The proton pumping bo oxidase from Vitreoscilla

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.

: Sequence alignment of ATP synthase c subunits.
Sequence analysis of the c subunit of Vitreoscilla ATP synthase revealed none of the typical motifs identified in ATP synthases known to pump sodium. Acetobacterium woodii, Fusobacterum nucleatum, Ilyobacter tartaricus, and Propionigenium modestum all harbor sodium pumping ATPases having the characteristic Q at position 32 (A. woodii numbering, orange), as well as the AE(S/T)xxY motif (green) after the RQP motif in the loop (yellow). Except for the RQP loop motif, in the proton pumping enzymes from Escherichia coli, Vibrio Cholerae, and Vitreoscilla these motifs are not conserved (purple).  This figure depicts a 12% SDS-PAGE of purified vbo 3 expressed in E. coli C43 Δcyo cells from a pET-17b vector harbouring the vbo 3 operon containing a His 9 -tag N-terminal to cyoC. The loaded amount was normalized to SUI/cyoB.
Reduced minus oxidized difference spectrum was obtained by subtracting the ferricyanide oxidized absorbance spectrum from the dithionite reduced absorbance spectrum. Enzyme concentration in the sample was about 1 μM. The sample was completely oxidized by adding 5μM ferricyanide and incubation at room temperature for 15 minutes, reduction of the sample was achieved by adding a few grains of sodium dithionite. Spectra were recorded with the Cary 60 UV-Vis spectrophotometer from Agilent Technologies.   The fully oxidized enzyme (O 0 ) is reduced to R 2 during the reductive cycle of the enzyme, which is less well understood. Naturally, the enzyme takes up only two electrons to the catalytic site, before the reaction starts. Under reductive and anaerobic conditions in the laboratory, however, the enzyme is able to take up more electrons and become fully reduced 5 . In the scheme above and in the following description, only the latter conditions is described.
The oxidative cycle then starts with oxygen (green) binding to the R 2 state, which is rapidly converted to the A 2 state. In presence of a pre-reduced heme b (as in our experiments), internal electron transfer from heme b to the catalytic site takes place to form state P 3 , which is able to take up a proton from solution to form state F 3 . This proton uptake is accompanied by a proton pumped across the membrane. Finally, the forth electron is transferred to catalytic site to from O 4 , again accompanied by uptake of one proton for oxygen reduction and one proton pumped across the membrane.  Changes of absorbance over time measured at 430nm upon dissociation of CO from the reaction center in the presence of 100 μM quinol Q 1 (10 times excess to vbo 3 , orange trace), in the presence of 25 μM HQNO (blue trace), or as it was purified (red trace).

Supplementary Table 2: Summary of mass spectrometric analysis of the SDS-PAGE bands of purified vbo 3 .
For the mass spectrometry, the following bands were excised from the SDS-PAGE and analyzed: 100kDa, 55kDa, 36kDa and 23kDa. Traces of the protoheme farnesyltransferase were found in the different bands as well. Subunit D was not picked up by mass spectrometry since the smallest 11kDa band was not sent for analysis. Total % Coverage: The percentage of the protein sequence covered by identified peptides.