First evidence for existence of an uphill electron transfer through the bc(1) and NADH-Q oxidoreductase complexes of the acidophilic obligate chemolithotrophic ferrous ion-oxidizing bacterium Thiobacillus ferrooxidans

Abstract

The energy-dependent electron transfer pathway involved in the reduction of pyridine nucleotides which is required for CO(2) fixation to occur in the acidophilic chemolithotrophic organism Thiobacillus ferrooxidans was investigated using ferrocytochrome c as the electron donor. The experimental results show that this uphill pathway involves a bc(1) and an NADH-Q oxidoreductase complex functioning in reverse, using an electrochemical proton gradient generated by ATP hydrolysis. Based on these results, a model is presented to explain the balance of the reducing equivalent from ferrocytochrome c between the exergonic and endergonic electron transfer pathways.

FIG. 1

(A) Oxidation of ferrocytochrome c (cyt. c) (10 μM) measured at 550-540 nm with T. ferrooxidans spheroplasts (spher.). Effects of the various bc₁ (antimycin, HQNO, funiculosin, myxothiazol, and stigmatellin) and NDH-1 (rotenone, amobarbital, and atabrine) complex inhibitors (inhib.) and of KCN addition are shown. (B) Oxidation of ferrocytochrome c (10 μM) measured at 550-540 nm with T. ferrooxidans spheroplasts in the presence of KCN. Effects of protonophores (CCCP and DNP), oligomycin, and ATP are shown. The protein (4 mg/ml) was suspended in 20 mM β-alanine–H₂SO₄ buffer, pH 3.5. See the text for inhibitor, ATP, and uncoupler concentrations. A, absorbance; s, seconds.

FIG. 2

The uphill electron transfer chain in T. ferrooxidans. Effects of the various bc₁ complex inhibitors on the oxidation of ferrocytochrome c (cyt. c) in spheroplasts (spher.), in the presence of KCN, are shown. (A) Q_n site inhibitors; (B) Q_p site inhibitors. Experimental conditions are as described in the legend to Fig. 1 and in the text. Protein concentration, 5 mg/ml. A, absorbance; s, seconds.

FIG. 3

The uphill electron transfer chain in T. ferrooxidans. Effects of the various NDH-1 complex inhibitors on the oxidation of ferrocytochrome c (cyt. c) in spheroplasts (spher.), in the presence of KCN, are shown. Experimental conditions are as described in the legend to Fig. 1 and in the text. Protein concentration, 5 mg/ml. A, absorbance; s, seconds.

FIG. 4

Model for the balance of reducing equivalents from ferrocytochrome c between the exergonic cytochrome oxidase and the endergonic bc₁ and NDH-1 pathways. The Q_p (center P) and Q_n (center N) sites are defined in the text.