The Gram-negative acidophile Acidithiobacillus ferrooxidans is capable of exocellular iron reduction through an electron transfer chain that spans both membranes and the periplasm. Four redox proteins are required to transfer electrons to Fe(III): (i) the inner membrane-anchored c-type cytochrome CycA, (ii) the periplasmic c-type cytochrome Cyc1, (iii) the periplasmic blue copper protein rusticyanin (Rus), and (iv) the outer membrane c-type cytochrome Cyc2. To investigate the Fe(III) reduction functionality of this pathway in neutrophilic hosts, it was reconstructed in three different Escherichia coli strains and Vibrio natriegens Vmax X2. The periplasmic proteins Rus and Cyc1 were only produced as membrane-associated proteins in E. coli and no holo-CycA was detected in E. coli cell extracts. V. natriegens produced all four holo-proteins simultaneously while the periplasmic proteins Rus and Cyc1 remained soluble. UV/Vis spectra of soluble cell extracts of V. natriegens showed typical absorbance maxima for heme c. The c-type cytochromes produced by V. natriegens could also be reduced and oxidized. Therefore, V. natriegens seems to be a very promising host for the production of soluble periplasmic as well as membrane-anchored proteins from an extreme acidophile.