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. Author manuscript; available in PMC: 2019 Nov 1.
Published in final edited form as: Biochim Biophys Acta Bioenerg. 2018 Aug 22;1859(11):1191–1198. doi: 10.1016/j.bbabio.2018.08.002

Mechanism of proton transfer through the KC proton pathway in the Vibrio cholerae cbb3 terminal oxidase

Young O Ahn 1,2, Ingrid Albertsson 1, Robert B Gennis 2, Pia Ädelroth 1,*
PMCID: PMC6260837  NIHMSID: NIHMS1505766  PMID: 30251700

Abstract

The heme-copper oxidases (HCuOs) are terminal components of the respiratory chain, catalyzing oxygen reduction coupled to the generation of a proton motive force. The C-family HCuOs, found in many pathogenic bacteria under low oxygen tension, utilize a single proton uptake pathway to deliver protons both for O2 reduction and for proton pumping. This pathway, called the KC-pathway, starts at Glu-49P in the accessory subunit CcoP, and connects into the catalytic subunit CcoN via the polar residues Tyr-(Y)-227, Asn (N)-293, Ser (S)-244, Tyr (Y)-321 and internal water molecules, and continues to the active site. However, although the residues are known to be functionally important, little is known about the mechanism and dynamics of proton transfer in the KC-pathway. Here, we studied variants of Y227, N293 and Y321. Our results show that in the N293L variant, proton-coupled electron transfer is slowed during single-turnover oxygen reduction, and moreover it shows a pH dependence that is not observed in wildtype. This suggests that there is a shift in the pKa of an internal proton donor into an experimentally accessible range, from >10 in wildtype to ~8.8 in N293L. Furthermore, we show that there are distinct roles for the conserved Y321 and Y227. In Y321F, proton uptake from bulk solution is greatly impaired, whereas Y227F shows wildtype-like rates and retains ~50% turnover activity. These tyrosines have evolutionary counterparts in the K-pathway of B- family HCuOs, but they do not have the same roles, indicating diversity in the proton transfer dynamics in the HCuO superfamily.

Keywords: oxygen reduction, bioenergetics, electron transfer, liposomes, proton pumping

Graphical Abstract

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1. Introduction

Aerobic organisms extract the energy required for sustaining life using the reactions that constitute aerobic respiration. The membrane-bound heme-copper oxygen reductases (HCuOs) catalyse the final step of respiration; reduction of oxygen to water. The energy thus released is used to produce and maintain a transmembrane electrochemical pH gradient, used e.g. by ATP synthase to produce ATP. In HCuOs, the gradient is formed both by using protons for water formation exclusively from the inside (bacterial cytoplasm or mitochondrial matrix), and by proton pumping. Protons are transferred through proton input pathways, from the cytoplasmic surface to the buried active site as well as all across the protein. Such proton transfer pathways are composed of hydrogen-bonded chains of protonatable and/or polar residues as well as water molecules, and the transfer of protons across such chains generally follows the Grotthuss mechanism which involves structural rearrangements in the hydrogen-bonded networks (reviewed in [1]). Based on sequence homology and the pattern of conserved amino-acids in the proton pathways, the O2-reducing HCuOs form three major families: the A-, B- and C-families [2]. A-family HCuOs, the most abundant, are found in bacteria, archaea and eukaryotes. B-family HCuOs (e.g. Thermus thermophilus ba3) are present only in bacteria and archaea. The C-family HCuOs (i.e. cytochrome cbb3) have so far been reported only in bacteria, including a number of pathogens, and are the most distant relatives to mitochondrial A-type enzyme.

The A-, B- and C- family HCuOs all have a catalytic subunit that shares a binuclear center consisting of a high-spin heme and a Cu ion (CuB), which has a redox-active tyrosine residue covalently linked to one of its histidine ligands [3, 4]. A low-spin heme is also present in all catalytic subunits of the HCuOs. Although most residues comprising the proton pathways are located in the catalytic subunit in all HCuOs, they have a different number and composition of their proton pathways (reviewed in [5]), indicating diversity in the detailed proton transfer mechanisms.

The A-family HCuOs have two pathways called the D- and K-pathways, whereas both the B- and C-family HCuOs contain only one functional proton pathway (called KB- and KC-pathway, respectively) that is spatially analogous to the KA-pathway [6–9]. Despite being spatially equivalent, residues lining the KB- and KC-pathways are not identical to the KA-pathway or to each other. In the A-family HCuOs, the D-pathway is used for all pumped protons and most substrate protons, while the K-pathway is only used for transfer of 1–2 substrate protons during the reductive phase of the catalytic cycle [10, 11]. The D-pathway starts at D132 (R. sphaeroides aa3 numbering) at the N-side surface and ends at E286 near the active site. E286 is a branching point directing protons either to the active site for oxygen reduction or onwards for proton pumping [12–14].

In the C-family HCuOs, electron input from soluble cytochrome c occurs first to the CcoP subunit which contains two hemes c (see Fig. 1), and then sequentially to the heme c in the CcoO subunit. From there, electrons are delivered to the active site via the low-spin heme b in CcoN (subunit I). The KC-pathway, being the only proton pathway, must conduct protons for both pumping and O2–reduction. Recent studies showed that the KC-pathway starts from the E49P (subunit CcoP, V. cholerae cbb3 numbering) [6, 9], and connects via polar residues and water molecules in CcoN, to Y321 (see Fig. 1) near the active site [6, 15]. However, a residue equivalent to the Glu-286 at the ‘top’ of the D-pathway in the A-family, is absent in the B- and C-family HCuOs. Instead, the Y321Vc was suggested to form such a branch point [6, 8, 16]. Y321Vc, although not conserved to the B-family, is spatially equivalent to Y244Tt of the KB-pathway [15] (Thermus thermophilus ba3 numbering). There is only one conserved residue between the KC-pathway and KB-pathways: Y227Vc (C-type) equivalent to Y248Tt (B-type, See Fig. 1C), both important for catalysis [6–8]

Figure 1. Structure of cytochrome cbb3 from P. stutzeri.

Figure 1.

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(A) Ribbon structure with the CcoN in pink, CcoO in green and CcoP in blue, respectively (from PDB ID: 3MK7 [15]). Heme b, b3 and the Ca2+ bound at the heme propionates are highlighted along with the residues in the KC-pathway. (B) Enlargement of the KC-pathway region (C) Structural overlay of the KC-pathway (using V. cholerae cbb3 numbering) in yellow with the KB-pathway from the T. thermophilus ba3 (B-family, PDB ID: 3EH5 [3]) in grey. The structural alignment was made using the MultiSeq module of VMD [17].

The reaction of fully reduced A-type HCuOs with O2 has been extensively investigated by the flow-flash technique. In this technique, the kinetics of oxygen binding and subsequent reactions is monitored by optical spectroscopy after a short laser flash dissociates the photolabile CO bound to the fully reduced enzyme. O2 binding, forming the so-called A intermediate, is followed by O-O bond breaking by electron transfer from the binuclear center and heme a, forming the peroxy (PR) intermediate (time constant (τ)~30 μs, numbers from the reaction in R. sphaeroides aa3 [18]). Next, the ferryl (F) intermediate is formed by internal proton transfer (τ ~100 μs), a transition linked to proton uptake and proton pumping [19, 20]. The F intermediate decays (τ~1 ms) to form the oxidized (O) state by electron transfer from CuA together with a second proton uptake and this transition is also linked to proton pumping [19, 20].

In the flow-flash reaction of the fully reduced cytochrome cbb3 enzyme (C-family, see [21]), the enzyme is reduced at all six redox centers (heme b3, CuB, heme b, and three hemes c (in CcoO and CcoP) and thus capable of fully reducing O2 to 2 H2O, leaving two electrons still in the enzyme. Previously, we have shown that in wildtype cbb3, all hemes show (partial) oxidation with a time constant of ~1 ms, with no detectable intermediates after formation of the O2-adduct [6, 21]. In the variant E49PA, with the Glu at the entrance of the KC-pathway (see Fig. 1) exchanged, proton uptake through the KC-pathway is severely slowed and so is the coupled heme c oxidation, whereas the oxidation of heme b remained fast [6, 9]. This leads to the formation of a partly reduced intermediate not observed with wildtype.

To examine the mechanism of proton transfer in the KC-pathway in more detail, here we further investigated variants of conserved key residues in the C-family HCuO from V. cholera: Y227, N293 and Y321, by the flow-flash technique. Our results show that exchanging N293 by leucine slows heme c oxidation and that it also induces a change in the pKa of an internal proton donor, resulting in possible uncoupling of proton pumping. Furthermore, we report that proton uptake in Y227VcF occurred only slightly slower than in the wildtype whereas proton uptake in Y321VcF was severely inhibited. The phylogenetic and functional importance of these two tyrosine residues (Y227Vc and Y321Vc) shared also to the K-pathway of B-family HCuOs are discussed.

2. Materials and Methods

2.1. Site-directed Mutagenesis and Purification of cbb3 Oxidase

The mutations were constructed using QuikChange site-directed mutagenesis kits from Stratagene. DNA oligonucleotides were synthesized at Integrated DNA Technologies. Sequence verification of the mutagenesis product was performed at the Biotechnology Center at the University of Illinois at Urbana-Champaign. The expression, purification, and characterization of the V. cholerae cbb3 wildtype and mutants were performed as previously described [6, 8].

2.2. Steady-state Activity

Steady-state activity was measured with a YSI model 53 oxygen monitor as in [6]. For the TMPD oxidase activity, the reaction mixture contained buffer (50 mM NaPi, 100 mM NaCl and 0.05% DDM at pH 6.5), 10 mM ascorbate and 500 μM TMPD as previously described [6]

2.3. Reconstitution of the V. cholerae cbb3 enzyme in the absence or in the presence ATP synthase into phospholipid vesicles

Reconstitution of the V. cholera cbb3 and ATP synthase into liposomes was done by mixing lipid vesicles containing ~1% sodium cholate with the V. cholera cbb3 alone (as in [22]) or together with ATP synthase as in [23]. After incubation for 30 min at room temperature with occasional gentle shaking, the sodium cholate was removed by addition of Bio-beads (BioRad) [22] or gel filtration (on a PD-10 column, GE Healthcare).

2.4. Proton pumping and ATP synthesis

The respiratory control ratio (RCR) was obtained by measuring the ratio of the TMPD oxidase activity of the cbb3 variants reconstituted in proteoliposomes in the presence of uncoupler (uncontrolled activity) to the activity in the absence of uncoupler (controlled activity). RCR values>1 indicate a proton motive force generated across the vesicle membrane by the cbb3 enzymes oriented ‘right-side-out’ since TMPD electron donation is only efficient from the outside.

In the liposomes containing both ATP synthases and cbb3 oxidases, only liposomes in which the cbb3 is (predominantly) oriented such that protons are pumped to the inside of the liposomes can synthesize ATP because ATP synthases orient with the F1 headpiece facing the outside (‘inside-out’ orientation). Turnover of the V. cholera cbb3 oxidase was initiated by addition of the electron donor ascorbate and the membrane-permeable mediator PMS. The resulting increase in ATP synthesis was monitored essentially as in [23] using a luminometer with the luciferin/luciferase system. Buffer (460 μl) containing 20 mM Tris-phosphate, pH 7.4 and 5 mM MgCl2 was mixed with 20 μl luciferase/luciferin solution (10 mg/ml in ddH2O). Time spans of 30 s (1 data point/s) were measured between different additions. 20 μl of the liposome sample and 80 μM ADP were added to the reaction chamber in this order. Standard curves were collected as a control by addition of 5 nM ATP (1μl 25 μM ATP stock solution) before the addition of the electron donors. 200 μM ascorbate and 40 μM PMS were added to the liposome sample mixture. The light intensity increased linearly with time indicating that ATP was being produced continuously. As a control, KCN (a HCuO inhibitor) was added, resulting in an impaired reaction.

2.5. Flash Photolysis

The enzyme samples were prepared as previously described [6, 22]. Briefly, the enzyme at a concentration of ~5 μM was transferred to a modified anaerobic cuvette and the atmosphere was exchanged to N2 on a vacuum line. The enzyme was completely reduced by addition of 2 mM ascorbate and 2 μM phenazine methosulphate (PMS). Then, N2 was exchanged for CO resulting in formation of fully reduced CO-bound cbb3 oxidase. The CO-ligand was photolyzed by a 10 ns laser flash at 532 nm (Brilliant B, Quantel), followed by detection of absorbance changes on set-up from Applied Photophysics essentially as in [24].

2.6. Flow-Flash measurements

Flow-flash measurements were performed using a modified stopped-flow apparatus (Applied Photophysics, Surrey, U.K.) essentially as in [24] and data analyzed as described in [6, 22]. The enzyme solution was rapidly mixed in a 1:5 ratio with an O2-saturated buffer, and the reaction with O2 was initiated by flash photolysis of the CO-enzyme complex approximately 200 ms after mixing. The flow-flash experiments were performed at different pH values (pH 6, 7.4, 9 and 10). Buffer conditions: 50 mM MES, 100 mM KCl and 0.05% DDM for pH 6.0, 50 mM Hepes, 50 mM KCl, 0.03% DDM and 50 μM EDTA at pH 7.4, 50 mM CHES, 100 mM KCl and 0.05% DDM for pH 9.0, and 100 mM CAPS, 50 mM KCl and 0.05% DDM for pH 10.0.

Proton uptake measurements during the reaction of the fully reduced cbb3 with O2 was performed as described in [6, 21].

2.7. Flow-flash data handling and analysis

The time-resolved absorbance changes were recorded at single wavelengths, and ~8 traces averaged. About 106 data points were initially collected and reduced to ~103 by averaging over a progressively larger time interval. The resulting traces at each wavelength were fitted either separately or globally to a series of irreversible reaction using the Pro-K software (Applied Photophysics, U.K.) in order to extract the rate constants and amplitudes and/or kinetic difference spectra. The values reported in the results are averages of 4~5 separate measurements, performed on several different preparations of the variants.

The pH dependence in the N293L variant was fitted to a titration with a single pKa with the equation kobs=αXHxkmax, where αXH=1/(1+10pH-pK(XH)) with kobs being the obtained rate constant at a certain pH and kmax the maximum rate constant at low pH. A small background rate k0 of ~100 s−1 (at high pH) was also added.

3. Results

3.1. CO-recombination kinetics and reaction of fully reduced V. cholerae cbb3 variants with O2:

The integrity of the active site in the cbb3 variants was probed by studying the UV-Vis spectra as well as the recombination kinetics of the inert CO ligand after laser flash photolysis. Y227F, Y321F and N293L showed normal UV-visible absorption spectra (Figure S1A) [6]. In the CO-recombination kinetics, wildtype (Wt) V. cholerae cbb3 shows two kinetic components (described in ref [22], see also Figure S1B); a rapid phase (τ ~60 μs) attributed to CO rebinding to one of the c-type hemes, and a slower phase (τ= 2.2 ms) associated with CO rebinding to the heme b3. Both these phases are present in all three variants, with similar time constants as in Wt. Also, the relative amplitudes of the two phases are similar between wildtype and the variants except in Y227F (Figure S1B), where the faster phase has a somewhat larger relative amplitude.

These results enabled us to further investigate the flow-flash reaction, which can only be done when the active site is not significantly perturbed. Here, the fully reduced CO-bound cbb3 variant is rapidly mixed with O2, and the reaction initiated by a short (10 ns) laser pulse. O2 binding and heme oxidation reactions are then followed optically and time-resolved. The kinetics were monitored at 420 nm (mainly heme c), 430 nm (mainly hemes b and b3), 550 nm (heme c), and 560 nm (heme b). We also studied proton uptake from solution by adding a pH-sensitive dye in the absence of buffer.

With wildtype V. cholerae cbb3 (and similarly in R. sphaeroides cbb3 [21]), as previously reported [6], oxidation of all (types of) hemes is observed in a single phase with a rate constant k∼3000 s−1 (τ ~0.3 ms, see Figure 2), and we assume (as in [6, 21]), that oxidation of heme b3, CuB, heme b, and one or more equivalents of heme c take place in the reaction. Proton uptake is observed with the same rate constant (coupled) k ∼3000 s−1 (τ~0.3 ms). At 430 nm, an additional faster reaction is observed with k∼20000 s−1 (τ∼50 μs), attributed to O2 binding to heme b3 [6].

Figure 2. Absorbance changes during the reaction of the fully reduced V. cholerae cbb3 wildtype (WT, black trace), Y227F (blue) and Y321F (red) variants with O2.

Figure 2.

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The absorbance changes were monitored at (A) 420 nm, (B) 430 nm, (C) 550 nm (heme c) and (D) 560 nm (heme b). (E) Absorbance increase of the pH sensitive dye phenol red at 570 nm due to proton uptake. Experimental conditions: A-D; ~1–2 μM reacting cbb3, 50 mM Hepes at pH 7.4, 50 mM KCl, 0.03% DDM and 1 mM O2 at 298 K. In (E) 40 μM phenol red was added and Hepes was omitted. The data shown in (E) are the traces obtained after subtracting the traces obtained upon addition of Hepes. The amplitudes in A-D are normalized to the same total absorbance change at 20 ms for clarity. In E, the signals obtained were normalized with the same scaling factor as used for the ΔACO at 420 nm. A laser artifact at t=0 has been truncated for clarity.

During the same flow-flash reaction in Y227F, the kinetics of oxidation of heme b/b3 is biphasic where the faster phase is somewhat faster than in WT (k ~5000 s−1, τ ~0.2 ms) and there is an additional slower phase with k ~200 s−1 (Figure 2B and 2D). The faster phase is presumably caused by contribution from O2 binding observed also at 430 nm (as in Wt, see above), and the data at 560/430 nm was better fitted with three exponentials (k1~20000 s−1, k2 ~3000 s−1, k3~200 s−1), but this fit is then not unique (i.e. other combinations of rate constants can equally well fit the data). The subsequent heme c oxidation in Y227F also exhibits biphasic kinetics where the first phase (k ~3400 s−1, τ~0.3 ms) is as fast as in Wt, and the second phase slower (rate constant of ~200 s−1, τ~5.0 ms), concomitant with the slower phase of heme b oxidation (Figure 2A and 2C). In Y321F, the rate constant for heme c oxidation is ~1500 s−1 (τ ~0.7 ms), i.e. slower than in Wt (Figure 2A and 2C), while oxidation of heme b/heme b3 (k~3400 s−1, τ ~0.3 ms) occurs with similar rate constant as in Wt (Figure 2B and 2D).

In N293L, heme b is oxidized with k ~2900 s−1 (τ ~0.3 ms), which is similar to Wt (Figure 3B and 3D). However, heme c oxidises more slowly with a rate constant of ~1400 s−1 (τ ~0.6 ms) (Figure 3A and 3C).

Figure 3. The pH dependence of the reaction of the fully reduced wildtype and N293L cbb3 with O2.

Figure 3.

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Data obtained at (A) 420 nm, (B) 430 nm, (C) 550 nm (D) 560 nm and (E) 570 nm as in Fig. 2. The amplitudes were normalized to the same total absorbance change at 100 ms for better comparison. A laser artifact at t=0 has been truncated for clarity. (A-E) Conditions the same as in Fig. 2 except for the buffers used in A-D; 100 mM MES (pH 6.0), 50 mM Hepes (pH 7.4), 100 mM CHES (pH 9.0), 100 mM CAPS (pH 10.0) and in E) the trace at pH 8.0 was obtained with cresol red instead of phenol red. Color-coding indicated in the figure.

3.2. Proton uptake during reaction of fully reduced V. cholerae cbb3 variants with O2:

Proton uptake from solution was investigated by using the pH-sensitive dye phenol red (pH 7.4) or m-cresol red (pH 8.0). In Y227F, biphasic kinetics is observed for proton uptake at 570 nm, where the faster phase (k1 ~3500 s−1 (τ ~0.3 ms)) is similar to Wt, and the slower phase (k2 ~200 s−1, τ~5.0 ms) is about 15-fold slower than in Wt (Figure 2E). These proton uptake rate constants are the same as those observed for heme c oxidation in this variant (k1 ~3400 s−1, k2 ~200 s−1).

In Y321F, proton uptake occurs with only a very small amplitude, about 10% of that observed with Wt. The rate constant (~1500 s−1, τ ~0.7 ms) is slower than in Wt (Figure 2E) and concomitant with heme c oxidation. There is also a slower proton uptake phase in Y321F (k ~30 s−1, τ ~30 ms), but it overlaps with pH drifts and could not be assigned with certainty. The results suggest that alteration of Y321 causes severe inhibition of proton uptake through the KC-pathway.

In N293L at pH 7.4, proton uptake is slower (k ~500 s−1; τ ~1.9 ms) than in the wildtype (k ~3000 s−1, τ ~0.3 ms) (Figure 3E).

We note that although proton uptake and heme c oxidation are both slowed in N293L compared to Wt, they are not fitted to the same rate constant (at pH 7.4). This might be due to the ‘forced’ one-exponential fit of the heme c oxidation trace, since if we include a second component, this rate constant (k2=200 s−1, see Table S1) is more similar to that for proton uptake (~500 s−1), see Discussion.

Thus, in both N293L and Y321F, proton uptake and electron transfer from heme c to the active site are both slowed without perturbing the fast initial electron transfer from heme b. The rapid phase in these variants thus brings the active site to an intermediate on the same reduction level as the PR intermediate in A-type HCuOs (see e.g. [25]), qualitatively similar to what is observed in E49P variants [6] (see Fig. 1).

3.3. pH-dependence of transitions during O2 reduction in WT and variant cbb3

Because the rate constants of proton uptake and heme c oxidation were affected in the variants, we explored the pH dependence of the kinetics in Wt, Y227, Y321F and N293L variants. In Wt, the heme oxidation rate constants are independent of pH, with only a slight decrease observed at pH 10.0 (Figure S2). Similarly, neither Y227F nor Y321F exhibits any significant pH dependence (Figure S2).

In N293L however, the rate of heme c oxidation decreases with increasing pH (Figure 3C). The kinetics were fitted with a single exponential, giving satisfactory fits except at pH 10 which could only be well fitted by two exponents (see Table S1).

The rate constants were plotted (Figure 4) and a tentative pKa fitted (using data up to pH=9) to ~8.8. The results suggest that the apparent pKa associated with this transition has been lowered from >10 in Wt to ~8.8 in N293L (see Discussion).

Figure 4. The pH dependence of the rate constant for heme c oxidation in N293L (grey) and WT (black).

Figure 4.

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The line shown for N293L is a fit to a pKa of ~8.8 not using the data point at pH 10 as explained in the text.

In addition, proton uptake with N293L at pH 8.0 occurs with k ~400 s−1 (τ ~2.5 ms, Figure 3E), slower than at pH 7.4, whereas proton uptake with Wt at pH 8.0 occurs as fast as at pH 7.4 (Figure S2C).

3.4. RCRs and Proton Pumping

The turnover activities of the V. cholerae cbb3 Wt and variants reconstituted in proteoliposomes were measured in the absence (coupled) or presence (uncoupled) of a protonophore (CCCP). The ratio of uncoupled versus coupled activity, called the respiratory control ratio (RCR), was ~6 for Wt. Y227F, N293L and Y321F also retained RCRs higher than 1 (~2.7, 1.1 and 1.6, respectively) indicating that they generate a membrane potential (Table 1), although possibly to a lower extent than in Wt. We note that the decrease in RCR is more severe in N293L compared to Y321F, although the turnover activity is higher (see Discussion).

Table 1.

Comparison of respiratory control ratio (RCR) and turnover activities of cbb3 wildtype and variants.

Turnover (in detergent), % of Wt (e s−1), (from ref [6]) RCR (In vesicles)
WT 100 (200) 5.7±0.7
Y227F 52 2.7±0.7
N293L 7 1.1±0.2
Y321F <1 1.6±0.9
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Vectorial proton transfer by Wt and variants was also studied after co-reconstitution with the ATP synthase (as in [23]). The activity of cbb3 was initiated by addition of the electron mediator PMS and ascorbate. Proton pumping as well as proton uptake to form H2O establishes a proton motive force across the membrane, which is used by the ATP synthase for synthesis of ATP. This assay will lead to ATP production only in those vesicles where the cbb3 is oriented ‘wrong-side-out’ since the ATPase always orients with the large ATP-synthesizing F1 ‘head’ on the outside of the liposomes [23]. In Figure S3, ATP synthesis is observed when the Wt reduces O2, and impaired upon addition of potassium cyanide (KCN), an inhibitor of HCuOs. Likewise, Y227F activity is also linked to production of ATP, although more slowly compared to Wt. In contrast, with N293L as well as Y321F, there is no ATP synthesis. Again, we note that the effects on generation of a transmembrane gradient are as severe (or more) in N293L compared to Y321F although the turnover activity is considerably higher (see Discussion).

3.5. Site-directed mutagenesis of the N293-E49 pair in the KC-pathway

In A-family HCuOs, upon alteration of the D132 residue (R. sphaeroides aa3 numbering) at the mouth of the D-pathway [6, 9], proton uptake from the bulk solution is impaired [26]. However, this Asp can be ‘swapped’ (double mutant D132N-N139D), moving the negative charge up the pathway to N139, which restores rapid proton uptake and proton pumping to near wildtype values [27]. Based on the analogy to the N293 and E49P in the KC-pathway, we prepared double variants of various N293-E49P pairs (Table S2). The double variants all have the activities expected based on the properties of the single mutant components of the pair (Table 2). This indicates that it is not possible to swap the roles of these residues in the KC-pathway.

4. Discussion

Previous studies established that C-family HCuOs utilize a single proton pathway, the socalled KC-pathway, extending from the acidic E49P (V. cholerae cbb3 numbering throughout) in CcoP on the cytosolic side [6, 8, 9, 15] to Y255 at the catalytic site, cross-linked to the CuB-ligand H211 [4, 28]. The pathway extends through a hydrogen-bonded network of internal water molecules and the polar residues S244, Y227, N293 and Y321 in CcoN (subunit I) (Figure 1) [6].

4.1. Proton transfer and the internal proton donor XH in C-type HCuOs

In the C-type HCuOs, mechanistic information is lagging significantly behind, partly because in the flow-flash reaction, there are no clearly observable intermediates between the O2-bound A-state and the (partly) oxidized enzyme [6, 9, 21, 22]. In the E49PA variant however, proton uptake and oxidation of the c-type hemes is significantly slowed while rapid oxidation of heme b is maintained, leading to an increased life-time (not observed in wildtype) of an intermediate on the same reduction level (with three electrons arrived at the active site) as PR (or F, see below) in A-type HCuOs [25]. The PR state has the O-O bond broken, which requires a proton, that presumably comes from the Tyr-255 [28] in analogy with what was shown for A-type HCuOs [29].

N293, located 8 Å above E49P and fully conserved (~99%) in the C-family [6, 8, 15], is involved in a polar cluster in the lower region of the KC-pathway together with Y227, S244 and H247 (Figure 1). Variants at N293 all severely reduce enzyme activity to less than ~10% (Table 1, and [6]), supporting its functional importance. As in the E49PA variant, in N293L there is also fast oxidation of heme b but slowed proton uptake and oxidation of hemes c (Figure 3), and we thus assume that there is an increased lifetime of an intermediate on the PR reduction level also in N293L, although the retardation of heme c oxidation is much less severe than in E49PA.

Furthermore, in N293L, in contrast to Wt, the rate constant for heme c oxidation is pH-dependent. This electron transfer is coupled to proton uptake from the bulk with the same rate constant (see Figure 3A-E), and we model the pH dependence as a reaction that is rate-limited by proton transfer from an internal group XH, in rapid equilibrium with bulk protons. The rate constant saturates at low pH at ~1500 s−1, and starts titrating at pH>8, and we fitted it with a tentative pKa (for XH) of ~8.8 (Figure 4). This model fits the data well except at pH 10 (see Fig. 4), which we interpret to mean that at very high pH, the assumption of a rapid equilibrium with bulk protons is no longer valid. This would lead to biphasic kinetics where the enzyme fraction that has XH protonated shows kobs=kmax=1500 s−1, and the fraction with deprotonated XH takes up protons (and thus oxidizes hemes c) with a slower rate constant (similar behaviour to what we observed also in variants of an NO reductase [30]). In Wt, oxidation of the heme(s) c is also coupled to proton uptake from bulk solution, but the rate constant is independent of pH. This can be explained by titration of XH as above, but with a pKa outside of the measurable range, >10. It is also possible that the reaction in Wt is coupled to and rate-limited by another (pH-independent) reaction, such as a conformational change. In this scenario, in N293L, proton transfer has become slow enough to limit the rate which then also becomes pH dependent. Thus, the intrinsic rates of proton transfer can be significantly more perturbed than the changes in overall rate constants observed. We find, however, the first explanation with a pKa >10 for XH in Wt more likely since the overall rate constant is not highly perturbed in N293L. Alteration of N293, located ~20 Å below the active site thus substantially affects the pKa of an internal proton donor XH (Figure 5), giving us a first indication of the involvement of such a site and its pKa in C-type HCuOs.

Figure 5. Proposed proton transfer events during O2-reduction in cbb3.

Figure 5.

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XH denotes an internal proton donor, and the residues specifically investigated in this work are in bold. In wildtype, during proton-coupled electron transfer (from heme c), XH is in rapid equilibrium with bulk protons (severely slowed in E49 variants, see [6, 9]). We also presume that the PLS (proton loading site in the heme propionate area) is protonated through the same pathway concomitantly with (or before) reprotonation of XH. In N293L, the pKa of XH is altered, leading to slowed reprotonation and (partial) decoupling of the pump. In Y321F proton uptake from bulk is severely inhibited. In Y227F, the effects on proton transfer are mild. The water molecules shown are those observed in previous MD simulations [6]. We suggest that XH is either the Y321 itself or a cluster of residues and water between Y321 and N293.

In A-family HCuOs, there is also a proton donor that titrates during oxygen reduction in a similar way; the Glu-286 (R. sphaeroides (Rs) aa3 numbering) and its apparent pKa is 9.4 [31]), similar to what we assigned to XH (pKa ~8.8). A high pKa proton donor might thus be a general element providing fast and essentially irreversible reprotonation during the catalytic mechanism of the HCuOs. The Glu-286 in A-type (at the end of the D-pathway close to heme a3-CuB) also functions as the branching point for protons to be used in O2 reduction and those to be pumped. In the E286Q variant, the catalytic cycle essentially stops at the PR state [32, 33] with no protons taken up, whereas in the variant D132NRs (D132 is at D-pathway entrance) the PR → F transition still takes place, requiring a proton, but since no bulk proton uptake occurs, that presumably comes internally from E286 [26]. Thus, in C-type HCuOs, the internal proton donor XH could have a similar role in regulating the flow of protons (Figure 5).

The alteration of the pKa of XH in N293L is presumably also the reason for the decrease in the pumping efficiency as indicated by the low RCR and ATP synthesis rates (Table 1 and Figure S3), which is (for RCR) lower in N293L than in Y321F, although activity is higher. Since even if two variants have the same stoichiometry of protons taken up/pumped per electrons transferred to O2, a lower activity could lead to a lower RCR since vectorial proton transfer always competes with back-leaks over the membrane. This result thus indicates that there is a more severe decoupling of the pump in N293L than in Y321F.

N293 is the only variant in this study that exhibits a pH dependence of heme c oxidation, thus N293 is more important for controlling the pKa of, or lies more directly in the proton path to, XH than Y227 does. For Y321F, the situation is different since essentially no protons are taken up (Figure 2E and below). The identity of the internal donor XH is still not known, but we find it likely that it is either Y321 itself (see below), or a cluster of groups, including N293, Y321 and water.

4.2. Exclusive role of N293 and E49P for proton transfer in cbb3

The acidic residue (Asp or Glu) at the entrance of the proton pathway is necessary for fast proton uptake in both the A-family D-pathway as well as in the B- and C-family K-pathway analogues [6, 7, 9, 26, 34, 35]. Interestingly, the effects of alteration of N293, located above the entry point of the KC-pathway, appear to be similar to the N139Rs of the A-type D-pathway in terms of the linkage to internal proton donors (see [36]), although there is no evolutionary relationship. In the D-pathway, the roles of D132 and N139 can be ‘swapped’ by the double mutation D132N-N139D which rescues the lower activity and slower proton transfer in the single variants [27]. As shown in Table S2, however, our results clearly show that this is not the case for N293 and E49P in the KC-pathway. Instead, N293 is tightly linked to the internal proton donor and the E49P is absolutely required to shuttle protons from the bulk solution into the continuation of the KC-pathway (Figure 1), the latter point consistent with our earlier MD simulations [6].

4.3. Role of Tyrosines 227 and 321 in the V. cholerae KC-pathway

The variants of the conserved Y227 (Y227A/H/F) exhibited a broad range of enzyme activities (12%, 18% and 52%, respectively) [6]. During the reaction of fully reduced Y227F with O2, the differences to wildtype are subtle compared to the other variants studied here. Although heme c oxidation and proton uptake are biphasic in Y227F, the faster phase of both heme c oxidation and proton uptake are as fast as in Wt (Figure 2). The slower heme c oxidation phase in Y227F occurs with k~200 s−1, and we interpret the biphasic nature as a heterogeneity in Y227F, where the ‘non-wildtype’ like population has undergone conformational changes slowing proton transfer. These results suggest that neighboring water molecules might take over the role of the Y227, similar to the situation in S244A (see Figure 1), which retains full enzymatic activity. Presumably, multiple proton pathways are possible in this lower KC-region [6, 8].

Y321 is further up the KC-pathway and is a suggested branch point through which all pumped and chemical protons are transferred [16, 37], and previous mutagenesis studies verified its importance for catalytic turnover (Table 1) [6, 8]. Here, we showed that in single-turnover O2 reduction, oxidation of heme b in Y321F was maintained as fast as in Wt, whereas oxidation of heme c was about 2-fold slower. The most striking difference to wildtype is however that proton uptake in Y321F occurred with only a very small amplitude (Figure 2).

The severe reduction of steady-state activity (and proton pumping, see below) is thus presumably linked to the inhibition of proton uptake from bulk in Y321F. Since there is partial oxidation of the hemes c in Y321F without concomitant proton uptake (this occurs also in E49PA but to a smaller extent [6]), we assume that there is internal transfer of a proton from either within the KC-pathway or ‘back’ from the loading site for protons to be pumped (PLS, see Figure 5) in Y321F. As a possible proton bifurcation point, electrostatic and/or structural changes of Y321 might be tightly connected to proton transfer to/from both the internal proton donor (XH) and the PLS. Presumably relative pKas of the branch point/internal donor and the PLS has to be fine-tuned in order to achieve tight coupling of the pump [36]. In A-type HCuOs, the same transitions that are linked to release of pumped protons from the PLS are also linked to proton uptake to the O2 intermediate at the catalytic site, whereas in B-type HCuOs, these two processes are separated in time. Thus, in B-type, the first proton uptake observed during O2 reduction actually goes to the PLS and only later is the (PR in this case) intermediate protonated [38]. This separation in time was suggested to be linked to the challenge of synchronizing uptake/pumping events when having only one proton pathway. Since C-type HCuOs also have only the KC-pathway, we should consider the same scenario possible also here.

A scheme of proposed proton transfer events in C-type HCuOs is shown in Figure 5 based on our current and previous data. O2 binding and oxidation of heme b forms an intermediate, which could accept a proton from the internal donor XH and thereby transition to the F state. Alternatively, forming the PR intermediate might trigger proton transfer from XH to the PLS via Y321. Then, an external proton uptake (to XH or the PLS) through E49P at the entrance drives the oxidation of heme c. In Wt, all transitions, (and proton uptake) after O2 binding occur simultaneously and no further intermediates are observed. Presumably there is both uptake and release of pumped protons in this transition.

In the Y321F variant there is more substantial oxidation of heme c without a corresponding uptake of protons from the bulk than what occurs in E49P variants. This might be due to a change in the pKa of the PLS thus using a PLS proton for O2 reduction chemistry (‘slipping’ of the pump, see dashed arrow in Fig. 5). A related change was observed in MD simulations of effects of Y321 to F exchange (Y317 in their work) as it lead to a less hydrated (less connected) path towards the BNC but a more hydrated path towards the proton exit route [37]. In this scenario, proton pumping would be abolished in Y321F, consistent with our ATP synthesis data (Figure S3) as well as the low RCR, although the slow turnover in Y321F makes detailed analysis difficult.

Taken together, our data supports the suggested role for Y321 in controlling whether protons are delivered to the active site or towards the PLS as well as preventing proton back-flow.

4.4. Functional divergence of tyrosine residues in the KB and KC pathways

Although both B- and C-family HCuOs utilize a single proton pathway spatially analogous to the K-pathway of A-type HCuOs, key conserved residues lining the pathways are phylogenetically divergent in each family. All K-pathways have a Glu residue at the cytoplasmic surface, which is the only residue conserved between the KA- and KB-pathways. These glutamates are also the only residues of the K pathways that are not residing in the catalytic subunits (SU-I or CcoN) but in an accessory subunit (SU-II in A and B-type [3, 7], CcoP in C-type [6, 15]). Subunit II of A- and B-type HCuOs and CcoP of C-type are unrelated proteins. This form of ‘convergent evolution’ in HCuOs is observed also for the ‘proton-collecting antenna’ around this Glu [39] and the crosslinked Tyr at the active site which is conserved in space but not in sequence [4, 28]. The relative location of the entry-point Glu is different in KB- and KC-pathways, which means that the region above have to be different as well in order to provide connectivity into the subunit I (or CcoN) part of the pathways (discussed also in [35]). The KB and KC pathways are more similar to each other in composition (lacking the lysine, having mostly Tyr and Thr residues) than they are to the KA-pathway, but the only residue conserved between them is the Y227 (see Fig. 1), equivalent to Y248 in the KB-pathway of the T. thermophilus ba3 enzyme [8, 15]. However, these Tyr must connect differently to the entry-point Glu (see Fig. 1C), ~15 Å away, and thus do not necessarily play the same role in the two pathways. Variants of Y227Vc exhibit varying enzyme activity (up to 52%) [6], whereas the Y248Tt in B-type is crucial for catalytic function [7, 35]. The Y248FTt variant further shows perturbations in internal pKas and proton transfer sequences [35], suggesting that the hydrogen bonded network around this Y248 is less flexible than around the Y227 in the KC-pathway.

Variants of the Y321Vc (Fig. 1 and 5), are largely inactive and although not conserved in sequence, Y321 is spatially equivalent to Y244Tt in the KB-pathway (Fig. 1C) [3, 8, 15]. The Y244FTt variant, where ~15% turnover activity remains, still retains proton pumping ability [7], although the variant did show alterations in timing of proton transfer to the PLS area [40]. Thus, the importance of the Tyr residues (Y248Tt in KB and Y227Vc in KC, versus Y244Tt in KB and Y321Vc in KC) is switched in the KB and KC pathways.

4.5. Summary and conclusions

We have identified an internal proton donor XH in the KC-pathway with a high pKa that presumably functions as a branching point for chemical and pumped protons, similar to the Glu-286 in the A-family. We also show that the detailed roles of the two Tyr residues in the KB- and KC– pathways appear to be different and specific to the conformation of the pathway, pointing to unique solutions for each family of HCuOs.

Supplementary Material

1
2

Highlights.

  • We studied variants with the KC proton pathway modified; Y227F, N293L and Y321F.

  • In N293L, proton-coupled electron transfer is slowed.

  • Further, in N293L we could observe titration of an internal proton donor XH.

  • In Y321F, proton uptake from bulk solution is greatly impaired.

  • We suggest that Y321 is among the candidates for the important proton donor XH.

Acknowledgements

Y.O.A was supported by a post-doctoral stipend from the Carl Trygger foundation (CTS). The study was supported by grants from the Carl Trygger foundation and from the Faculty of Science at Stockholm University to PÄ as well as by grant number HL16101 from the NIH to RBG. We are grateful to Christoph von Ballmoos (University of Bern) for help with the coupled ATP synthesis measurements.

Abbreviations:

TMPD

N,N,N’,N’-tetramethyl-p-phenylenediamine

PMS

phenazine methosulphate

HCuO

heme-copper oxidase, Unless otherwise indicated, residues/mutations are in subunit I or CcoN. The superscript “P” denotes the residue/mutation is in subunit CcoP

“Vc”

“Rs” and “Tt” superscripts indicate Vibrio cholerae, Rhodobacter sphaeroides, and Thermus thermophilus, respectively.

Footnotes

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NIHMS1505766-supplement-1.pdf (4.5MB, pdf)
2
NIHMS1505766-supplement-2.docx (426.1KB, docx)

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