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A structural and functional perspective on the evolution of the heme–copper oxidases
FEBS Letters 588 (2014) 3787–3792
journal homepage: www.FEBSLetters.org
Hypothesis
A structural and functional perspective on the evolution of the heme–copper oxidases Vivek Sharma a,⇑, Mårten Wikström b a b
Department of Physics, Tampere University of Technology, PO Box 692, FI-33101 Tampere, Finland Helsinki Bioenergetics Group, Institute of Biotechnology, University of Helsinki, FI-00014 Helsinki, Finland
a r t i c l e
i n f o
Article history: Received 15 August 2014 Revised 10 September 2014 Accepted 11 September 2014 Available online 26 September 2014 Edited by Peter Brzezinski Keywords: Electron transfer Cytochrome c oxidase Nitric oxide reductase Proton pumping
a b s t r a c t The heme–copper oxidases (HCOs) catalyze the reduction of O2 to water, and couple the free energy to proton pumping across the membrane. HCOs are divided into three sub-classes, A, B and C, whose order of emergence in evolution has been controversial. Here we have analyzed recent structural and functional data on HCOs and their homologues, the nitric oxide reductases (NORs). We suggest that the C-type oxidases are ancient enzymes that emerged from the NORs. In contrast, the A-type oxidases are the most advanced from both structural and functional viewpoints, which we interpret as evidence for having evolved later. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction 1.1. Heme–copper oxidases The emergence of molecular oxygen (O2) on Earth significantly perturbed the processes by which organisms generate energy. Early on, many organisms respired anaerobically, and were dependent on alternative means to fulfill their energy requirements [1]. This scenario changed dramatically with the increase in O2 concentration, and facultative or strictly aerobic organisms evolved [2]. During these transitions, and distributed over hundreds of millions of years, one class of enzymes that has undergone major structural changes are the heme–copper oxidases (HCOs) [3]. The HCOs are characterized by a unique active site structure in which a high-spin heme is magnetically coupled to a copper ion, CuB, that has three histidine ligands and a tyrosine cross-linked to one of them [4,5]. In addition, they employ a low-spin heme in the immediate vicinity, which acts as an electron donor to the active site [4,5] (Fig. 1). The HCOs catalyze the four-electron reduction of O2 to water (Eo0 = 815 mV at 300 K), and couples the free energy of this reaction to proton pumping across either the inner membrane of mitochondria or the plasma membrane of bacteria [4,5]. The
Abbreviations: HCO, heme–copper oxidase; NOR, nitric oxide reductase; BNC, binuclear center ⇑ Corresponding author. E-mail address: vivek.sharma@tut.fi (V. Sharma).
proton electrochemical gradient established across the membrane is utilized for the synthesis of ATP, or for active transport [5]. The HCOs are mainly divided into three distinct subfamilies, A, B and C ([6], but see also [7]). Mitochondrial or bacterial A-type oxidases have been studied in great depth, and their molecular mechanism of oxygen reduction and proton pumping is relatively well understood [5,8]. A combination of structural, functional and computational investigations has shed light on the coupling between the two reactions [5]. Even though corresponding data on the B- and C-type oxidases is relatively scarce, recent experimental highlights indeed show similarities between the molecular mechanisms of B- and A-type oxidases [9]. 1.2. Nitric oxide reductases The nitric oxide reductases (NORs) are structurally related to the HCOs. Instead of a copper ion, they employ a non-heme iron (FeB) in their active site, which is magnetically coupled to the high-spin heme [10,11]. NORs catalyze the reduction of NO to N2O (Eo0 = 1.175 V) [10,11]. However, they do not seem to conserve the free energy of the reaction to proton pumping or to any other endergonic process. The NORs are classified into cNORs, qNORs and qCuANORs, depending upon the electron donor, that is either cytochrome (c) or a quinol (q) [10]. The recently solved crystal structures of cNOR and qNOR [12,13] have stimulated further experimentation on the molecular mechanisms [14].
http://dx.doi.org/10.1016/j.febslet.2014.09.020 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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argued for an independent emergence of C- and B-type oxidases in bacterial and archaeal phyla, respectively. Thus, phylogenetic analyses have led to diametrically opposite views on the evolutionary history of the HCOs and NORs (cf. [21]). Here, we have deliberately avoided the molecular phylogenetics approach. Based purely on recent structural and functional data, we propose that the C- and B-type HCOs evolved from the NORs prior to the A-type enzymes, which are by far the most sophisticated HCOs with regard to the efficiency of energy transduction. 2. C-type oxidases and NORs
Fig. 1. Cytochrome c oxidase immersed in a membrane-solvent environment. Subunit I (blue), II (red) and III (orange) are shown as transparent ribbons. The Dand K-channels of proton transfer, based on D91 (red) and K319 (blue), respectively, are shown with blue arrows. Electron transfer from CuA to the heme a3-CuB BNC (via heme a) is shown with a red arrow. Crystallographically observed water molecules (ice-blue), hemes (green), copper atoms (orange), Na+ (yellow) and Cl (cyan) ions are also displayed.
1.3. Controversial phylogeny of HCOs and NORs Evolutionary aspects pertaining to HCOs and NORs were first analyzed by Saraste et al. in the early 1990s [3,15]. Based on the limited sequence data available at the time, they proposed that the HCOs were present in the last common ancestor of archaea and bacteria, and that the C-type oxidases, which are known to possess a high binding affinity for oxygen [16], were present prior to the great divergence. The high sequence similarity between the C-type oxidases and the NORs led to the proposal that cellular respiration emerged later than the events of denitrification [3,15]. Subsequent studies by Myllykallio et al. [17] and Pereira et al. [6] challenged the idea of the pre-divergent presence of C-type oxidases, based on the fact that these enzymes are absent in archaea, and that they are only confined to some lesser-deep branches of the proteobacterial domain. This work led to the suggestions that the C-type oxidases appeared later in the bacterial domain, whereas the genes of the B-type oxidases, which are well represented in the archaeal domain, were suggested to have been transferred horizontally from the bacterial domain owing to their higher sequence similarities with their bacterial counterparts [6,18]. In 2008, Ducluzeau et al. re-analyzed a much larger amount of sequence data, and instead advocated for the ancestral origin of the C-type oxidases due to their presence in some of the deepest branches of the bacterial domain [19]. The subsequent studies by Brochier-Armanet et al. [20] provided an alternative view that, due to the presence of A-type oxidases in all three domains of life, these enzymes would have been the first to appear (even before the NORs), and were already present prior to the great divergence. These latter authors further
From the available crystal structures it is evident that the C-type oxidases are much more closely related to the cNORs than to the A- and B-type oxidases. Both the C-type oxidases and the cNORs contain similar catalytic cores that comprise two subunits – N and O in C-type oxidases, b and c in cNORs [13,22]. Moreover, there are structural similarities at the local level, viz. a unique Ca++ ion binding site involving the D-ring propionates of the two adjacent heme b groups (Fig. 2), analogous proteinaceous interaction partners of the heme propionate groups [22–24], and the relative orientations of the heme groups in the catalytic subunit [13,22]. Besides this, functional similarities are also apparent, such as the capability to reduce both NO and O2, [25,26], and a low redox potential of the heme group in the BNC [27,28]. On this basis it is tempting to suggest that the C-type HCOs have evolved from the NORs in the bacterial lineage, enabling organisms to conserve free energy from O2 reduction under conditions of low oxygen tension, and perhaps at the same time to effectively scavenge the toxic O2. Indeed, bioinformatics [15,21], biochemical and geochemical arguments [29], as well as the presence of NORs in archaea [21,30,31], corroborate their presence prior to the great divergence. On the other hand, representatives of C-type oxidases are not (yet) found in archaea, but are present in some of the deepest branches of bacteria [19]. The C-type oxidases may thus indeed have evolved from the NORs, but their genes may have been rapidly lost in the archaeal and mitochondrial lineages during evolution, concomitant with the increase in [O2] (see also [32]). The evolution of the C-type oxidases from the NORs may have occurred by multiple modifications in the protein architecture, such as (a) a change of the BNC metal ion from FeB to CuB [13,22], (b) emergence of a cross-linked histidine-tyrosine ligand of CuB [22,23,33,34], (c) introduction of a proton ‘‘channel’’ in the region surrounding helices VI-VIII (a K-channel analogue) [22,23], (d) incorporation of the non-catalytic subunit P to enable efficient electron and proton transfer [22,35], (e) emergence of a second Ca++ ion site in C-type oxidases (Ca++ ion ligated by Asp131, Pseudomonas stutzeri numbering) in place of a conserved Lys in helix IV of cNOR (Lys144 Pseudomonas aeruginosa numbering) [13,22] and (f) other structural perturbations around the active site (e.g. a glutamate near the proximal histidine ligand of the high-spin heme in the C-type oxidases) [27,36]. It is likely that structural changes in and around the active site contributes to the proton pumping capability of C-type oxidases [37], which is absent in NORs [38]. However, it should also be noted that the number of sequenced genomes of bacteria far surpasses the sequenced genomes of archaea (6833 vs 229, based on Genomes Online Database (GOLD) [39]). Hence, C-type oxidases might still be found in archaea, which would further support their ancient origin together with the NORs. 3. B-type oxidases Many B-type oxidases are commonly found in the archaeal domain, and are capable of energy conservation even at high
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temperatures (70 °C). Factors responsible for this could be the tighter packing of the protein structure together with the cofactors [40]. Horizontal gene transfer of B-type oxidases from the bacterial to the archaeal domain, or vertical inheritance from the possibly lost C-type genes in the archaeal domain, are two possibilities through which archaea may have acquired B-type oxidases. In any case, some structural and functional similarities between the B- and C-type oxidases (also possibly NORs) can be proposed. Both B- and C-type oxidases show much higher binding affinity for O2 relative to the A-type oxidases, either due to the presence of large hydrophobic cavities in the catalytic subunit [41], or may be due to thermodynamic/kinetic factors [36], and both are capable of reducing NO to N2O, which does not occur in the A-type oxidases [42,43]. Some local structural similarities in the ‘‘K-type’’ proton channels, and in the heme–propionate interactions are also present [23,24]. Hence, it is possible that the B-type oxidases evolved from the C-type oxidases in bacteria, and spread later on to the archaeal domain through horizontal gene transfers [6,18]. For the hypothetical transition of C- to B-type oxidase to happen, many structural modifications must have occurred. The Ca++ binding site (between the heme propionates) unique for the C-type oxidases and the NORs, was replaced by a double arginine motif (RR; q = +2e) from the loop between helices XI and XII (Fig. 2), which is characteristic of the A-type enzymes. Moreover, the loop between helices III and IV evolved from a (K/R)E(Y/F)AE motif in the C-type oxidases to a x(W/Y)xxYPPL motif in the B-type oxidases, again more reminiscent of the A-type oxidases (Fig. 2). We further propose that this transition from C- to B-type oxidase could have occurred by considerable shortening of the relatively long TM helices XI and XII in the former, together with the replacement of the well-conserved WR motif in the former to the RR motif in the B-type oxidases (Fig. 2). Such losses of metal-sites (here Ca++) and replacement or re-organization of surrounding segments has indeed been pointed out in other enzyme families [44]. Moreover, shortening of loops in the B-type oxidases may have to do with their capability to conserve energy at higher temperatures [40]. Variations in these segments are also apparent from the recently solved crystal structure of qNOR [12], where the TM helices XI and XII are of intermediate length (Fig. 2). The loop between helices XI and XII adopts a unique arrangement that is different from all the HCOs as well as the cNORs, but has a similar motif, namely (xR)(Fig. 2). 4. A-type oxidases The B- and A-type oxidases share many structural and functional properties, many more than shared between oxidases of C- and A-type (Fig. 2). It therefore seems logical to suggest that the A-type oxidases emerged from the B-type oxidases in both the archaeal and bacterial domains. The A-type oxidase from mitochondria are evolutionarily advanced; they possess additional nuclear-encoded subunits, which make them rather complex nanomachines regulated by various external stimuli, such as the [ATP]/[ADP] ratio in the cytoplasm or proton motive force [45]. The mitochondrial oxidase may function as a dimer in vivo, in which two monomers are glued together e.g. by the negatively charged cardiolipin [46], which may also function as a protoncollecting antenna. The A-type HCOs turn out to be by far the most efficient in energy transduction compared to their B- and C-type counterparts. They are capable of pumping nearly 1H+/e against a high pmf of 150 mV. In contrast, proton-pumping by C- and B-type oxidases has been reported to have very low stoichiometries [47,48], and particular care to minimize protonmotive force was required to show that the mechanistic stoichiometry is yet close to unity also in these cases [49]. Since the D-pathway of proton transfer (Fig. 1)
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is unique for the A-type enzymes, their high proton-pumping efficiency is most likely achieved by the kinetic gating properties of this structure [50–52]. The B- and C-type oxidases are devoid of such sophisticated mechanisms, and are therefore more prone to proton leakage under high proton motive force [49], as also exemplified by mathematical modelling of the oxidase proton pump mechanism [53]. The canonical K-channel of proton transfer in the A-type oxidases comprises a well-conserved lysine residue (Lys319 in Bos taurus enzyme). Its positively charged side chain is known to play a significant role in the reductive phase of the catalytic cycle [5,54–56]. The ‘K-type’ proton channels of the B- and C-type oxidases are known to be devoid of this residue, which may further contribute to their lower proton-pumping efficiency. Interestingly, the A-type oxidase from Aeropyrum pernix has a conserved D-channel, but the residues that are conserved in the canonical K-channel of A-type enzymes, or their analogues in B- and C-types, are absent [6,57]. A similar scenario also exists in cytochrome oxidases from Ferroplasma acidamanus and Acidithiobacillus ferrooxidans [58], and it would therefore be of great interest to assess the proton-pumping efficiency of these enzymes. The notion that all known heme–copper oxidases conserve the free energy of oxygen reduction by pumping protons across the membrane, whereas cNORs are non-electrogenic, raises the intriguing question of whether a member of the family would exist which would generate protonmotive force purely due to uptake of electrons and protons from the opposite sides of the membrane (a Mitchellian redox loop), but which would not function as a proton pump. Such an oxygen reductase indeed exists in the form cytochrome bd [59], which is however completely unrelated to the HCO superfamily. Interestingly, the presence of a ‘‘K-type’’ proton uptake channel in the quinol dependent NOR has indeed been suggested [12]. If this turns out to be true, this enzyme may be a ‘‘missing link’’ between the NORs and the HCOs. 5. Emergence of the cross-linked tyrosine Multiple sequence alignments of the catalytic subunit of the HCOs show that in some of the sequences the cross-linked tyrosine (Tyr244 in Bos taurus enzyme) is neither conserved in helix VI nor in helix VII [29]. However, 3D modeling of the sequence data reveals that the functionally critical role of this tyrosine may be substituted by one of the conserved tyrosines in the vicinity of the BNC [29]. Our own modeling data shows that a ‘‘K-type’’ proton channel and propionate–protein interactions analogous to the B-type oxidases are also conserved in these enzymes, which advocates for the possibility that these sequences correspond to HCOs. However, they may nevertheless have a weak NOR activity, as observed for the B-type oxidases [42]. Multiple origins of the cross-linked tyrosine in different parts of the catalytic subunit could therefore have occurred as a response to the increase in oxygen concentrations, thereby making these enzymes genuine O2 reductases. Furthermore, the position of the cross-linked tyrosine in helix VI of the evolutionarily advanced A-type oxidases may represent a catalytically beneficial location, e.g. compared to the C-type oxidases, in which it is located in helix VII and most likely acts as a primary proton donor [36,60]. It is nevertheless possible that these enzymes lack the critical tyrosine in the active site; structural data is required to resolve this point. Even in such a case they may nevertheless be labeled as oxygen reductases, albeit primitive, in which the fourth electron required to complete the oxygen reduction chemistry cannot be delivered directly from a tyrosine in a semi-concerted fashion. Our modeling data of these enzymes indirectly supports this view, and shows a conserved aspartate residue located on the proximal
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Fig. 2. Arrangement of loops between helices III and IV, and XI and XII in cbb3 oxidase (A, PDB: 3MK7), qNOR (B, PDB: 3AYF), ba3 oxidase (C, PDB: 3S8F) and aa3 oxidase (D, PDB: 1V54). Acidic (red), basic (blue), polar (green) and non-polar (white) amino acid residues are shown in licorice representation. Hemes (orange), Cu (purple), Ca (yellow) and Zn (green) are also shown.
Fig. 3. D-channel area in the A- and B-type oxidases (A and B, respectively). Acidic (red), basic (blue), polar (green) and non-polar (white) amino acid residues are shown in licorice representation. Hemes and CuB are shown in orange and purple, respectively. Crystallographic water molecules (red) and modeled water molecules (light green) are shown as spheres.
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side of the high-spin heme, akin to the glutamate described for the C-type HCOs [27,60]. This may result in a low redox potential of the high-spin heme, so that the low-spin heme will always be reduced when the high-spin heme is reduced and binds O2 [36,60]. 6. Emerging proton channel The loops between helices III and IV, and between helices XI and XII, and the crystallographically visible cluster of water molecules just above the high-spin heme, are all known to play critical roles in the proton-pumping mechanism of the A-type oxidases [5]. It seems likely that the functional D-channel in the A-type oxidases correlates with these structural motifs. Whilst the B-type oxidases have these segments conserved (Figs. 2 and 3), a functional D-channel is nevertheless lacking [61]. However, the recent high resolution crystal structure of the B-type oxidase from T. thermophilus shows water molecules and polar residues in the D-channel area that could well be a ‘‘developing D-channel’’ in this organism, and thus a ‘‘snapshot of evolution’’ (Fig. 3) [62]. Indeed, many of the residues forming part of this structure (Fig. 3) are either partially conserved or are replaced by other polar residues based on the alignment described in Chang et al [61]. Nevertheless, the absence of ‘proton-uptake and release’ sites (such as Asp91 and Glu242 in the bovine enzyme, respectively) renders this structure non-functional as a proton transfer pathway, and supports the idea that the A-type oxidases may have evolved from the B-type oxidases. The increase in O2 concentration together with higher energy demands may have led to higher rates of horizontal gene transfer, and thus to the large spread of the energetically efficient A-type oxidases in all three domains of life. Acknowledgements VS acknowledges postdoctoral researcher funding from the Academy of Finland, and research grants from the Center of Excellence in Biomembrane Research (Academy of Finland). MW acknowledges research grants from the Academy of Finland, the Sigrid Jusélius Foundation and Biocentrum Helsinki. References [1] Canfield, D.E., Rosing, M.T. and Bjerrum, C. (2006) Early anaerobic metabolisms. Philos. Trans. R. Soc. B. 361, 1819–1836. [2] Flück, M., Webster, K.A., Graham, J., Giomi, F., Gerlach, F. and Schmitz, A. (2007) Coping with cyclic oxygen availability: evolutionary aspects. Integr. Comp. Biol. 47, 524–531. [3] Castresana, J. and Saraste, M. (1995) Evolution of energetic metabolism: the respiration-early hypothesis. Trends Biochem. Sci. 20, 443–448. [4] Ferguson-Miller, S. and Babcock, G.T. (1996) Heme copper terminal oxidases. Chem. Rev. 96, 2889–2908. [5] Kaila, V.R.I., Verkhovsky, M.I. and Wikström, M. (2010) Proton-coupled electron transfer in cytochrome oxidase. Chem. Rev. 110, 7062–7081. [6] Pereira, M.M., Santana, M. and Teixeira, M. (2001) A novel scenario for the evolution of haem–copper oxygen reductases. Biochim. Biophys. Acta 1505, 185–208. [7] Hemp, J. and Gennis, R.B. (2008) Diversity of the heme–copper superfamily in archaea: insights from genomics and structural modeling. Results Probl. Cell Differ. 45, 1–31. [8] Wikström, M. (2012) Active site intermediates in the reduction of O2 by cytochrome oxidase, and their derivatives. Biochim. Biophys. Acta, 1817468– 1817475. [9] von Ballmoos, C., Ädelroth, P., Gennis, R.B. and Brzezinski, P. (2012) Proton transfer in ba3 cytochrome c oxidase from Thermus thermophilus. Biochim. Biophys. Acta 1817, 650–657. [10] Watmough, N.J., Field, S.J., Hughes, R.J. and Richardson, D.J. (2009) The bacterial respiratory nitric oxide reductase. Biochem. Soc. Trans. 37, 392–399. [11] Berks, B.C., Ferguson, S.J., Moir, J.W. and Richardson, D.J. (1995) Enzymes and associated electron transport systems that catalyse the respiratory reduction of nitrogen oxides and oxyanions. Biochim. Biophys. Acta 1232, 97–173. [12] Matsumoto, Y., Tosha, T., Pisliakov, A.V., Hino, T., Sugimoto, H., Nagano, S., Sugita, Y. and Shiro, Y. (2012) Crystal structure of quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus. Nat. Struct. Mol. Biol. 19, 238–245.
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[43] Brudvig, G.W., Stevens, T.H. and Chan, S.I. (1980) Reactions of nitric oxide with cytochrome c oxidase? Biochemistry 19, 5275–5285. [44] Torrance, J.W., MacArthur, M.W. and Thornton, J.M. (2008) Evolution of binding sites for zinc and calcium ions playing structural roles. Proteins 71, 813–830. [45] Susanne, A. (2012) The power of life – cytochrome c oxidase takes center stage in metabolic control, cell signalling and survival. Mitochondrion 12, 46–56. [46] Paradies, G., Paradies, V., Benedictis, V.D., Ruggiero, F.M. and Petrosillo, G. (2014) Functional role of cardiolipin in mitochondrial bioenergetics. Biochim. Biophys. Acta 1837, 408–417. [47] Kannt, A., Soulimane, T., Buse, G., Becker, A., Bamberg, E. and Michel, H. (1998) Electrical current generation and proton pumping catalyzed by the ba3-type cytochrome c oxidase from Thermus thermophilus. FEBS Lett. 28, 17–22. [48] Han, H. et al. (2011) Adaptation of aerobic respiration to low O2 environments. Proc. Natl. Acad. Sci. U.S.A. 108, 14109–14114. [49] Rauhamäki, V. and Wikström, M. (2014) The causes of reduced protonpumping efficiency in type B and C respiratory heme–copper oxidases, and in some mutated variants of type A. Biochim. Biophys. Acta 1837, 999– 1003. [50] Wikström, M., Verkhovsky, M.I. and Hummer, G. (2003) Water-gated mechanism of proton translocation by cytochrome c oxidase. Biochim. Biophys. Acta 1604, 61–65. [51] Siegbahn, P.E.M. and Blomberg, M.R. (2007) Energy diagrams and mechanism for proton pumping in cytochrome c oxidase. Biochim. Biophys. Acta 1767, 1143–1156. [52] Kaila, V.R.I., Verkhovsky, M.I., Hummer, G. and Wikström, M. (2008) Glutamic acid 242 is a valve in the proton pump of cytochrome c oxidase. Proc. Natl. Acad. Sci. U.S.A. 105, 6255–6259. [53] Kim, Y.C. and Hummer, G. (2012) Proton-pumping mechanism of cytochrome c oxidase: a kinetic master-equation approach. Biochim. Biophys. Acta 1817, 526–536.
[54] Bloch, D., Belevich, I., Jasaitis, A., Ribacka, C., Puustinen, A., Verkhovsky, M.I. and Wikström, M. (2004) The catalytic cycle of cytochrome c oxidase is not the sum of its two halves. Proc. Natl. Acad. Sci. U.S.A. 101, 529–533. [55] Konstantinov, A.A., Siletsky, S., Mitchell, D., Kaulen, A. and Gennis, R.B. (1997) The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer. Proc. Natl. Acad. Sci. U.S.A. 94, 9085–9090. [56] Ädelroth, P., Gennis, R.B. and Brzezinski, P. (1998) Role of the pathway through K(I-362) in proton transfer in cytochrome c oxidase from R. sphaeroides. Biochemistry 37, 2470–2746. [57] Kawarbayashi, Y. et al. (1999) Complete genome sequence of an aerobic hyper-thermophilic crenearchaeon, Aeropyrum pernix K1. DNA Res. 6, 83–101. [58] Ferguson, S.J. and Ingledew, W.J. (2008) Energetic problems faced by microorganisms growing or surviving on parsimonious energy sources and at acidic pH: I. Acidithiobacillus ferrooxidans as a paradigm. Biochim. Biophys. Acta 1777, 1471–1479. [59] Giuffrè, A., Borisov, V.B., Mastronicola, D., Sarti, P. and Forte, E. (2012) Cytochrome bd oxidase and nitric oxide: from reaction mechanisms to bacterial physiology. FEBS Lett. 586, 622–629. [60] Sharma, V., Wikström, M. and Kaila, V.R.I. (2010) Redox-coupled proton transfer in the active site of cytochrome cbb3. Biochim. Biophys. Acta 1797, 1512–1520. [61] Chang, H.Y., Hemp, J., Chen, Y., Fee, J.A. and Gennis, R.B. (2009) The cytochrome ba3 oxygen reductase from Thermus thermophilus uses a single input channel for proton delivery to the active site and for proton pumping. Proc. Natl. Acad. Sci. U.S.A. 106, 16169–16173. [62] Tiefenbrunn, T., Liu, W., Chen, Y., Katritch, V., Stout, C.D., Fee, J.A. and Cherezov, V. (2011) High resolution structure of the ba3 cytochrome c oxidase from Thermus thermophilus in a lipidic environment. PLoS ONE 6, e22348.
journal homepage: www.FEBSLetters.org
Hypothesis
A structural and functional perspective on the evolution of the heme–copper oxidases Vivek Sharma a,⇑, Mårten Wikström b a b
Department of Physics, Tampere University of Technology, PO Box 692, FI-33101 Tampere, Finland Helsinki Bioenergetics Group, Institute of Biotechnology, University of Helsinki, FI-00014 Helsinki, Finland
a r t i c l e
i n f o
Article history: Received 15 August 2014 Revised 10 September 2014 Accepted 11 September 2014 Available online 26 September 2014 Edited by Peter Brzezinski Keywords: Electron transfer Cytochrome c oxidase Nitric oxide reductase Proton pumping
a b s t r a c t The heme–copper oxidases (HCOs) catalyze the reduction of O2 to water, and couple the free energy to proton pumping across the membrane. HCOs are divided into three sub-classes, A, B and C, whose order of emergence in evolution has been controversial. Here we have analyzed recent structural and functional data on HCOs and their homologues, the nitric oxide reductases (NORs). We suggest that the C-type oxidases are ancient enzymes that emerged from the NORs. In contrast, the A-type oxidases are the most advanced from both structural and functional viewpoints, which we interpret as evidence for having evolved later. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction 1.1. Heme–copper oxidases The emergence of molecular oxygen (O2) on Earth significantly perturbed the processes by which organisms generate energy. Early on, many organisms respired anaerobically, and were dependent on alternative means to fulfill their energy requirements [1]. This scenario changed dramatically with the increase in O2 concentration, and facultative or strictly aerobic organisms evolved [2]. During these transitions, and distributed over hundreds of millions of years, one class of enzymes that has undergone major structural changes are the heme–copper oxidases (HCOs) [3]. The HCOs are characterized by a unique active site structure in which a high-spin heme is magnetically coupled to a copper ion, CuB, that has three histidine ligands and a tyrosine cross-linked to one of them [4,5]. In addition, they employ a low-spin heme in the immediate vicinity, which acts as an electron donor to the active site [4,5] (Fig. 1). The HCOs catalyze the four-electron reduction of O2 to water (Eo0 = 815 mV at 300 K), and couples the free energy of this reaction to proton pumping across either the inner membrane of mitochondria or the plasma membrane of bacteria [4,5]. The
Abbreviations: HCO, heme–copper oxidase; NOR, nitric oxide reductase; BNC, binuclear center ⇑ Corresponding author. E-mail address: vivek.sharma@tut.fi (V. Sharma).
proton electrochemical gradient established across the membrane is utilized for the synthesis of ATP, or for active transport [5]. The HCOs are mainly divided into three distinct subfamilies, A, B and C ([6], but see also [7]). Mitochondrial or bacterial A-type oxidases have been studied in great depth, and their molecular mechanism of oxygen reduction and proton pumping is relatively well understood [5,8]. A combination of structural, functional and computational investigations has shed light on the coupling between the two reactions [5]. Even though corresponding data on the B- and C-type oxidases is relatively scarce, recent experimental highlights indeed show similarities between the molecular mechanisms of B- and A-type oxidases [9]. 1.2. Nitric oxide reductases The nitric oxide reductases (NORs) are structurally related to the HCOs. Instead of a copper ion, they employ a non-heme iron (FeB) in their active site, which is magnetically coupled to the high-spin heme [10,11]. NORs catalyze the reduction of NO to N2O (Eo0 = 1.175 V) [10,11]. However, they do not seem to conserve the free energy of the reaction to proton pumping or to any other endergonic process. The NORs are classified into cNORs, qNORs and qCuANORs, depending upon the electron donor, that is either cytochrome (c) or a quinol (q) [10]. The recently solved crystal structures of cNOR and qNOR [12,13] have stimulated further experimentation on the molecular mechanisms [14].
http://dx.doi.org/10.1016/j.febslet.2014.09.020 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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argued for an independent emergence of C- and B-type oxidases in bacterial and archaeal phyla, respectively. Thus, phylogenetic analyses have led to diametrically opposite views on the evolutionary history of the HCOs and NORs (cf. [21]). Here, we have deliberately avoided the molecular phylogenetics approach. Based purely on recent structural and functional data, we propose that the C- and B-type HCOs evolved from the NORs prior to the A-type enzymes, which are by far the most sophisticated HCOs with regard to the efficiency of energy transduction. 2. C-type oxidases and NORs
Fig. 1. Cytochrome c oxidase immersed in a membrane-solvent environment. Subunit I (blue), II (red) and III (orange) are shown as transparent ribbons. The Dand K-channels of proton transfer, based on D91 (red) and K319 (blue), respectively, are shown with blue arrows. Electron transfer from CuA to the heme a3-CuB BNC (via heme a) is shown with a red arrow. Crystallographically observed water molecules (ice-blue), hemes (green), copper atoms (orange), Na+ (yellow) and Cl (cyan) ions are also displayed.
1.3. Controversial phylogeny of HCOs and NORs Evolutionary aspects pertaining to HCOs and NORs were first analyzed by Saraste et al. in the early 1990s [3,15]. Based on the limited sequence data available at the time, they proposed that the HCOs were present in the last common ancestor of archaea and bacteria, and that the C-type oxidases, which are known to possess a high binding affinity for oxygen [16], were present prior to the great divergence. The high sequence similarity between the C-type oxidases and the NORs led to the proposal that cellular respiration emerged later than the events of denitrification [3,15]. Subsequent studies by Myllykallio et al. [17] and Pereira et al. [6] challenged the idea of the pre-divergent presence of C-type oxidases, based on the fact that these enzymes are absent in archaea, and that they are only confined to some lesser-deep branches of the proteobacterial domain. This work led to the suggestions that the C-type oxidases appeared later in the bacterial domain, whereas the genes of the B-type oxidases, which are well represented in the archaeal domain, were suggested to have been transferred horizontally from the bacterial domain owing to their higher sequence similarities with their bacterial counterparts [6,18]. In 2008, Ducluzeau et al. re-analyzed a much larger amount of sequence data, and instead advocated for the ancestral origin of the C-type oxidases due to their presence in some of the deepest branches of the bacterial domain [19]. The subsequent studies by Brochier-Armanet et al. [20] provided an alternative view that, due to the presence of A-type oxidases in all three domains of life, these enzymes would have been the first to appear (even before the NORs), and were already present prior to the great divergence. These latter authors further
From the available crystal structures it is evident that the C-type oxidases are much more closely related to the cNORs than to the A- and B-type oxidases. Both the C-type oxidases and the cNORs contain similar catalytic cores that comprise two subunits – N and O in C-type oxidases, b and c in cNORs [13,22]. Moreover, there are structural similarities at the local level, viz. a unique Ca++ ion binding site involving the D-ring propionates of the two adjacent heme b groups (Fig. 2), analogous proteinaceous interaction partners of the heme propionate groups [22–24], and the relative orientations of the heme groups in the catalytic subunit [13,22]. Besides this, functional similarities are also apparent, such as the capability to reduce both NO and O2, [25,26], and a low redox potential of the heme group in the BNC [27,28]. On this basis it is tempting to suggest that the C-type HCOs have evolved from the NORs in the bacterial lineage, enabling organisms to conserve free energy from O2 reduction under conditions of low oxygen tension, and perhaps at the same time to effectively scavenge the toxic O2. Indeed, bioinformatics [15,21], biochemical and geochemical arguments [29], as well as the presence of NORs in archaea [21,30,31], corroborate their presence prior to the great divergence. On the other hand, representatives of C-type oxidases are not (yet) found in archaea, but are present in some of the deepest branches of bacteria [19]. The C-type oxidases may thus indeed have evolved from the NORs, but their genes may have been rapidly lost in the archaeal and mitochondrial lineages during evolution, concomitant with the increase in [O2] (see also [32]). The evolution of the C-type oxidases from the NORs may have occurred by multiple modifications in the protein architecture, such as (a) a change of the BNC metal ion from FeB to CuB [13,22], (b) emergence of a cross-linked histidine-tyrosine ligand of CuB [22,23,33,34], (c) introduction of a proton ‘‘channel’’ in the region surrounding helices VI-VIII (a K-channel analogue) [22,23], (d) incorporation of the non-catalytic subunit P to enable efficient electron and proton transfer [22,35], (e) emergence of a second Ca++ ion site in C-type oxidases (Ca++ ion ligated by Asp131, Pseudomonas stutzeri numbering) in place of a conserved Lys in helix IV of cNOR (Lys144 Pseudomonas aeruginosa numbering) [13,22] and (f) other structural perturbations around the active site (e.g. a glutamate near the proximal histidine ligand of the high-spin heme in the C-type oxidases) [27,36]. It is likely that structural changes in and around the active site contributes to the proton pumping capability of C-type oxidases [37], which is absent in NORs [38]. However, it should also be noted that the number of sequenced genomes of bacteria far surpasses the sequenced genomes of archaea (6833 vs 229, based on Genomes Online Database (GOLD) [39]). Hence, C-type oxidases might still be found in archaea, which would further support their ancient origin together with the NORs. 3. B-type oxidases Many B-type oxidases are commonly found in the archaeal domain, and are capable of energy conservation even at high
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temperatures (70 °C). Factors responsible for this could be the tighter packing of the protein structure together with the cofactors [40]. Horizontal gene transfer of B-type oxidases from the bacterial to the archaeal domain, or vertical inheritance from the possibly lost C-type genes in the archaeal domain, are two possibilities through which archaea may have acquired B-type oxidases. In any case, some structural and functional similarities between the B- and C-type oxidases (also possibly NORs) can be proposed. Both B- and C-type oxidases show much higher binding affinity for O2 relative to the A-type oxidases, either due to the presence of large hydrophobic cavities in the catalytic subunit [41], or may be due to thermodynamic/kinetic factors [36], and both are capable of reducing NO to N2O, which does not occur in the A-type oxidases [42,43]. Some local structural similarities in the ‘‘K-type’’ proton channels, and in the heme–propionate interactions are also present [23,24]. Hence, it is possible that the B-type oxidases evolved from the C-type oxidases in bacteria, and spread later on to the archaeal domain through horizontal gene transfers [6,18]. For the hypothetical transition of C- to B-type oxidase to happen, many structural modifications must have occurred. The Ca++ binding site (between the heme propionates) unique for the C-type oxidases and the NORs, was replaced by a double arginine motif (RR; q = +2e) from the loop between helices XI and XII (Fig. 2), which is characteristic of the A-type enzymes. Moreover, the loop between helices III and IV evolved from a (K/R)E(Y/F)AE motif in the C-type oxidases to a x(W/Y)xxYPPL motif in the B-type oxidases, again more reminiscent of the A-type oxidases (Fig. 2). We further propose that this transition from C- to B-type oxidase could have occurred by considerable shortening of the relatively long TM helices XI and XII in the former, together with the replacement of the well-conserved WR motif in the former to the RR motif in the B-type oxidases (Fig. 2). Such losses of metal-sites (here Ca++) and replacement or re-organization of surrounding segments has indeed been pointed out in other enzyme families [44]. Moreover, shortening of loops in the B-type oxidases may have to do with their capability to conserve energy at higher temperatures [40]. Variations in these segments are also apparent from the recently solved crystal structure of qNOR [12], where the TM helices XI and XII are of intermediate length (Fig. 2). The loop between helices XI and XII adopts a unique arrangement that is different from all the HCOs as well as the cNORs, but has a similar motif, namely (xR)(Fig. 2). 4. A-type oxidases The B- and A-type oxidases share many structural and functional properties, many more than shared between oxidases of C- and A-type (Fig. 2). It therefore seems logical to suggest that the A-type oxidases emerged from the B-type oxidases in both the archaeal and bacterial domains. The A-type oxidase from mitochondria are evolutionarily advanced; they possess additional nuclear-encoded subunits, which make them rather complex nanomachines regulated by various external stimuli, such as the [ATP]/[ADP] ratio in the cytoplasm or proton motive force [45]. The mitochondrial oxidase may function as a dimer in vivo, in which two monomers are glued together e.g. by the negatively charged cardiolipin [46], which may also function as a protoncollecting antenna. The A-type HCOs turn out to be by far the most efficient in energy transduction compared to their B- and C-type counterparts. They are capable of pumping nearly 1H+/e against a high pmf of 150 mV. In contrast, proton-pumping by C- and B-type oxidases has been reported to have very low stoichiometries [47,48], and particular care to minimize protonmotive force was required to show that the mechanistic stoichiometry is yet close to unity also in these cases [49]. Since the D-pathway of proton transfer (Fig. 1)
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is unique for the A-type enzymes, their high proton-pumping efficiency is most likely achieved by the kinetic gating properties of this structure [50–52]. The B- and C-type oxidases are devoid of such sophisticated mechanisms, and are therefore more prone to proton leakage under high proton motive force [49], as also exemplified by mathematical modelling of the oxidase proton pump mechanism [53]. The canonical K-channel of proton transfer in the A-type oxidases comprises a well-conserved lysine residue (Lys319 in Bos taurus enzyme). Its positively charged side chain is known to play a significant role in the reductive phase of the catalytic cycle [5,54–56]. The ‘K-type’ proton channels of the B- and C-type oxidases are known to be devoid of this residue, which may further contribute to their lower proton-pumping efficiency. Interestingly, the A-type oxidase from Aeropyrum pernix has a conserved D-channel, but the residues that are conserved in the canonical K-channel of A-type enzymes, or their analogues in B- and C-types, are absent [6,57]. A similar scenario also exists in cytochrome oxidases from Ferroplasma acidamanus and Acidithiobacillus ferrooxidans [58], and it would therefore be of great interest to assess the proton-pumping efficiency of these enzymes. The notion that all known heme–copper oxidases conserve the free energy of oxygen reduction by pumping protons across the membrane, whereas cNORs are non-electrogenic, raises the intriguing question of whether a member of the family would exist which would generate protonmotive force purely due to uptake of electrons and protons from the opposite sides of the membrane (a Mitchellian redox loop), but which would not function as a proton pump. Such an oxygen reductase indeed exists in the form cytochrome bd [59], which is however completely unrelated to the HCO superfamily. Interestingly, the presence of a ‘‘K-type’’ proton uptake channel in the quinol dependent NOR has indeed been suggested [12]. If this turns out to be true, this enzyme may be a ‘‘missing link’’ between the NORs and the HCOs. 5. Emergence of the cross-linked tyrosine Multiple sequence alignments of the catalytic subunit of the HCOs show that in some of the sequences the cross-linked tyrosine (Tyr244 in Bos taurus enzyme) is neither conserved in helix VI nor in helix VII [29]. However, 3D modeling of the sequence data reveals that the functionally critical role of this tyrosine may be substituted by one of the conserved tyrosines in the vicinity of the BNC [29]. Our own modeling data shows that a ‘‘K-type’’ proton channel and propionate–protein interactions analogous to the B-type oxidases are also conserved in these enzymes, which advocates for the possibility that these sequences correspond to HCOs. However, they may nevertheless have a weak NOR activity, as observed for the B-type oxidases [42]. Multiple origins of the cross-linked tyrosine in different parts of the catalytic subunit could therefore have occurred as a response to the increase in oxygen concentrations, thereby making these enzymes genuine O2 reductases. Furthermore, the position of the cross-linked tyrosine in helix VI of the evolutionarily advanced A-type oxidases may represent a catalytically beneficial location, e.g. compared to the C-type oxidases, in which it is located in helix VII and most likely acts as a primary proton donor [36,60]. It is nevertheless possible that these enzymes lack the critical tyrosine in the active site; structural data is required to resolve this point. Even in such a case they may nevertheless be labeled as oxygen reductases, albeit primitive, in which the fourth electron required to complete the oxygen reduction chemistry cannot be delivered directly from a tyrosine in a semi-concerted fashion. Our modeling data of these enzymes indirectly supports this view, and shows a conserved aspartate residue located on the proximal
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Fig. 2. Arrangement of loops between helices III and IV, and XI and XII in cbb3 oxidase (A, PDB: 3MK7), qNOR (B, PDB: 3AYF), ba3 oxidase (C, PDB: 3S8F) and aa3 oxidase (D, PDB: 1V54). Acidic (red), basic (blue), polar (green) and non-polar (white) amino acid residues are shown in licorice representation. Hemes (orange), Cu (purple), Ca (yellow) and Zn (green) are also shown.
Fig. 3. D-channel area in the A- and B-type oxidases (A and B, respectively). Acidic (red), basic (blue), polar (green) and non-polar (white) amino acid residues are shown in licorice representation. Hemes and CuB are shown in orange and purple, respectively. Crystallographic water molecules (red) and modeled water molecules (light green) are shown as spheres.
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side of the high-spin heme, akin to the glutamate described for the C-type HCOs [27,60]. This may result in a low redox potential of the high-spin heme, so that the low-spin heme will always be reduced when the high-spin heme is reduced and binds O2 [36,60]. 6. Emerging proton channel The loops between helices III and IV, and between helices XI and XII, and the crystallographically visible cluster of water molecules just above the high-spin heme, are all known to play critical roles in the proton-pumping mechanism of the A-type oxidases [5]. It seems likely that the functional D-channel in the A-type oxidases correlates with these structural motifs. Whilst the B-type oxidases have these segments conserved (Figs. 2 and 3), a functional D-channel is nevertheless lacking [61]. However, the recent high resolution crystal structure of the B-type oxidase from T. thermophilus shows water molecules and polar residues in the D-channel area that could well be a ‘‘developing D-channel’’ in this organism, and thus a ‘‘snapshot of evolution’’ (Fig. 3) [62]. Indeed, many of the residues forming part of this structure (Fig. 3) are either partially conserved or are replaced by other polar residues based on the alignment described in Chang et al [61]. Nevertheless, the absence of ‘proton-uptake and release’ sites (such as Asp91 and Glu242 in the bovine enzyme, respectively) renders this structure non-functional as a proton transfer pathway, and supports the idea that the A-type oxidases may have evolved from the B-type oxidases. The increase in O2 concentration together with higher energy demands may have led to higher rates of horizontal gene transfer, and thus to the large spread of the energetically efficient A-type oxidases in all three domains of life. Acknowledgements VS acknowledges postdoctoral researcher funding from the Academy of Finland, and research grants from the Center of Excellence in Biomembrane Research (Academy of Finland). MW acknowledges research grants from the Academy of Finland, the Sigrid Jusélius Foundation and Biocentrum Helsinki. References [1] Canfield, D.E., Rosing, M.T. and Bjerrum, C. (2006) Early anaerobic metabolisms. Philos. Trans. R. Soc. B. 361, 1819–1836. [2] Flück, M., Webster, K.A., Graham, J., Giomi, F., Gerlach, F. and Schmitz, A. (2007) Coping with cyclic oxygen availability: evolutionary aspects. Integr. Comp. Biol. 47, 524–531. [3] Castresana, J. and Saraste, M. (1995) Evolution of energetic metabolism: the respiration-early hypothesis. Trends Biochem. Sci. 20, 443–448. [4] Ferguson-Miller, S. and Babcock, G.T. (1996) Heme copper terminal oxidases. Chem. Rev. 96, 2889–2908. [5] Kaila, V.R.I., Verkhovsky, M.I. and Wikström, M. (2010) Proton-coupled electron transfer in cytochrome oxidase. Chem. Rev. 110, 7062–7081. [6] Pereira, M.M., Santana, M. and Teixeira, M. (2001) A novel scenario for the evolution of haem–copper oxygen reductases. Biochim. Biophys. Acta 1505, 185–208. [7] Hemp, J. and Gennis, R.B. (2008) Diversity of the heme–copper superfamily in archaea: insights from genomics and structural modeling. Results Probl. Cell Differ. 45, 1–31. [8] Wikström, M. (2012) Active site intermediates in the reduction of O2 by cytochrome oxidase, and their derivatives. Biochim. Biophys. Acta, 1817468– 1817475. [9] von Ballmoos, C., Ädelroth, P., Gennis, R.B. and Brzezinski, P. (2012) Proton transfer in ba3 cytochrome c oxidase from Thermus thermophilus. Biochim. Biophys. Acta 1817, 650–657. [10] Watmough, N.J., Field, S.J., Hughes, R.J. and Richardson, D.J. (2009) The bacterial respiratory nitric oxide reductase. Biochem. Soc. Trans. 37, 392–399. [11] Berks, B.C., Ferguson, S.J., Moir, J.W. and Richardson, D.J. (1995) Enzymes and associated electron transport systems that catalyse the respiratory reduction of nitrogen oxides and oxyanions. Biochim. Biophys. Acta 1232, 97–173. [12] Matsumoto, Y., Tosha, T., Pisliakov, A.V., Hino, T., Sugimoto, H., Nagano, S., Sugita, Y. and Shiro, Y. (2012) Crystal structure of quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus. Nat. Struct. Mol. Biol. 19, 238–245.
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