The proton pumping bo oxidase from Vitreoscilla

Simone Graf,Christoph Von Ballmoos,Peter Brzezinski

doi:10.1038/s41598-019-40723-2

Simone Graf, Christoph Von Ballmoos + Show 1 more

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https://doi.org/10.1038/s41598-019-40723-2

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Journal: Scientific Reports	Publication Date: Mar 18, 2019
Citations: 7	License type: open-access

Affiliation: University of Bern, Stockholm University

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.

Highlights

The cytochrome bo[3] quinol oxidase from Vitreoscilla catalyses oxidation of ubiquinol and reduction of O2 to H2O
We found that the enzyme pumps protons with a stoichiometry of 1 H+/e− and is functionally indistinguishable from the E. coli counterpart
The Vitreoscilla cytochrome cyo operon has been sequenced earlier[35], showing that it encodes for subunits I-IV and the protoheme IX farnesyltransferase that is important for heme o biosynthesis[36,37]

Summary

Introduction

The cytochrome bo[3] quinol oxidase from Vitreoscilla (vbo3) catalyses oxidation of ubiquinol and reduction of O2 to H2O. Heme-copper oxidases catalyze the terminal step reducing oxygen to water This highly exergonic electron transfer is coupled to proton pumping that maintains an electrochemical proton gradient across the membrane utilizing two mechanisms. The cytochrome bo[3] oxidase from E. coli (ecbo3) is a quinol dependent oxidase which pumps four protons across the lipid bilayer for each O2 reduced to two H2O8 These electron and proton-transfer reactions are very fast (ns to ms) and cannot be studied with conventional spectroscopic techniques. In a series of reports, 22Na-uptake experiments in native Vitreoscilla membranes and with purified enzyme reconstituted into liposomes indicated a Na+-dependent oxidase activity and the ability of the enzyme to pump sodium ions instead of protons[32,33]. We found that the enzyme pumps protons with a stoichiometry of 1 H+/e− and is functionally indistinguishable from the E. coli counterpart

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