The inhibitory binding site(s) of Zn2+ in cytochrome c oxidase

FEBS Letters 581 (2007) 611–616

The inhibitory binding site(s) of Zn2+ in cytochrome c oxidase Francesco Franciaa, Lisa Giachinib,c, Federico Boscherinib,c, Giovanni Venturolia,c, Giuseppe Capitaniod, Pietro Luca Martinod, Sergio Papad,e,* a

Department of Biology, University of Bologna, Bologna, Italy Department of Physics, University of Bologna, Bologna, Italy c Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia (CNISM), Bologna, Italy Department of Medical Biochemistry, Biology and Physics, University of Bari, Policlinico, P.zza G. Cesare, 70124 Bari, Italy e Institute of Bioenergetics and Biomembranes, CNR, Bari, Italy b

d

Received 4 December 2006; revised 22 December 2006; accepted 9 January 2007 Available online 18 January 2007 Edited by Peter Brzezinski

Abstract EXAFS analysis of Zn binding site(s) in bovine-heart cytochrome c oxidase and characterization of the inhibitory effect of internal zinc on respiratory activity and proton pumping of the liposome reconstituted oxidase are presented. EXAFS identifies tetrahedral coordination site(s) for Zn2+ with two N-histidine imidazoles, one N-histidine imidazol or N-lysine and one O–COOH (glutamate or aspartate), possibly located at the entry site of the proton conducting D pathway in the oxidase and involved in inhibition of the oxygen reduction catalysis and proton pumping by internally trapped zinc.  2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Cytochrome c oxidase; Protein Zn binding; Proton pumping

1. Introduction Zn2+ interaction with respiratory chain was first observed by Skulachev et al. [1], who reported its inhibitory effect on the cytochrome bc1 complex. This finding was confirmed and extended by others [2,3]. It has been found that Zn2+ also inhibits cytochrome c oxidase [4–10]. Cytochrome c oxidase has four redox centers: a binuclear CuA, bound to subunit II, heme a, heme a3 and CuB, all bound to subunit I [11]. Cytochrome c, at the outer (P) side of the coupling membrane, delivers electrons to CuA, heme a transfers electrons from CuA to the heme a3/CuB binuclear center, where O2 is reduced to 2H2O with consumption of four protons from the inner (N) space, thus directly resulting in the generation of transmembrane DlH+ [12,13]. In addition up to four protons are pumped from the N to the P space in the reduction of one molecule of O2 to 2H2O [14,15]. Investigations on cytochrome c oxidase reconstituted in liposomes (COV) in the presence of ZnCl2, revealed an uncoupling effect exerted by the metal on the proton pumping activity of the oxidase [5]. Uncoupling was associated with * Corresponding author. Address: Department of Medical Biochemistry, Biology and Physics, University of Bari, Policlinico, P.zza G. Cesare, 70124 Bari, Italy. Fax: +39 080 5448538. E-mail address: [email protected] (S. Papa).

Abbreviation: CCCP, carbonyl cyanide m-chlorophenylhydrazone

inhibition of intermediate steps of the catalytic cycle of oxygen reduction to water [5–7]. A slow [8] inhibitory effect exerted by externally added zinc to preformed COV on the oxidase activity, dependent on the presence of a membrane potential [9], has also been observed [8–10]. In this paper, we present an EXAFS study of Zn binding site(s) in bovine heart cytochrome c oxidase and characterization of the impact of internal zinc on respiratory and proton pumping activities of the reconstituted oxidase.

2. Materials and methods 2.1. Materials Horse heart cytochrome c, valinomycin, phospholipid (L -a-lecithin, L -a-phosphatidylcholine, type II-S: from soybean), CCCP from Sigma Chemical Co., hexammineruthenium (II) chloride from Sigma–Aldrich. 2.2. Enzyme preparation, liposome reconstitution and functional analysis Purified bovine heart cytochrome c oxidase (COX) contained 10 nmoles of heme a + a3/mg protein, SDS–PAGE analysis revealed the complete set of 13 subunits [16]. Respiratory activity was measured polarographically in 40 mM KCl, 10 mM Hepes, 0.1 mM EDTA, pH 7.4, 40 nM aa3, 50 lM cytochrome c, 25 mM ascorbate at 25 C. Oxidase vesicles (COV), were prepared by cholate dialysis as in [16]. Where indicated, ZnCl2 (200 lM) was added to the sonication medium and to the first two dialysis solutions, but omitted from the third. Respiratory rates, orientation of the oxidase in COV, passive proton conduction, residual soluble COX, COX absorbance changes and pH changes were measured as in [16]. 2.3. EXAFS analysis Zn K-edge-extended X-ray absorption spectroscopy (EXAFS) measurements were performed on samples of bovine COX characterized by 1, 1.5 and 2 zinc atoms per complex respectively. The latter two samples were obtained by incubating the protein with sub-stoichiometric amounts of ZnSO4 (20 h on ice) in order to maximize the occupancy of high affinity binding site(s). Metal content, measured by inductively coupled plasma-atomic emission spectroscopy (ICP-AES), showed that the preparation was exempt from contaminating Zn. Fe content revealed by ICP-AES was consistent with the heme content of the complex evaluated spectrophotometrically. For EXAFS measurements ZnCOX suspensions were embedded in dry PVA films [17]. EXAFS measurements were performed at the BM8 ‘‘GILDA’’ beam-line of the European Synchrotron Radiation Facility (ESRF) in Grenoble. Measurements are described in [18]; data analysis was performed using ARTEMIS package [19]. Final fitting was performed directly in k space (with a k weight of 3), ˚ 1, using all paths involving up to five scattering in the range 2.5–10 A ˚ and adopting a rigid body processes with an effective path length 65 A refinement approach [20]. As free parameters we used a single,

0014-5793/$32.00  2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.01.017

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common shift in the energy origin, the first ligand distances, the bending angle between the Zn–Ne bond and the imidazole ring of histidines (keeping Zn coplanar with the imidazole ring). Debye–Waller factors were grouped and fixed to 5 values accordingly to Ref. [21].

3. Results The presence of ZnCl2 during liposome reconstitution of cytochrome c oxidase had no effect on the sidedness of the incorporated COX, neither increased the residual amount of soluble COX. Zn2+ had no significant effect on the respiratory activity in the controlled state (no ionophore added) but inhibited respiration in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) and valinomycin (Table 1). It can be noted that the COV used in our studies [see also Ref. 16] are tightly coupled, i.e. there is negligible passive back leakage of pumped protons. This is reflected in the respiratory control ratio (I.C.R.) which is as high as 30 (see Table 1). It is likely that the inhibition of electron flow in the controlled state by internally trapped Zn2+ [5] or externally added Zn2+ [9], observed in reconstituted bacterial cytochrome c oxidase, which exhibited much lower respiratory control ratio (I.C.R. 4.5–

6.2, see Refs. [5,9]), was due to depression by Zn2+of passive proton leakage. Zn2+ had no effect on passive proton conduction of COV (data not shown). Proton pumping was measured in a turnover of the oxidase, i.e. oxidation by a stoichiometric amount of oxygen of the fully reduced oxidase followed by direct re-reduction of the enzyme (R fi O fi R transition). Valinomycin was present to collapse the membrane potential so to disclose the maximal pumping activity, amounting to @4H+ released in the medium per COX molecule [see also 16,22] (Fig. 1). Zn2+ caused depression of proton pumping (Table 1, Fig. 1), which reached 40% at 200 lM ZnCl2 present during COV reconstitution. ZnCl2 up to 200 lM had no effect on passive backflow of pumped protons. This shows that the depression of proton release from COV in the R fi O fi R transition is due to a direct inhibitory effect of Zn2+ on the pump. X-ray crystallographic analysis has shown the existence in bovine heart cytochrome c oxidase of an endogenous Zn2+ bound to subunit Vb [23]. The structure of this site, to which Zn2+ is coordinated by four cysteine residues, has been studied by EXAFS [24]. We examined three samples: sample a, exempt of exogenous zinc, characterized by a stoichiometry of 1.0 Zn2+ per complex; samples b and c, characterized by a stoichi-

Table 1 Effect of internal Zn2+ on respiratory activity and proton pumping in cytochrome c oxidase reconstituted in liposomes (COV)

Control +ZnCl2 100 lM +ZnCl2 200 lM

(a) % Right side out COV

(b) % Soluble COX

82 ± 1.9 N.D. 87 ± 3.6

1.3 ± 0.4 1.3 2.0 ± 0.3

Respiratory rate (lM e s1 COX1) (c) No additions

(d) +CCCP, valinomycin

16.2 ± 1.7 13.6 14.1 ± 0.8

446 ± 19.7 398 347 ± 20.7

(e) ICR

(f) H+pumping H+/COX RfiOfiR

(g) t1/2 (s) H+ backflow

29.6 ± 3.4 29.2 25.5 ± 1.1

3.7 ± 0.09 3.3 ± 0.07 2.4 ± 0.08

>30 24 ± 3.4 >30

The orientation of COX in liposomes (a), the residual soluble COX (b), the respiratory rate in the absence (c) and presence of valynomicin plus CCCP (d), the respiratory control ratio (e = d/c), the extent of proton release per oxidase molecule in the R fi O fi R transition (f) were determined as in Section 2 and in Fig. 1. (g) Half-time of decay of the proton gradient.

Fig. 1. Proton release and redox changes of aa3 in the R fi O fi R transition of cytochrome c oxidase. COV, 1 lM aa3, were suspended in 150 mM KCl supplemented with 4 lM valinomycin, pH 7.2, 25 C. Where indicated COV were prepared in the presence of ZnCl2. The anaerobic reduction of cytochrome c oxidase, supplemented with 1.5 lM cytochrome c, was attained with the addition of an excess of hexammineruthenium (II) and the oxidation, re-reduction cycle (R fi O fi R transition) was produced by the addition of an amount of water containing O2 electron equivalents slightly substoichiometric with those of the four redox centers of the oxidase. The H+/COX ratio was obtained by dividing the protons released by the amount of O2 added. For details see Section 2.

F. Francia et al. / FEBS Letters 581 (2007) 611–616

ometry of 1.5 and 2.0 Zn ions per complex, respectively (see Section 2). The EXAFS functions extracted from the data and the corresponding Fourier transforms are shown in Fig. 2. The results of first shell analysis of sample a (see fit in Fig. 2B) are consistent with a Zn2+ coordination by four sul˚ in agreement phur atoms with a bond length of 2.33 ± 0.01 A with previous EXAFS [24] and crystallographic analysis [23].

Fig. 2. (A) k3-weighted v(k) EXAFS signals obtained from samples characterized by different Zn/COX ratios. (B) The corresponding magnitude of Fourier transform (FT). From top to bottom: trace a, sample a, characterized by 1.0 Zn/complex; trace b, sample b, 1.5 Zn/ complex; trace c, sample c, 2.0 Zn/complex. The first-shell fit to the FT of sample a is shown with empty squares in Fig. 2B. The vertical dashed line indicates the position of the peak attributed to the binding of exogenous zinc. The amplitude of this peak increases from sample b to sample c, reflecting the progressive occupancy of a new Zn2+ binding site. Trace d in panel A is the EXAFS signal of the site to which exogenous Zn2+ binds, extracted from trace c by subtracting trace a with the appropriate weights (i.e. according to Eq. (2)). The fit corresponding to cluster 1 of Table 2 (i.e. three histidines and one aspartate or glutamate residue) is shown as a dashed line. See text for details. Trace d in panel B is the correspondent FT.

613

Fig. 2B shows that, going from sample a to samples b and c, a second peak at shorter distances appears in the radial distribution function (its position is indicated by a vertical dashed line in Fig. 2B). The amplitude of this second peak increases at increasing Zn/complex ratios (from sample b to c), being comparable to that of the peak of the endogenous Zn binding site. This strongly suggests that a second, high affinity binding site becomes progressively populated. The EXAFS signal for this site (vsite2) can be extracted by subtracting the signal of sample a (va) from those, appropriately weighted, of sample b (vb), or sample c (vc) according to: vsite2 ¼ ð1:5vb  va Þ=0:5

ð1Þ

vsite2 ¼ 2vc  va

ð2Þ

The EXAFS signal obtained from the sample characterized by 2 Zn/complex using Eq. (2), is shown in Fig. 2A (trace d). The similarity of the EXAFS signal extracted from sample b or from sample c using Eq. (1) or Eq. (2) (not shown) clearly indicates that a unique, additional high affinity site is occupied in our samples. First-shell analysis indicates a coordination number of four and the best fit was obtained for a cluster type formed by 3N at ˚ and 1O at 2.03 ± 0.05 A ˚ . Among nitrogen/oxy1.98 ± 0.01 A gen ligands of zinc in proteins, the Nd1/Ne2 of the histidine imidazole ring and the Od1/Od2 of the aspartate and glutamate residues are the most common [25]. Coordination of Zn2+ by O from water has also been observed, particularly for non-structural zinc [25]. The mean Zn–N distance resulting from first shell analysis is however slightly shorter than the typical bond ˚ for Zn–Ne and 2.14 A ˚ length reported for histidine, i.e. 2.05 A for Zn–Nd bonds [25]. This suggests the presence of at least one unusually shorter Zn–Ne bond or that one of the coordinating N atoms belongs to a lysine, whose Zn–N bond length is typ˚ [25] and that the coordination with the histidine ically 1.94 A residues involves Ne. The EXAFS signal was therefore fitted to a series of putative binding clusters formed by the –C– COO of an aspartate or glutamate residue or the O atom of a water molecule and three imidazoles or two imidazoles plus a lysine. As shown in Table 2, which summarizes the binding clusters considered, except those including a water molecule, an excellent fit is obtained in all cases. The corresponding clusters in which the aspartate or glutamate residue was replaced by water yielded equivalent fits, but the best fitting Zn–O distances were systematically shorter (1.98– ˚ ) than the typical coordination distance derived from a 2.02 A large protein data set of tetrahedral Zn2+ binding sites ˚ for catalytic and 2.15 A ˚ for structural sites [25], see (2.12 A also http://tanna.bch.ed.ac.uk/). The presence of a water molecule seems therefore unlikely. Using as a goodness of fit index the R factor (the sum of the squares of the differences between each experimental point and fit normalized to the sum of the squares of the experimental points), it appears that the clusters including more than one imidazole Nd1 as ligand are unfavoured.

4. Discussion In the steady-state turnover the catalytic cycle of cytochrome c oxidase starts with 2e reduction of the a3–CuB binuclear center of the oxidized (O) enzyme; two protons from the

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Table 2 Structural parameters obtained for the different clusters examined ˚) ˚) Cluster Residues Zn–N (his) (A Zn–N (lys) (A 1 2 3 4 5 6 7

3his(3Ne2)1glu/asp 3his(1Nd1, 2Ne2)1glu/asp 3his(2Nd1, 1Ne2)1glu/asp 3his(3Nd1)1glu/asp 2his(2Ne2)1lys1glu/asp 2his(1Nd1, 1Ne2)1lys1glu/asp 2his(2Nd1)1lys1glu/asp

1.97(1) 1.97(1) 1.97(1) 1.97(2) 2.00(3) 2.00(3) 2.00(3)

1.93(5) 1.93(5) 1.93(5)

˚) Zn–O (Asp/Glu) (A

Bending angle (His) ()

R factor

2.03(3) 2.03(3) 2.03(3) 2.01(3) 2.02(5) 2.02(5) 2.02(5)

3(4) 4(3) 4(3) 4(3) 3(7) 4(5) 5(4)

14 14 15 17 15 15 16

The 1r error on the least significant figure is reported in brackets.

N space are consumed and the mixed valence (MV) intermediate is generated. MV reacts with O2 giving rise to the peroxy (PM) intermediate. PM is then converted to the ferryl (F) intermediate, and (F) to (O), by two successive 1e reduction steps each associated with consumption of 1H+ from the N space and final production of 2H2O [16,26]. Mutational analysis [26,27] and X-ray crystallographic structures of the oxidase [23,28,29] have provided evidence that the protons consumed in the reduction of O2 to 2H2O, are translocated from the N space to the binuclear center by two pathways. A K pathway used by the two protons consumed in the O fi MV transition, and a D pathway used by the two protons consumed, one in each of the P fi F and F fi O transitions [26,30,31]. It is debated whether pumped protons are translocated also by the D pathway [29–31] or by alternative pathways [32–34]. By EXAFS analysis we have extracted a signal attributable to a site which binds exogenous Zn2+ with a tetrahedral geometry, formed by a carboxylate residue (glutamate or aspartate) and three histidines (clusters 1–4) or two histidines plus a lysine (clusters 5–7). We cannot discriminate between these two possibilities, since the R factor favours cluster 1, while including a lysine led to a Zn–NLys and Zn–NHis distance that

agrees better with the expected values reported in Ref. [25] (see Table 2). The fit corresponding to cluster 1 is shown as a dashed line in Fig. 2A. An internal binding site whose occupancy by Zn results in inhibition of the P fi F and F fi O transitions can be conceived to be located at the entry mouth of the D channel. Guided by the EXAFS characterization of the possible Zn coordinating residues, we have inspected, by RasMol analysis of the X-ray crystallographic structure of bovine cytochrome c oxidase [23], the position of candidate residues exposed at the N surface at the entrance site of the D pathway. As shown in Fig. 3A, there are His 503 and Glu 506 of subunit I, His 2, Glu 5 and Lys 9 of subunit VIIc and His 3 of subunit III, which could be candidates, in various combinations, for tetrahedral coordination of Zn2+. Lys 13 of subunit I is close to the Dpathway entrance and could, even if not exposed to the N-surface in the crystal structure, participate in Zn2+ coordination (Fig. 3B). Since all the residues indicated in Fig. 3A are located in non-structured segments of the protein backbone, conformational perturbation induced by the binding of Zn2+ itself are possible. Our Zn–COX complexes are embedded in a weakly interacting matrix as PVA; this situation differs from the crystallographic one, in which conformational movements

Fig. 3A. Space-filled view of the mass of the 13 subunits bovine cytochrome c oxidase protruding at the N side of the membrane. The picture was elaborated with the Rasmol 2.6 program using the 2OCC atomic coordinates from the PDB data bank of cytochrome c oxidase [32]. With their respective numbering the putative Zn binding residues emerging at the N surface of subunits I (red), subunit VIIc (green) and subunit III (yellow) and Asp91-I (blue) of the D channel are shown.

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Fig. 3B. View parallel to the membrane of the location in subunits I (black) and VIIc (green) of the oxidase of the putative Zn2+ coordinating residues. The picture was elaborated as in Fig. 3A. It can be noted that the putative Zn coordinating residues, except Lys 13-I, belong to the carboxyterminal segment of subunit-I and the NH2-terminal segment of subunit VIIC respectively.

can be partially hindered. A Zn2+ coordination involving two His N-imidazols, one Glut O-carboxylate, and one N-histidine (or one N-lysine) seems to be favoured by the EXAFS data. Recent X-ray crystallographic analysis shows Zn2+ coordination to His 503-I, in different combinations with residues of subunit VIIc and III shown in Fig. 3 (Shinya Yoshikawa, personal communication). Zn tetrahedral coordination with these residues at/around the entrance of the D pathway can explain the reported inhibition of the P fi F and F fi O transitions of the catalytic cycle. Studies on bacterial photosynthetic reaction centers (RC) have revealed that Zn2+ inhibits proton transfer from the surface to the final electron acceptor by competing with protons for the same binding site which involves two histidines plus one aspartate [35]. As shown in [5] for P. Denitrificans cytochrome c oxidase and in this work for the bovine oxidase, internal Zn2+ binding, possibly at the site described here, results also in inhibition of proton pumping. The site is thus involved in the coupling of proton pumping with the reduction of oxygen to water. Pumped protons could be translocated by the D-pathway up to a bifurcation point where a switch at Glut 242 separates scalar from pumped protons [26,36]. Alternatively, redox-linked proton-transfer events at the entrance of the D-pathway could be associated, through allosteric conformational changes, with proton transfer by a distinct pathway [32–34].

Acknowledgements: This work was supported by Grants from: MIURPRIN ‘‘Molecular Mechanisms, Physiology and Pathology of Membrane Bioenergetics Systems’’ No. 2005052128, and Progetto Fondazione Cassa di Risparmio di Puglia, Bari. Authors acknowledge the GILDA staff at the Grenoble Synchrotron for excellent assistance. Measurement at ESRF were supported by the public user program.

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