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.

Posttranslational modification and sequence variation of redox-active proteins correlate with biofilm life cycle in natural microbial communities

“…Mono-and diheme cytochromes have been shown to transfer electrons from rusticyanin to cytochrome c oxidase and cytochrome bc1 complex in A. ferrooxidans (Castelle et al, 2008) (Taha et al, 2008 (Figure 10b) (Ram et al, 2005). In late developmental-stage biofilms, the lowering of the redox potential of Cyt 579 may represent an alteration of the pathway to enable the use of different electron acceptors (Figure 10c).…”

“…Redox measurements and poising were performed in 10 mM NH 4 OAc pH 4.3 with 50 mM each of 1, 4-benzoquionone, N,N,N 0 ,N 0 -tetramethyl-p-phenylenediamine, ferrocenecarboxylic acid and 1,1 0 -ferrocenedicarboxylic acid used as redox mediators (Castelle et al, 2008). Concentrated solutions of sodium dithionite were used as the reductant and sodium hexachloroiridate was used as the oxidant.…”

“…Our working model positions Cyt 572 as the Fe(II) oxidase on the outer membrane of LeptoII cells, which oxidizes Fe(II) to Fe(III) and transfers electrons to Cyt 579 . This scheme is analogous to the proposed role of an outer membranebound c-type cytochrome, Cyc2, and rusticyanin, a periplasmic Cu protein, in Acidithiobacillus ferrooxidans, an acidophilic Fe(II)-oxidizing bacterium found in AMD environments (Castelle et al, 2008).…”

The Multicenter Aerobic Iron Respiratory Chain of Acidithiobacillus ferrooxidans Functions as an Ensemble with a Single Macroscopic Rate Constant

“…A preparation of "respirasomes" that included all four proteins in a stable super-complex was obtained from cell-free extracts of At. ferrooxidans (25). This respirasome was shown to catalyze the iron-dependent reduction of molecular oxygen.…”

New Functional Sulfide Oxidase-Oxygen Reductase Supercomplex in the Membrane of the Hyperthermophilic Bacterium Aquifex aeolicus

“…The blots were then processed and developed as described previously (21). Primary antibodies used were directed against Sqr from A. aeolicus or against subunit 2 (CoxB) of cytochrome c oxidase from the bacterium Acidithiobacillus ferrooxidans, which we have shown here to cross-react with the terminal oxidase of Aquifex.…”