Heme-copper terminal oxidase using both cytochrome c and ubiquinol as electron donors

Abstract

The cytochrome c oxidase Cox2 has been purified from native membranes of the hyperthermophilic eubacterium Aquifex aeolicus. It is a cytochrome ba(3) oxidase belonging to the family B of the heme-copper containing terminal oxidases. It consists of three subunits, subunit I (CoxA2, 63.9 kDa), subunit II (CoxB2, 16.8 kDa), and an additional subunit IIa of 5.2 kDa. Surprisingly it is able to oxidize both reduced cytochrome c and ubiquinol in a cyanide sensitive manner. Cox2 is part of a respiratory chain supercomplex. This supercomplex contains the fully assembled cytochrome bc(1) complex and Cox2. Although direct ubiquinol oxidation by Cox2 conserves less energy than ubiquinol oxidation by the cytochrome bc(1) complex followed by cytochrome c oxidation by a cytochrome c oxidase, ubiquinol oxidation by Cox2 is of advantage when all ubiquinone would be completely reduced to ubiquinol, e.g., by the sulfidequinone oxidoreductase, because the cytochrome bc(1) complex requires the presence of ubiquinone to function according to the Q-cycle mechanism. In the case that all ubiquinone has been reduced to ubiquinol its reoxidation by Cox2 will enable the cytochrome bc(1) complex to resume working.

Fig. 1.

Identification of a sequencing error for the deposited Cox2 subunit II (CoxB2). (A) MS spectrum of the trypsin digested Cox2 subunit II (band in Fig. S1C). Masses and sequence regions of assigned signals are labeled. Asterisks mark peaks verifying the new sequence regions. The bar plot aligns identified peptide mass fingerprint (PMF) fragments to the corresponding protein sequence regions. The arrow indicates the start of the incorrectly annotated Cox2 subunit II sequence; (B–D) MS/MS spectra of 1,014.6, 1,947.9, and 2,246.1. The manually interpreted peptide sequence is indicated (N-terminal b-ions, C-terminal y-ions, immonium ions). A differentiation between leucine/isoleucine (L/I) and lysine/glutamine (K/Q) is not possible. Because of the C-terminal specificity of the trypsin digest at lysine/arginine (K/R), the C terminus is solely indicated as K in C and D.

Fig. 2.

UV-Vis difference spectra of Cox2 and the supercomplex. The spectra were recorded at room temperature. (A) Dithionite reduced minus air oxidized Cox2 (lower) and supercomplex (upper). Dotted lines show the absorption signals with a magnification of 5× in the range of 490–600 nm. The spectra of reduced enzymes were recorded after 10 s when 200 mM dithionite were added into the cuvette. (B) decylubiquinol reduced minus air oxidized Cox2 (lower) and supercomplex (upper). Both spectra were recorded after proteins were incubated with 45 μM decylubiquinol in the presence/absence of 1 mM potassium cyanide for 30 min. (C) TMPD/ascorbate reduced minus air oxidized Cox2 (lower) and supercomplex (upper). The reducing spectra were recorded after 10 s when 0.3 mM TMPD/3 mM ascorbate and 1 mM potassium cyanide were applied. (D) Incubation of Cox2 with decylubiquinol and cyanide (solid lines) or cyanide alone (dotted lines). It shows that cyanide alone cannot induce the 595 nm absorption of Cox2 within 30 min.

Fig. 3.

Oxygen consumption by Cox2 and the supercomplex in the presence of decylubiquinol and DTT. Reactions were recorded using an oxygen electrode at 40 °C. (A) Oxygen consumption by Cox2. The reaction was inhibited by 1 mM potassium cyanide. (B) Oxygen consumption by the supercomplex. This reaction was not inhibited by 40 μM stigmatellin, but inhibited by 1 mM potassium cyanide. (C) Oxygen consumption by the supercomplex. This reaction could be accelerated by addition of air oxidized horse heart cytochrome c (60 μM). The cytochrome c has to be injected together with the protein to avoid direct reduction by DTT. The scale bars indicate a change in the dioxygen concentration by micromole per liter per minute.

Fig. 4.

The effect of increasing decylubiquinol concentrations on the supercomplex activities. All reactions were measured spectrophotometrically at 80 °C. (A) Decylubiquinol oxidation in the presence (dashed line) and absence (solid line) of 1 mM potassium cyanide. (B) Cytochrome c reduction (dashed line) and decylubiquinol oxidation (solid line) in the presence of 60 μM horse heart cytochrome c and 1 mM potassium cyanide. (C) Cytochrome c reduction (dashed line) and decylubiquinol oxidation (solid line) in the presence of 60 μM horse heart cytochrome c. Error bars indicate the standard errors of the mean from three experiments. The y axis shows the enzyme-specific activity in percent.

Fig. 5.

Cytochrome c reduction of Cox2 in the presence of 1 mM potassium cyanide and 45 μM decylubiquinol. The reaction was measured spectrophotometrically at 80 °C as described in SI Materials and Methods for cytochrome c reduction. A specific activity of 0.53 μmol/ min per milligram was calculated for the Cox2 ubiquinol∶cytochrome c oxidoreductase activity. For the blank measurements buffer without Cox2 was used.