A new iron-oxidizing/O2-reducing supercomplex spanning both inner and outer membranes, isolated from the extreme acidophile Acidithiobacillus ferrooxidans

Abstract

The iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans involves various metalloenzymes. Here we demonstrate that the oxygen reduction pathway from ferrous iron (named downhill pathway) is organized as a supercomplex constituted of proteins located in the outer and inner membranes as well as in the periplasm. For the first time, the outer membrane-bound cytochrome c Cyc2 was purified, and we showed that it is responsible for iron oxidation and determined that its redox potential is the highest measured to date for a cytochrome c. The organization of metalloproteins inside the supramolecular structure was specified by protein-protein interaction experiments. The isolated complex spanning the two membranes had iron oxidase as well as oxygen reductase activities, indicating functional electron transfer between the first iron electron acceptor, Cyc2, and the Cu(A) center of cytochrome c oxidase aa(3). This is the first characterization of a respirasome from an acidophilic bacterium. In Acidithiobacillus ferrooxidans,O(2) reduction from ferrous iron must be coupled to the energy-consuming reduction of NAD(+)(P) from ferrous iron (uphill pathway) required for CO(2) fixation and other anabolic processes. Besides the proteins involved in the O(2) reduction, there were additional proteins in the supercomplex, involved in uphill pathway (bc complex and cytochrome Cyc(42)), suggesting a possible physical link between these two pathways.

FIGURE 1.

Analyses of solubilized membrane from A. ferrooxidans by absorption spectra (A) and Western blots (B). A, proteins (2.2 mg/ml in buffer A, 3 mg of DDM/mg of proteins) were analyzed as prepared (dashed line) and reduced by sodium dithionite (solid line). Signals arising from c-type cytochromes are identified by at 418, 523, and 552 nm (c), from b-type cytochromes at 560 nm (b), and from cyt c oxidase by at 440 and 597 nm (aa₃). B, solubilized membranes (85 μg in each lane) were separated by a 12% denaturing gel. Proteins were stained with Coomassie Blue (lane 2) and revealed on nitrocellulose membrane with anti-RcY and anti-Cyc1 antibodies (lanes 3 and 4, respectively). Molecular weight markers (lane 1) are indicated in kDa.

FIGURE 2.

Absorption spectra of 18–20% fraction from glycerol density gradient. Proteins (2.9 mg/ml in buffer A, 0.1% DDM) were analyzed as prepared (dashed line) and reduced by sodium dithionite (solid line). Signals arising from c-type cytochromes are identified by at 418, 523, 548 and 552 nm (c), from b-type cytochromes at 560 nm (b), and from cyt c oxidase at 440 and 597 nm (aa₃).

FIGURE 3.

Oxidation of exogenous ferrocytochrome c by the 18–20% glycerol gradient fraction. Kinetics of oxidation of 100 μm of ferrocytochrome c was monitored at 552 nm after addition of 100 μg of proteins from the 18–20% glycerol gradient fraction as prepared (solid line), with 10 μm stigmatellin (dashed line), and with 10 μm stigmatellin + 100 μm KCN (dotted line).

FIGURE 4.

Low temperature absorption spectrum of the purified supercomplex. Proteins (2 mg/ml in buffer A, 0.1% DDM) were reduced by sodium dithionite. Signals arising from c-type cytochromes are identified at 415, 525, 545, and 548 nm (c), from b-type cytochromes at 427, 528, and 560 nm (b), and from cyt c oxidase by at 439, 447, and 596 nm (aa₃).

FIGURE 5.

Supercomplex subunit composition. A, analysis of the purified supercomplex by a 5–15% BN gel associated with Coomassie Blue staining (lane 1), TMBZ staining (lane 2), cyt c oxidase activity staining (lane 3), and bc complex heme-specific staining (lane 4). On the left, arrows indicate band 1 and band 2, corresponding to multiprotein complexes. B, analysis of the purified supercomplex by a 10% SDS gel associated with Coomassie Blue staining (lane 1) and Western blotting with antibodies raised against RcY, Cyc2, subunit II of the cyt c oxidase (CoxB), and Cyc1 (lane 2). Molecular mass markers are indicated in kDa.

FIGURE 6.

EPR spectra in the region of the g_z peak recorded on enriched cytochrome Cyc2 from A. ferrooxidans. Proteins are in buffer B, without any treatment (spectrum A) and reduced with 2 mm FeSO₄ (spectrum B). Instrument settings were as follows: temperature, 15 K; microwave frequency, 9.47 GHz; microwave power, 64 milliwatts; modulation amplitude, 1.72 mT.

FIGURE 7.

Protein-protein interactions within the supercomplex analyzed by far-Western blotting experiments. Purified prey proteins (Cyc1, Cyc2, RcY, ORF1, and cyt c oxidase, 10 μg each) were separated on a 12.5% denaturing gel and transferred on nitrocellulose membrane, incubated with purified RcY (A) and Cyc1 (B). After washing, bound proteins were detected with anti-RcY antibody (A) and anti-Cyc1 (B) antibody. Bovine serum albumin (BSA) serves as a negative control and bait proteins (RcY and Cyc1) as positive controls. The arrowheads point to extra bands corresponding to the binding of RcY to Cyc1 and to Cyc2 (A) and the binding of Cyc1 to ORF1, to RcY, and to subunit II (CoxB) of cyt c oxidase (B). Molecular mass markers are indicated in kDa.

FIGURE 8.

Structural organization of metalloproteins involved in downhill pathway (from molecular Fe(II) to O₂) in the A. ferrooxidans iron respiration model. This representation, based on the proteins present in the supercomplex as well as on data from literature, takes no account of the stoichiometry of the various proteins. Dashed arrows indicate that electron transfer needs to be demonstrated. •, copper; ♦, Fe/S cluster; +, heme; Q, quinone.