Observación número 15/AOP/M04/2006:

When the stoichiometry assay was performed with the 4H mutant COVs, the extruded pH profile displayed a small acidification phase followed by a large alkalisation phase (Figure 6-11). However, the coupling stoichiometry could not be estimated accurately, based solely on the extent of acidification relative to a calibrating acid pulse. This is because the rapid proton re-entry rate into the vesicles due to poor coupling competed with the rate of acidification. Hence, an alternative approach was used in which the profile was calculated iteratively, to give a best fit to data and provide an estimate of the H+/e- stoichiometry. This fit took into account (1) the measured rate constant of the influx of protons, (2) the rate constant of alkalinisation phase, after the initial acidification caused by the calibrating acid pulse (Figure 6-6) and (3) the measured rate constant of cyt c oxidation, which for a 1 H+/e- stoichiometry, will be equivalent to the rate constant of proton extrusion. The coupling stoichiometry (given by the ratio of rate constant of cyt c oxidation, to the rate constant of proton extrusion) was varied until the best fit of the extent and kinetics of the extruded protons was reached. This approach was tested with bovine COVs, where the estimated stoichiometry of 1.0 ±0.1 was consistent (Figure 6-10) with the total extents approach (Figure 6-4). For the 4H mutant, the best fit to the data was obtained with a coupling stoichiometry of 1 ±0.07 H+/e-.

6.4.4 Interpretation of findings

The results show that the yeast 4H mutant with four hydrophilic to hydrophobic replacements, along the H channel, still retained proton translocating activity. Thus it can be concluded that the H channel is not critical for proton translocation in yeast CcO. This provides the first preliminary evidence against the proposal [38-40,87] that the H channel provides the route for translocated protons in a mitochondrial form of CcO. Instead it is likely that the D channel provides this route in yeast CcO as has been shown by mutagenesis in bacterial CcOs [84,91,92].

An alternative proposal is that the H channel functions as a dielectric channel [78], which could facilitate rapid electron transfer into/out of haem a (the nearest metal centre), by modulating the dielectric strength around an otherwise deeply buried site of the enzyme. The H channel is a candidate for this role since the hydrophilic residues connect the region around haem a to the aqueous phases (IMS/matrix), where surface changes and re-orientation of dipoles along the H channel, can influence the properties of haem a, which could include minimisation of net charge changes and the energy barrier for electron transfer. Although not a complete pathway in bacterial CcOs, it is also evident, and may have the same role. An interesting observation that may support such a function is the altered kinetic properties of the 4H mutant compared to WT (Figure 6-3), that had a 2-fold smaller apparent Km value compared to the WT (Table

6-1 and Figure 6-3C) that could point towards an activity modulating role of the H channel. It is well known that the electrostatic binding/unbinding of cyt c to its site on subunit II is influenced by ionic strength [42,43,258]. In WT yeast CcO, this leads to a marked decrease in turnover activity between 10-40 mM KCl concentrations (Figure 6-3B). However no decrease in turnover activity was observed in this concentration range for all the H channel mutants, and in fact, the activity stayed the same. This finding suggests that the H channel may either have an allosteric effect on the cyt c binding site, or that the dielectric properties of the H channel have been changed by the mutations, in such a way that it results in an alteration of the activity profile with KCl concentration.

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6.5 Conclusions

The activity profiles of the WT and mutant mitos allowed us to identify the optimal activity conditions. The reconstitution protocol produced well-coupled bovine COVs that had a measured H+/e- stoichiometry of 1.0 ±0.1, consistent with literature values [178,249,251,252]. Various factors were tested to improve the reconstitution behaviour of WT yeast CcO, but to date, the COVs produced were too poorly coupled or did not incorporate CcO to allow measurement of their H+/e- stoichiometries. COVs with a measurable, but low, RCR were produced with the 4H mutant CcO. This gave the first H+/e- stoichiometry data that show definitively, that the proton translocating activity of 1 ±0.07 H+/e is retained in this mutant. The H channel has alternatively been proposed to act as a dielectric channel [78], and the altered kinetics of the H channel mutants could possibly be explained by a modulation of the H channel dielectric properties.

6.6 Future work

The quality of the reconstituted COVs may be affected by as yet unknown factors associated with the purified preparations of the enzyme. Factors related to the composition/nature of the purified enzyme preparations will be tested, to see whether any affect the reconstitution behaviour (e.g. dimer versus monomer state, alternative detergents). Initially the standard method of purification of yeast CcO involved two chromatography steps; nickel affinity, and DEAE column chromatography, the resulting fractions containing CcO as determined by visible spectroscopy were pooled together, concentrated, and stored at -80 oC [118]. CcO preparations of WT and 4H mutant CcO purified this way were used for membrane reconstitution. Since then efforts were made to improve the purification procedure that employed the use of automated FPLC, and replacing DEAE column chromatography with gel filtration. Gel filtration was an important control in determining the homogeneity of the preparation. It showed that WT CcO was present as a mixture of trimer/dimers/monomers and included a large excluded front of lipid/CcO/detergent aggregates. Fractions were collected of the mostly dimeric state and their membrane reconstitution was tested (Table 6-2), however the RCR was not significantly improved. However, this method of purification, and the use of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS PAGE) will provide useful controls to test various factors, with the aim of producing a homogeneous preparation with a single elution peak from gel filtration. Factors that could be tested include for example, changing the detergent from DDM (12 carbon alkyl chain length) to, n-decyl-β-D-maltopyranoside (DM) (10 carbons) or n-octyl-β-D- maltopyranoside (OM) (8 carbons) or n-octyl-β-D-glucopyranoside. Glycerol/ethylene glycol could be added to the running buffers to avoid aggregation. EDTA could be added to chelate any traces of metal (Ni ions) that could have eluted from the Ni column. Yeast mitochondrial lipids could be added to the running buffers, and to the columns to stabilise CcO. The pH, ionic strength and the buffer used could also be optimised. So far the most optimal conditions have been 50 mM HEPES, 500 mM NaCl, 0.05 % DDM at pH 7.4.

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7 Comparisons of subunit 5A and 5B isozymes of