechanism of 0 2 reduction A one-electron reduction of 0 2, the triplet dioxygen, is well known to be energetically unfavorable, whereas a two-electron reduction is quite favorable [21]. This property of Oz provides the stability for the oxygenated forms of hemoglobin and myoglobin, in which sequestering the FeZ+-O2 inside the protein provides protection from the two-electron reduction. Thus, CUB placed near the Oz-binding site must trigger the reduction of 0 2 or Fea3 z+ via the two-electron process to the level of per~,xide. This process is likely to be limited by the election transfer across a short distance of approximately 3,~, from CUB 1÷ to 0 2 on Fea32+. T h e process is expected to be very r a p i d - - o f the order of a picosecond. Unexpectedly, however, time-resolved resonance Raman spectroscopy detects the existence of the oxygenated form of the enzyme (Fe-O z) with a fairly long life time, tl/z =0.1 msec at 4°C, during the reaction of the fully reduced enzyme with O 2 [22]. T h e isotopic shifts of the Fe-O 2 vibration determined by exchanging 1 6 0 2 with 1 8 0 2 and 1 6 0 1 8 0
Beef heart cytochrome c oxidase Yoshikawa
indicate that O 2 binds to Fe z+ in an end-on fashion similar to its binding in hemoglobin [4°',23]. The resonance Raman results suggest a structure at the active site that is consistent with a diminished electron-transfer rate from CUB to the bound O 2. Currentl3, only the crystal structure of cytochrome c oxidase in the fully oxidized state has been reported. Crystal structures of many other metalloproteins indicate that the conformational differences induced by a change in the oxidation state at the metal site are very small. Thus, the structure of the O 2 reduction site in the fully reduced state of cytochrome c oxidase is likely to closely resemble the structure in the fully oxidized state. When O z initially binds to the Fea3 z+ in an end-on fashion without any interaction occurring with CuB, coordination
577
of CUBI+ is trigonal planar, with the three histidine imidazoles as the ligands. The Cu I+ in such a coordination is very stable [24t, and this stability limits the electron transfer from CUB 1+ to the 02 bound to Fea3 2+. Thus, the second electron is likely to come from heme a. The crystal structure indicates that Tyr244 hydrogen bonds to the bound O 2 concomitantly with a small conformational change [25]. Formation of this hydrogen bond is likely to promote the reduction of the bound O2 to a hydroperoxy species. The formation can not be very rapid because a conformational change of Tyr244, albeit small, takes place. In peroxidase, the hydroperoxo intermediate very rapidly decomposes to compound I, which is an oxoferryl intermediate (Fe4+-O) with one extra oxidation equivalent, concomitantly with the release of HeO [26]. By analogy,
Figure 4
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Schematic representations of two possible channels for proton pumping. Shaded ovals, dotted lines and dotted lines with arrows denote cavities, hydrogen bonds, and possible hydrogen-bond configurations, respectively. The cavities contain no electron density but are large enough for keeping randomly oriented or mobile water molecules. A pair of amino acid sidechains in a possible hydrogen-bond configuration are placed too far apart with each other to form a hydrogen bond; however, a small conformational change in the pair without movement of peptide bond could provide a new hydrogen bond. The matrix side of each network is shown at the bottom and the cytosolic side at the top. Both (a) and (b) show only the structure connecting the matrix port to the cytosolic port. No dead-end branch is shown except for a branch leading to heme a in the channel B. Both channels occur in subunit I, therefore, all the amino acid residues shown belong to subunit I.
578
Membrane proteins
a similar process is likely to occur for the hydroperoxy intermediate of cytochrome c. Recent resonance Raman evidence suggests that the extra oxidation equivalent is on the iron atom as FeS+-O [4"]. CUB, with its extremely high tedox potential, would be able to reduce the Fea3 at this high oxidation state during enzymic turnover.
Mechanism of proton pumping The coupling of the 0 2 reduction with proton translocation has been carefully and ingeniously investigated by examining the effect of the electrochemical potential on the reverse of the Oz reduction - - t h e oxidation of water [5]. These investigations have demonstrated that only two out of four electron transfer steps during the 0 2 reduction to water are driven in the reverse direction when an electrochemical potential opposite in polarity to that of physiological conditions is applied. Papa and coworkers [27] have reported that the efficiency of proton uptake is dependent on the electron transfer rate, which suggests an indirect coupling of the Oz reduction with proton translocation. One year before the crystal structures of cytochrome c oxidase appeared, Wikstrs6m et al. [28] proposed a mechanism for redox-coupled proton pumping on the basis of the coordination structure of the 0 2 reduction site deduced from mutagenesis results [28]. In their mechanism, one of the three histidine ligands of Cu B acts as a unidirectional proton shuttle by dissociating itself from Cu B. The dissociation is dependent on protonation of the histidine imidazole as well as on the oxidation and ligand-binding states of the O 2 reduction site. The mechanism seems consistent with the crystal structure of the azide-bound fully oxidized bacterial cytochrome c oxidase [7,29°]. One of the three histidine imidazoles of this enzyme is missing in the crystal structure which does suggest the mobility of this residue. In the mechanism of Wikstr/Sm et al. [28], this histidine imidazole forms the proton pumping site in addition to being a part of the 0 2 reduction site. Thus, this proton pumping site must keep the protons completely away from the dioxygen reduction site, othei~visc the pumping protons are likely to be taken by O Z reduction intermediates that form during the O 2 reduction and that have an extremely high affinity to protons. Such proton trapping would ultimately short circuit the proton pumping mechanism [28,30]. As mentioned above, the lack of one of the histidine of Cu B ligands in the crystal structure of bacterial enzyme suggests a mobility of the imidazole; however, it would appear to be very difficult for such a mobile imidazole to keep the protons completely away from the intermediate species of O z reduction on Yea3. Two hydrogen-bond networks from the matrix side to the cytosolic side have been mapped. (Figure 4) [8°°]. These networks are probable conduits for proton pumping; both
are well separated from the redox-active metal site. No probable proton pumping pathways have been detected at the 0 2 reduction site in the beef heart crystal structure. For the elucidation of the proton pumping mechanism, the identification of redox-coupled conformational changes is of paramount importance. Redox-linked difference infrared spectra obtained from bacterial cytochrome c oxidasc suggest conformational and/or protonation changes at a carboxyl sidechain arc involved [31"]. In addition, redox-linked conformational changes have been suggested by the circular dichroism spectrum of beef heart enzyme [32].
Conclusions The crystal structure of beef heart cytochrome c oxidase in the fully oxidized form at 2.8 ~, resolution suggests that heine a, not CUB, is the donor of the second electron to reduce 0 2 bound at Fea32+. Tvr244 positioned close to the 0 2 reduction site is the proton donor for the production of water. Two possible proton pumping networks well separated from the O z reduction site have been identified within the crystal structure of beef heart enzyme. The crystal structures of the enzyme in various oxidation and ligand-binding states at the level of 2.0]~ resolution are desirable for further understanding the mechanism of the redox coupled proton pumping of this enzyme. Hopefull>, at such a high resolution, the 02 reduction intermediate species will be detectable at the Fea3-Cu B site. Beef heart cytochrome c oxidase crystals at 2.1-2.3 ~ resolution have been obtained; the crystal analysis of these as well as further improvement of the crystallization conditions are underwa.v
Acknowledgement I wish to thank Herbert [, Axclrod for his valuable discussion.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest • ,, of outstanding interest 1. Ferguson-Miller S, Babcock GT: Heme/copper terminal •. oxidases. Chem Rev 1996, 96:2889-290Z A comprehensive and well-organized review on the structure and function of cytochrome c oxidase. It is currently the best review of the present level of the mechanistic research of this enzyme. A comparison of the paragraphs in this review related to the 02 reduction with those of [4 °°] will be .'elpful for considering this intriguing enzyme reaction. 2.
Malmstr6m BG: Cytochrome c oxidase as a redox-linked pro, ,,1 pump. Chem Rev 1990, 90:1247-1260.
3.
Warburg O: Uber eisen, den sauerstoffiJbertragenden bestandteil des atmungsfermentes. Biochem Z 1924, 152:479494. [Title translation: Iron and oxygen transporters of the respiratory system.]
4. °,
Kitagawa T, Ogura T: Oxygen activation mechanism at the binuclear site of heme-copper oxidase superfamily as revealed by time-resolved resonance Raman spectroscopy, Progr/norg Chem 1997, 45:431-4'79. A good summary of resonance Raman studies on the 02 activation of cytochrome c oxidase, showing how powerful this spectroscopy is for investigating the 02 reduction mechanism. It includes a well-written introduction of
Beef heart cytochrome c oxidase Yoshikawa
the principles and practice of time-resolved resonance Raman spectroscopy, which is suitable for biochemists not familiar to this technique. 5.
18.
Yoshikawa S, Mochizuki M, Zhao X-J, Caughey WS: Effects of overall oxidation state on infrared spectra of heine a 3 cyanide in bovine heart cytochrome c oxidase. J Biol Chem 1995, 270:4270-4279.
19.
Calhoun MW, Thomas JW, Gennis RB: The cytochrome oxidase superfamily of redox-driven proton pumps. Trends Biochem Sci 1994, 19:325-330.
20.
Hill BC: Modeling the sequence of electron transfer reactions in the single turnover of reduced, mammalian cytochrome c oxidase. J Biol Chem 1994, 269:2419-2425.
21.
Caughey WS, Wallace WJ, Volpe JA, Yoshikawa S: Cytochrome c oxidase. In The Enzymes, edn 3. Edited by Boyer PD. New York: Academic Press; 1976:299-344.
22.
Ogura 1", Takahashi S, Shinzawa-ltoh K, Yoshikawa S, Kitagawa T: Time-resolved resonance Raman investigation of cytochrome oxidase catalysis: observation of a n e w oxygen-isotope sensitive Raman band. Bu//Chem Soc Jpn 1991, 64:2901-2907.
23.
Ogura T, Takahashi S, Hirota S, Shinzawa Itoh K, Yoshikawa S, Appelman EH, Kitagawa T: Time-resolved resonance Raman elucidation of the pathway for dioxygen reduction by cytochrome c oxidase. J Am Chem Soc t993, 115:8527-8536.
24.
Cotton FA, Wilkinson G: Chemistry of the transition elements, copper. In Advanced Inorganic Chemistry, edn 4. New York: John Wiley Sons; 1980:798-821.
WikstrSm M, Morgan JE: The dioxygen cycle. J Biol Chem 1992, 267:10266-10273
6.
7.
Tsukihara T, Aoyama H, Yamashita E, Tomizaki T, Yamaguchi H, Shinzawa-ltoh K, Nakashima R, Yaono R, Yoshikawa S: Structures of metal sites of oxidized bovine heart cytochrome c oxidase at 2.8 A. Science 1995, 269:1069-1074. Iwata S, Ostermeier C, Ludwig B, Michel H: Structure at 2.8 A resolution of cytochrome c oxidase from Paracoccus denitrificans. Nature 1995, 376:660-669.
8. •-
Tsukihara T, Aoyama H, Yamashita E, Tomizaki T, Yamaguchi H, Shinzawa-ltoh K, Nakashima R, Yaono R, Yoshikawa S: The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A. Science 1996, 272:1136-1144. The detailed crystal structures of 13-subunit beef heart cytochrome c oxidase are described at 2.8 ~ resolution in the fully oxidized state. Two hydrogen bond networks, which do not involve the 02 reduction site, are shown to be possible proton pumping sites. Phospholipids and ADP-binding sites in the crystal structure are also given.
579
9.
Kadenbach B, Ungibauer M, Jarausch J, Buge U, Kuhn-Nentwig L: The complexity of respiratory complexes. Trend Biochem Sci 1983, 8:398-400.
10.
Steffens GCM, Soulinane T, Wolff G, Buse G: Stoichiometry and redox behavior of metals in cytochrome-c oxidase. Eur J Biochem 1993, 213:1149-1157
25.
Verkhovsky MI, Morgan JE, Wikstr6m M: Oxygen binding and activation: early steps in the reaction of oxygen with cytochrome c oxidase. Biochemistry 1994, 33:3079-3086.
11.
Kroneck PMH, Antholine WA, Riester J, Zumft WG: The cupric site in nitrous oxide reductase contains a mixed-valence [Cu(ll), Cu(I)] binuclear center: a multifrequency electron paramagnetic resonance investigation. FEBS Let1 1988, 242:70-74.
26.
Kitagawa T, Mizutani Y: Resonance Raman spectra of highly oxidized metalloporphyrins and heine proteins. Coordination Chem Rev 1994, 135/136:685-735.
27.
Capitanio N, Capitanio G, Demarinis DA, De Nitto E, Massari S, Papa S: Factors affecting the H+/e - stoichiometry in mitochondrial cytochrome c oxidase: influence of the rate of electron flow and transmembrane ApH. Biochemistry 1996, 35:10800-10806.
28.
Wikstrom M, Bogachev A, Finel M, Morgan JE, Puustinen A, Raitio M, Verkhovskaya M, Verkhovsky MI: Mechanism of proton translocation by the respiratory oxidases. The histidine cycle. Biochim Biophys Acta 1994, 1187:106-111.
12.
Larsson S, K~llebring B, Wittung P, Malmstr6m BG: The Cu A center of cytochrome-c oxidase: electronic structure and spectra of models compared to the properties of Cu A domains. Proc Natl Acad Sci USA 1995, 92:7167-7171.
13.
Wimanns M, Lappalainen P, Kelly M, Sauer-Eriksson E, Saraste M: Crystal structure of the membrane-exposed domain from a respiratory quinol oxidase complex with an engineered dinuclear copper center. Proc Nat/Acad Sci USA 1995, 92:11955-11959.
14.
Anthony G, Reimann A, Kadenbach B: Tissue-specific regulation of bovine heart ¢ytochrome-c oxidase activity by ADP via interaction with subunit Via. Proc Nat/Acad Sci USA 1993, 90:1652-1656.
15.
Frank V, Kadenbach B: Regulation of the H+/e - stoichiometry of cytochrome c oxidase from bovine heart by intramitochondrial ATP/ADP ratios. FEBS Lett 1996, 382:121-124.
16.
Nicholls P, Wrigglesworth JM: Routes of cytochrome a 3 reduction. Ann New York Acad Sci 1988, 550:59-67.
17.
Jensen LH: The iron-sulfur proteins: an overview. In Iron-Sulfur Protein Research. Edited by Matsubara H, Katsube Y, Wade K. Berlin: Springer-Verlag; 1986:3-21.
29. Ostermeier C, Iwata S, Michel H: Cytochrome c oxidase. Curr * Opin Struct Bio11996, 6:460-466. A summary of the crystal structure of the bacterial enzyme and a discussion of the reaction mechanism. 30.
Williams RIP: Purpose of proton pathways. Nature 1995, 376:643.
31. •
Hellwig P, Rest B, Kaiser U, Ostermeier C, Michel H, M&ntele W: Carboxyl group protonation upon reduction of the Paracoccus denitrificans cytochrome c oxidase: direct evidence by FTIR spectroscopy. FEBS Lett 1996, 385:53-57. This is the first report showing redox-linked conformational and/or protonation changes of the carboxyl group of glutamate or aspartate. This finding may promote the real-time measurement of proton pump behavior. 32.
Wittung P, MalmstrSm BG: Redox-linked conformational changes in cytochrome c oxidase. FEBS Lett 1996, 388:47-49.