The proton pumping bo oxidase from Vitreoscilla

Abstract

The cytochrome bo3 quinol oxidase from Vitreoscilla (vbo3) catalyses oxidation of ubiquinol and reduction of O2 to H2O. Data from earlier studies suggested that the free energy released in this reaction is used to pump sodium ions instead of protons across a membrane. Here, we have studied the functional properties of heterologously expressed vbo3 with a variety of methods. (i) Following oxygen consumption with a Clark-type electrode, we did not observe a measurable effect of Na+ on the oxidase activity of purified vbo3 solubilized in detergent or reconstituted in liposomes. (ii) Using fluorescent dyes, we find that vbo3 does not pump Na+ ions, but H+ across the membrane, and that H+-pumping is not influenced by the presence of Na+. (iii) Using an oxygen pulse method, it was found that 2 H+/e− are ejected from proteoliposomes, in agreement with the values found for the H+-pumping bo3 oxidase of Escherichia coli (ecbo3). This coincides with the interpretation that 1 H+/e− is pumped across the membrane and 1 H+/e− is released during quinol oxidation. (iv) When the electron transfer kinetics of vbo3 upon reaction with oxygen were followed in single turnover experiments, a similar sequence of reaction steps was observed as reported for the E. coli enzyme and none of these reactions was notably affected by the presence of Na+. Overall the data show that vbo3 is a proton pumping terminal oxidase, behaving similarly to the Escherichia coli bo3 quinol oxidase.

Lipid Composition Affects the Efficiency in the Functional Reconstitution of the Cytochrome c Oxidase

“…Similar to previous studies on the enzymatic activity of the bo 3 quinol oxidase [ 28 , 29 ], a proteoliposome-based assay was used to quantify proton turnover of C c Os. To this end, C c Os were reconstituted into the membrane of large unilamellar vesicles (LUVs) and the lumen of formed proteoliposomes was filled with the pH sensitive dye 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS; Figure 1 a) [ 30 , 31 ].…”

“…Enzymatic activity of the C c O-containing proteoliposomes was first assessed using a cuvette-based bulk spectrofluorometer [ 29 ], in which the emission intensities I 405 and I 458 are averaged over all proteoliposomes present in the optical readout volume. This approach yields ensemble-averaged information on the luminal pH value and therefore cannot be used for exact quantification of the proton turnover rates.…”

“…Analysing pH changes on the level of single proteoliposomes using fluorescence microscopy allows for the determination of single-enzyme kinetics and thus yields information on the proton turnover rate of single C c O in dependence of its lipid environment. The results are cross-validated using a cuvette-based spectrofluorometric assay [ 29 ], which provides an ensemble-averaged recording of luminal pH values. In comparison to the microscopy-based assay, this approach offers a higher signal-to-noise ratio in the recording process but does not permit a direct extraction of the rate of proton turnover.…”

Bifunctional DNA Duplexes Permit Efficient Incorporation of pH Probes into Liposomes

“…Typically, after purification in detergent solution, membrane proteins are reconstituted into liposomes, imitating their native environment for functional analysis. Proton transport activities are measured with soluble pH‐sensitive dyes that are incorporated into the liposomes and detected either via absorbance or fluorescence measurements . After reconstitution, the non‐incorporated dye has to be removed by gel filtration or ultracentrifugation or a combination of both.…”

“…Proton transport activities are measured with soluble pH-sensitive dyes that are incorporated into the liposomes and detected either via absorbance or fluorescence measurements. [6][7][8][9] After reconstitution, the non-incorporated dye has to be removed by gel filtration or ultracentrifugation or a combination of both. While convenient, the method suffers from poor incorporation yield (< 5 %) of the dye which is not desirable if precious dyes are used.…”