Oxidation of endogenous substrates and ferrous iron in a chemoautotrophic bacterium Acidithiobacillus (Thiobacillus) ferrooxidans

Effects of uncouplers and electron transport inhibitors have been studied on the oxygen-coupled oxidation of Fe2*, endogenous substrates and formic acid by Acidithiobacillus ferrooxidans.Fe2+ oxidation was slightly stimulated by low concentrations of uncouplers within one minute indicating a normal respiratory control during Fe2+ oxidation, although higher concentrations of uncouplers were inhibitory.Complex I inhibitors rotenone, amytal and atabrine, and complex III inhibitors Abstract antimycin A, myxothiazol and2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), partially inhibited Fe2+ oxidation and the reduced activities by these inhibitors were stimutated by an uncoupler Z,4-dinitrophenol or carbonyl cyanide ru-chlorophenylhydrazone with the exception of atabrine-reduced activity.These results support the interpretation that some electrons were flowing along the uphill (endergonic) electron transport pathway to reduce NAD* during Fe2* oxidation.Complex I inhibitor piericidin A showed no effect on Fe2* oxidation and the reduced activity of Fe2* oxidation by atabrine could be further inhibited by the addition of an uncoupler.Some organic compounds such as ascorbic acid, propyl gallate (PG), salicylhydroxamic acid (SHAM), L-cysteine, glutathione and tiron can be oxidized by A. ferrooxidans using the Fe2*-oxidizing system.These compounds could chemically reduce Fe3* to Fe2* and their oxidation by the cells was greatly stimulated by the addition of FeCls.Their oxidation was also not sensitive to the inhibition by piericidin A but was inhibited by atabrine and the reduced activities by atabrine can also be further inhibited by an uncoupler.Other inhibitors and uncouplers showed qualitatively similar but quantitatively different effects on the oxidation of these organic compounds compared to those on Fe2* oxidation... 167 2-5 Effect of thiol agents NEM, HgCl2 and ANO on endogenous respiration ...... 168 2-6 Effect of arsenate (NazHAsOa) and its combination with KCN or rotenone on endogenous respiration ... .. 169 2-7 Complex I inhibitors on endogenous respiration . .... .. 169 2-8 Effect of KCN concentration on endogenous respiration ..... ...... I70 2-9 Effect of KCN on endogenous respiration in the presence of DNP ..... . . 170 2-10 Effect of KCN and chloramphenicol (Ch, 0.31 mM) on endogenous respiration in the presence and absence of CCCP ....... n 1 2-LlEffect of KCN and NaN3 on endogenous respiration in the presence of FeCl:... 171 2-12 Effect of NaN on endogenous respiration ... ....112 2-13 Effect of HQNO on endogenous respiration in 0.1 M F-alanine-HzSO buffer (a) and water (b) ... ...173 xix t64 r65 r66 r66 2-L4 Effect of HQNO and CCCP on endogenous respiration in the absence and presence of KCN and rotenone at pH 3.5 (a, b) .... .'.'..'..'...174 2-L5 Comparison of the effects of HQNO and CCCP on endogenous respiration in the presence of NaNs .. ... . . 17 52-16 Effect of TTFA on endogenous respiration in the absence and presence ofotherreagents ..115 2-17 Effect of ferrozine, oxalic acid and NaPzOz on endogenous respiration ..... .... 116 2-L8 Effect oftiron on endogenous respiration in the absence and presence of o-phenanthroline(O-phen.),2,2'-dipyridyl,DNPandrotenone'...176 2-19 Effect of SHAM on endogenous respiration in the absence and presence of rotenone, amytal, KCN, o-phenanthroline (O-phen.) and 2,2'-dipyridyt ... .... 177 2-20 Effect of SHAM on endogenous respiration in the absence and presence of (a) HQNO and KCN and (b) CCCP and KCN ........ 178 2-21 Effect of propyl gallate (PG) on endogenous respiration in the absence and presence of KCN or o-phenanthroline (O-phen.)..... I79 2-22 External pH change during endogenous respiration in cells of 100 mg / n[,... 180 2-23 External pH change during endogenous respiration in cells of 20 mg I mI' in the presence of different compounds . . . .... ' .180 2-24 02 consumption and CO2 production in the absence and presence of differentcompounds ........ 181 2-25 Effect of KCN and NaN: on Fe3* reduction by endogenous substrates atpH3.5... ............ 185 2-26 Flffect of amytal on Fe3* reduction by endogenous substrates at (a) pH2.3 and (b) pH 3.5 .......... 186 2-27 Effect of rotenone on Fe3* reduction by endogenous substrates at (a) pH 2.3 and (b) pH 3.5....187 2-28 F;ffect of piericid A (PA, 0.5 pM) and combination of PA, 0.1 mM rotenone (R) and 2 mM amytal (Am) on Fe3* reduction byendogenoussubstratesatpH3.5... .. 188 2-29 Effect of atabrine on Fe3* reduction by endogenous substrates at pH 2.3 and pH 3.5 .. 188 2fi0 Effect of antimycin A on Fe3* reduction by endogenous substrates at pH 3.5 ...189 2-31 Effect of myxothi azol, on Fe3+ reduction by endogenous substrates at pH 2.3 and pH 3.5 ... 189 2fl2 Effectof HQNO onFe3*reductionbyendogenous substrates.... ......... 190 2-33 Effect of TTFA on Fe3* reduction by endogenous substrates at pH 3.5 .......... 191 2-34 Effect of CCCP on Fe3* reduction by endogenous substrates at (a) pH 2.3 and (b) pH 3.5 ... ...192 2-35 Effect of DNP on Fe3* reduction by endogenous substrates at(a)pH2.3and(b)pH3.5... ........193 2-36 Effect of nigericin on Fe3* reduction by endogenous substrates in the absence and presence of complex I inhibitors at pH 3.5 ........ 194 2-37 Effect of valinomycin on Fe3* reduction by endogenous substrates in the absence and presence of complex I inhibitors at pH 3.5 .. I94 2-38 Effect of KSCN on Fe3* reduction by endogenous substrates at (a) pH 2.3 and (b) pH 3.5 ... ..195 239 Flffect of CHgCOOK on Fe3* reduction by endogenous substrates in the absence and presence of complex I inhibitors at pH 3.5 ....... 196 2-40 Effect of KF on Fe3* reduction by endogenous substrates in the absence and presence of complex I inhibitors at pH 3.5 ... 196 2-41 F;ffect of Na2-succinate and Naz-malonate on Fe3* reduction by endogenous substrates at pH 3.5 ... .... I97 2-42 Effect of NEM on Fe3* reduction by endogenous substrates in the absence and presence of complex I inhibitors at pH 3.5 ..... 198 2-43 Effect of HgCl2 and AgNO3 on Fe3+ reduction by endogenous substrates at pH 2.3 and pH 3.5 ..... 198 2-44 Effect of inhibitors of ATP synthase, DCCD (a) and oligomycin (b), on Fe3* reduction by endogenous substrates at pH 2.3 andpH 3.5 ..... g 2-45 Proposed model for electron transport pathways of oxidation of endogenous substrates in A. fetooxians .. .. 200 XXl oxidation of Fe2* (Beck and Shafia 1964;Ingledew et al.1917).Because the oxidation of Fe2* involves ATP generation (Adapoe and Silver 1975;Elbehti et al.Z}}};Ingledew 1982; Mignone and Donati 2004) and because of the controversy about the H* pumping during Fe2+ oxidation (Elbehti et al. 2000; Ingledew et aI.1977).the effect of uncouplers, CCCP and DNP, were re-investigated to see if a normal respiratory control exists during Fe2* oxidation.Elbehti et al. (2000) indirectly showed the existence of an uphill reaction from cytochrome c to NDH-I (NAD* reduction) in A. ferrooxidans (see Fig. 6) by testing the oxidation of external reduced cytochrome c afte..r cytochrome c oxidase was inhibited by KCN.However, no evidence has been reported to show electron transport along the uphill electron transport pathway to reduce NAD* during oxidation of Fe2* or other substrates used by this organism.This investigation has attempted to present such evidence by studying the effects of different electron transport inhibitors.

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