Observación número 14/AOP/M04/2006:

CCCP is a proton ionophore that collapses any protonmotive force by rapidly equilibrating the H+ concentration inside and outside the vesicles. Figure 6-5 shows the immediate proton consumption for water formation in response to a pulse of 1.35 µM cyt c2+ to bovine COVs, that were uncoupled with 2.5 µM CCCP. The size of the alkalinisation was equivalent to 1 ±0.1 H+ per electron.

Figure 6-5. External H+ concentration changes in response to the addition of cyt c2+ to uncoupled bovine COVs.

Bovine COVs were uncoupled by the addition of 2.5 µM CCCP (4 µL) in the same cuvette as Figure 6-4. The baseline was allowed to stabilise and the pH was adjusted to pH 7.4. 1.35 µM horse heart cyt c2+ (1.5 µL) was added at the time indicated. The size of a calibrating 1.35 µM acid pulse after addition of 2.5 µM CCCP is also shown. Absorbance changes of phenol red were measured at ΔA556.5-504.0 and converted to [H

+

] changes using a calibrating 1 µM acid pulse. Reaction was carried out at 14 oC.

185

6.3.5 Reconstitution of purified yeast CcO into phospholipid vesicles

It has not yet been possible to generate yeast WT COVs that were as tightly coupled as the bovine COVs when using the same protocol; the highest RCR value measured was very low (~1.2). The addition of 0.05 % (w/v) DDM to uncoupled COVs had no effect on the turnover activity, and in some cases it caused the activity to decrease. Nevertheless, the H+/e- stoichiometry assay was carried out. However, the traces following a cyt c2+ pulse, gave the same result as that of uncoupled COVs (immediate alkalinisation), with a 1 H+ consumed per electron stoichiometry (data not shown).

Hence, changes were made to the protocol that was used for bovine CcO reconstitution that is described in Section 2.21 to generate coupled yeast WT COVs. These conditions and the resulting RCR values are summarised in Table 6-2. Purified yeast CcO was prepared in 50 mM KPi, 250 mM NaCl, 0.01 % (w/v) DDM pH 8.0. The lipid composition was altered from all lecithin to 1:4 (w/w) ratio of cardiolipin to lecithin because cardiolipin is a mitochondrial inner membrane lipid. The pH of the entire reconstitution procedure was lowered from pH 7.4 to pH 6.9, which was near the optimum turnover activity. The enzyme to lipid ratio was halved to give 0.095 nmole CcO/mg of lipid. The divalent cation, Mg2+, was added to the enzyme/lipid preparation before dialysis, and to all the dialysis buffers at 10 mM MgCl2 [247].Octyl glucoside

(>99.5 %, Melford Laboratories Ltd, UK) detergent was tested instead of potassium cholate for promoting the association of CcO with the liposomes. Since the RCR of bovine CcO was variable across 5 different enzyme preparations, the effect of using different enzyme preparations was also tested. Hence, further WT yeast CcO preparations that had been purified in the presence of lipids, or in the presence of digitonin, instead of DDM were also tested, as was a 4H mutant CcO preparation (see below). The condition that gave the highest RCR was with digitonin-purified WT CcO with 3.9 pmole CcO/mg lecithin and extended dialysis times, and with all other conditions for reconstitution the same as for bovine CcO reconstitution. Larger volume preparations were also necessary for better coupling. An RCR of ~1.4 was achieved with WT CcO, but proton pumping experiments still showed the behaviour of uncoupled COVs.

Table 6-2. Summary of conditions for membrane reconstitution of yeast CcO (WT and 4Hmutant).

RCR repeats represent COVs that were prepared more than once. Abbreviations are; OG, octyl glucoside, w/o, without, PC, phosphatidylcholine, PI phosphatidylinositol, PE, phosphatidylethanolamine.

Dialysis protocol Condition tested Details RCR

Fast dialysis protocol.

The protocol that was used for bovine CcO; 2x1.5 h 250 mL 75 mM HEPES pH 7.4, 14 mM KCl, 0.1 % cholate, 2x1.5 h same buffer w/o cholate overnight, 500 mL 0.5 mM HEPES pH 7.4, 44.6 mM KCl, 43.4 mM sucrose overnight.

Effect of cardiolipin 40 mg/mL lecithin, 2 % (w/v) K+ cholate 1.21, 1.15, 1, 1 1:4 cardiolipin:lecithin (20 % w/w cardiolipin) No coupling

Effect of pH,

20% w/w cardiolipin

pH 7.4 No coupling

pH 6.9 No coupling

Effect of CcO:lipid ratio, pH 7.4, 20

%(w/w) cardiolipin

0.19 nmole CcO/mg of lipid No coupling

0.095 nmole CcO/mg of lipid No coupling

Effect of Mg2+, pH 7.4, 20 % (w/w)

cardiolipin

10 mM MgCl2 1.21, 1.15

w/o MgCl2 No coupling

Effect of OG, pH 7.4, 20 % (w/w) cardiolipin 10 mM MgCl2 2 % OG 1.04 10 mM MgCl2, 2 % K

+

cholate 1.21, 1.15

w/o 10 mM MgCl2, 2 % OG 1.16

Effect of 4H mutant preparation, pH 7.4,

20 % (w/w) cardiolipin, 10 mM MgCl2 4H mutant, 2 % K+ cholate 1.51, 1.40, 1.81 Prolonged dialysis protocol Dialysis was 3 h 500 mL 75 mM HEPES pH 7.4, 14 mM KCl, 0.1 % cholate,

1x500 mL same buffer w/o cholate overnight, 1x6 h 500 mL same buffer all day, 1x 1 L 0.5 mM HEPES pH 7.4, 44.6 mM KCl, 43.4 mM sucrose overnight Effect of different yeast CcO preparations, pH 7.4

Yeast WT CcO purified in the absence of lipids All the above

Yeast WT CcO presence of lipids (1:1:1) PC, PE, PI. COVs were prepared with 20 % w/w cardiolipin, 10 mM MgCl2 2 % K

+

cholate. (USED FAST DIALYSIS)

1.21, 1.18 WT CcO purified by FPLC in the absence of lipids, used fraction collected after passing

through Ni Column. COVs prepared with 40 mg/mL lecithin, w/o MgCl2, 2 % K +

cholate

1.26 Purified using FPLC absence of lipids, fraction collected after passing through Ni column and gel filtration. COVs prepared with 40 mg/mL lecithin, w/o MgCl2, 2 % K

+

cholate

1.18, 1.30, 1.33

Using 1 % digitonin solubilised WT mitos. COVs were prepared with 40 mg/mL lecithin, w/o MgCl2, 2 % K

+

cholate

1.40 Purified in the presence of 0.01 % (w/v) digitonin instead of 0.05 % (w/v) DDM using

fraction collected after Ni column. COVs were prepared with 40 mg/mL lecithin, w/o MgCl2, 2 % K

+

cholate

187 An observation worth noting was that the profiles of a calibrating acid pulse before and after CCCP addition were different. This is shown in Figure 6-6 for a blank COVs preparation (as a control), and for yeast COVs that exhibited an RCR of 1.4, and that were prepared using CcO that was purified in the presence of digitonin. An acid pulse in a low background concentration of CCCP, resulted in an acidification followed by an alkalinisation phase, this was due to the equilibration of protons into the interior of the vesicles until the pH had stabilised. In the presence of high [CCCP], the vesicles were uncoupled and the acid pulse equilibrated rapidly inside and outside the vesicles to the same final extent. By comparison to the blank COVs this evidence suggests that intact liposomes were indeed present, and that the method used to form liposomes worked well. Therefore the low RCR values obtained of yeast CcO and the subsequent absence of proton pumping, suggests that CcO did not fully incorporate into the liposomes.

Figure 6-6. Comparison of HCl pulses in coupled and uncoupled vesicles prepared in the (A) absence of CcO and (B) presence of yeast CcO.

A. 464 µL blank COVs in 2 mL of 50 µM phenol red, 44.6 mM KCl, 43.4 mM sucrose, 10 µg/mL carbonic anhydrase, 10 µM valinomycin and 10.5 nM CCCP. pH was adjusted to pH 7.4 and temperature was set to 25 oC. A pulse of 1 µM HCl was added (black trace). Once baseline had stabilised the blank COVs were uncoupled with 5 µM CCCP and the acid pulse was repeated (red trace). B. 150 µL of WT yeast COVs (RCR of 1.4) in 1.6 mL of the same buffer but with 4 nM background CCCP. A pulse of 2 µM HCl was added (black trace). The yeast COVs were uncoupled with 5 µM CCCP and the acid pulse was repeated (red). Absorbance changes of phenol red were measured at ΔA556.5-504.0.