Effect of uncouplers on endogenous respiration and ferrous iron oxidation in a chemolithoautotrophic bacterium Acidithiobacillus (Thiobacillus) ferrooxidans

Oxidation of ferrous iron (Fe2+) to ferric iron (Fe3+) with oxygen (O2) by Acidithiobacillus (Thiobacillus) ferrooxidans is considered to be inhibited by uncouplers. Oxidation of the endogenous substrates (presumably NADH) with O2 or Fe3+, on the other hand, was stimulated by uncouplers, 2,4-dinitrophenol (DNP) and carbonylcyanide-m-chlorophenyl-hydrazone (CCCP), as expected in respiratorily controlled mitochondria or heterotrophic bacteria. Amytal and rotenone were inhibitory. Fe3+ reduction by endogenous substrates was studied extensively and was found to be stimulated by a permeable anion, SCN- and weak acids, as well as the above uncouplers. Proton translocating properties of some of these stimulators were shown by following a pH change in the cell suspension. It was concluded that any compounds that destroy proton electrochemical gradient, Deltap, stimulated endogenous respiration. Stimulation of Fe2+ or ascorbate oxidation by lower concentrations of uncouplers was successfully demonstrated by shortening the reaction time, but only to a small extent. Uncouplers at concentrations stimulatory to endogenous respiration inhibited Fe2+ oxidation if present before Fe2+ addition. The inhibition by 10 microM CCCP was reversed by washing the cells in a buffer. Complex I inhibitors, atabrine, rotenone and amytal inhibited Fe2+ oxidation, more strongly in the presence of 0.1 mM DNP. It is proposed that Fe2+ oxidation required Deltap perhaps to climb an energetically uphill reaction or to reduce NAD+ to NADH by reversed electron flow for CO2 fixation. The latter interpretation implies some obligatory coupling between Fe2+ oxidation and NAD+ reduction.