Purification and Some Properties of Sulfur:Ferric IonOxidoreductase from Thiobacillus ferrooxidans
TSUYOSHI SUGIO,* WATARU MIZUNASHI, KENJI INAGAKI, AND TATSUO TANODivision ofBiological Function and Genetic Resources Science, Faculty ofAgriculture, Okayama University,
1-1-1 Tsushima Naka, Okayama 700, JapanReceived 12 February 1987/Accepted 27 June 1987
A sulfur:ferric ion oxidoreductase that utilizes ferric ion (Fe3+) as an electron acceptor of elemental sulfurwas purified from iron-grown Thiobacilus ferrooxidans to an electrophoretically homogeneous state. Underanaerobic conditions in the presence of Fe3+, the enzyme reduced 4 mol of Fe3+ with 1 mol of elemental sulfurto give 4 mol of Fe2' and 1 mol of sulfite, indicating that it corresponds to a ferric ion-reducing system (T.Sugio, C. Domatsu, 0. Munakata, T. Tano, and K. Imai, Appl. Environ. Microbiol. 49:1401-1406, 1985).Under aerobic conditions, sulfite, but not Fe2+, was produced during the oxidation of elemental sulfur by thisenzyme because the Fe2+ produced was rapidly reoxidized chemically by molecular oxygen. The possibility thatFe3+ serves as an electron acceptor under aerobic conditions was ascertained by adding o-phenanthroline,which chelates Fe2 , to the reaction mixture. Sulfur:ferric ion oxidoreductase had an apparent molecularweight of 46,000, and it is composed of two identical subunits (Mr = 23,000) as estimated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Sulfur oxidation by this enzyme was absolutely dependent on thepresence of reduced glutathione. The enzyme had an isoelectric point and a pH optimum at pH 4.6 and 6.5,respectively. Almost all the activity of sulfur:ferric ion oxidoreductase was observed in the osmotic shock fluidof the cells, suggesting that it was localized in the periplasmic space of the cells.
The iron-oxidizing bacterium Thiobacillus ferrooxidans isone of the most important microorganisms for the bacterialleaching of sulfide ores and for the cycling of inorganic sulfurcompounds and iron in natural environments. These abilitiesof T. ferrooxidans are due to their possession of uniqueenzyme systems that can oxidize both Fe2" and reducedsulfur compounds.The mechanism of sulfur oxidation of T. ferrooxidans had
been considered similar to that of other thiobacilli (3, 11, 12,22). We recently reported the presence of a ferric ion-reducing (FIR) system that catalyzes the reduction of Fe3+with elemental sulfur in the pure strain of T. ferrooxidansAP19-3, and proposed an alternative sulfur oxidation routethat is composed of both the FIR and iron-oxidizing systems(13). This proposition was strongly supported by the produc-tion of Fe2' as an intermediate during the aerobic oxidationof elemental sulfur by this strain (15). The involvement of theFIR system in sulfur oxidation was further supported bygrowth inhibition of this strain with heavy metals. Cupric ion(Cu2+) competed on the active site of the FIR system withFe3+, which is absolutely required as an electron acceptor ofelemental sulfur, and this competitive inhibition was sug-gested to be a main cause of the inhibition of sulfur oxidationby Cu2+ (19). Also, intact cells of this strain incubatedaerobically with elemental sulfur at a pH at which the ironoxidase of the cells barely operates (above pH 5.0) produceda large amount of sulfite, an intermediate of sulfur oxidationby the FIR system (16).
Sulfur:oxygen oxidoreductase, which utilizes molecularoxygen as an electron acceptor of elemental sulfur, waspartially purified from Thiobacillus thiooxidans (20) andThiobacillus thioparus (21). However, there have been noreports on the purification of an enzyme that is involved insulfur oxidation by T. ferrooxidans. This work shows thepurification of sulfur:ferric ion oxidoreductase, which plays
* Corresponding author.
a crucial role in sulfur oxidation of this strain, from iron-grown T. ferrooxidans AP19-3. It may be the first sulfur-oxidizing enzyme to be purified to an electrophoreticallyhomogeneous state from the iron-oxidizing bacterium T.ferrooxidans.
MATERIALS AND METHODSMicroorganism, medium, and conditions of cultivation. T.
ferrooxidans AP19-3 (14) was used throughout this study.The composition of a 10 times concentrated basal saltssolution was as follows: (NH4)SO4, 30 g; KCl, 1 g; K2HPO4,5 g; MgSO4 7H20, 5 g; Ca(NO3)2, 0.1 g; deionized water,1,000 ml; and concentrated H2SO4, 25 ml. Iron-salts mediumwas prepared by adding 1 liter of a 10 times concentratedbasal salts solution, 8 liters of deionized water, and 300 g ofFeSO4 7H20 to a 10-liter carboy. An active culture ofiron-salts-grown T. ferrooxidans AP19-3 (1 liter) was inocu-lated into the 9 liters of the iron-salts medium describedabove and cultured under aeration at 28°C for 144 h. Theculture from six carboys (ca. 60 liters) was filtered with Toyofilter paper (no. 2) to remove the bulk of ferric precipitatesand centrifuged with a Hitachi 18PR-52 continuous flowrotor (15,000 x g, with a flow rate of 200 ml/min) to yield ca.0.35 g of cell protein. Harvested cells were washed threetimes with 0.1 M P-alanine-SO42- buffer (pH 3.0) and threetimes with 0.1 M sodium phosphate buffer (pH 7.5).
Activity of sulfur:ferric ion oxidoreductase. The activity ofthe sulfur:ferric ion oxidoreductase that catalyzes the pro-duction of sulfite and Fe2+ from elemental sulfur and Fe3"was determined by measuring either sulfite or Fe2+ producedin the reaction mixture. Ferrous ion was determined colori-metrically by a modification of the o-phenanthroline methodas previously described (10). Sulfite was determined colori-metrically by the pararosaniline method (24).
Since Fe2+ produced by sulfur:ferric ion oxidoreductasewas rapidly oxidized under aerobic conditions at pH 6.5, thedetermination of Fe2+ in the reaction mixture was performed
SULFUR:FERRIC ION OXIDOREDUCTASE IN T. FERROOXIDANS
TABLE 1. Summary of purification procedure for sulfur:ferricion oxidoreductase from iron-grown T. ferrooxidans AP19-3
~~~~Total Total Sp acta~ Yield
Step protein activity (U/mg) ()(mg) (U)
Cell extract 744.3 7,443 10.0 100.0105,000 x g supernatant 364.5 7,946 21.8 106.8(NH4)2SO4 (O to 40o) 111.8 7,222 64.6 97.0Sephadex G-100 15.5 5,595 361.0 75.2FPLC (Mono Q)b 1.1 1,121 1,019.8 15.0PAGE-gel cutc 0.1 154.3 1,543.2 2.0
a The activity of sulfur:ferric ion oxidoreductase was determined by mea-suring sulfite produced in the reaction mixture under aerobic conditions at pH6.5 by the methods described in Materials and Methods.
b Purification with an anion-exchange resin or Mono Q column (0.5 by 5 cm)of a Pharmacia FPLC system.
c After PAGE, the enzyme fraction on the gel was cut off and extracted with0.1 M sodium phosphate buffer (pH 6.5) by the method described iti Materialsand Methods.
under anaerobic conditions in an atmosphere of nitrogen.The reaction mixture contained 4 ml of 0.05 M P-alanine (pH6.5), enzyme, 0.2 mg of bovine serum albumin, 100 mg ofelemental sulfur, and 20 ,umol of reduced glutathione (GSH;adjusted to pH 6.5 with dilute NaOH). All the componentsexcept GSH were put into a 30-ml flask. The reactionmixture was bubbled with nitrogen through a glass capillarytube for 15 min before the addition of GSH. The flask wasshaken vigorously for 10 s to mix the GSH, and the reactionwas carried out at 30°C with shaking.Under aerobic conditions, the activity of sulfur:ferric ion
oxidoreductase was measured by sulfite production in areaction mixture containing 4 ml of 0.1 M sodium phosphatebuffer (pH 6.5), enzyme, 0.2 mg of bovine serum albumin,100 mg of elemental sulfur, and 20 ,umol ofGSH (adjusted topH 6.5 with dilute NaOH). One unit of activity was definedas 1 nmol of sulfite produced in 1 ml of the reaction mixtureper mg of protein per min. Specific activity of sulfur:ferric ionoxidoreductase was defined as units per milligram of protein.
Activity of acid phosphatase. The activity of acid phospha-tase was determined by measuring the production ofp-nitro-phenol spectrophotometrically. The reaction mixture was asfollows: 4.0 ml of 0.1 M sodium acetate buffer (pH 4.3);disodium p-nitrophenylphosphate, 20 ,umol; enzyme. Totalvolume was 5.0 ml. A sample of the reaction mixture (1.0 ml)was centrifuged at 10,000 x g for 2 min, and the supernatantsolution (0.5 ml) was added to 2.5 ml of 0.1 M NaOH. Theyellow color developed was measured with a ShimadzuUV-140 spectrophotometer at 400 nm.
Purification of sulfur:ferric ion oxidoreductase. All steps ofthe purification process were done at 4°C unless indicatedotherwise. Iron-grown cells of T. ferrooxidans AP19-3washed three times with 0.1 M sodium phosphate buffer (pH7.5) were disrupted by passage two times through a Frenchpressure cell at 1,500 kg/cm2 and centrifuged at 12,000 x gfor 20 min. The supernatant solution (crude cell extract) wasfurther centrifuged at 105,000 x g for 60 min to obtain anorange supernatant solution (105,000 x g supernatant). Solidammonium sulfate was slowly added to the 105,000 x gsupernatant to give 40% saturation. The resulting precipitatewas collected by centrifugation at 10,000 x g for 20 min,redissolved in a minimal volume of 0.1 M sodium phosphatebuffer (pH 7.5), and dialyzed against 2 liters of the samebuffer for 24 h. A red-brown solution from the previous stepwas subsequently applied to a column (1.6 by 100 cm) ofSephadex G-100 equilibrated with the same buffer.An active fraction from the previous step was further
purified with a Pharmacia FPLC system. The active fractionwas dialyzed with 20 mM Tris hydrochloride buffer (pH 7.5),applied to an anion-exchange resin or Mono Q column (0.5by 5 cm) equilibrated with the same buffer, and then elutedwith a linear gradient of NaCl from 0 to 0.25 M (33 nml). Theactive fraction from a Mono Q column showed four proteinbands on the gel from a polyacrylamide disc gel electropho-retic. The part of the gel (5 mm) containing sulfur:ferric ionoxidoreductase was cut out, frozen at -20°C, disrupted in0.1 ml of 0.1 M sodium phosphate buffer (pH 6.5), andcentrifuged at 12,000 X g for 10 min. The supernatantsolution thus obtained (PAGE-gel cut) was stored at -20°C.
Molecular weight determination. The molecular weight ofthe native enzyme was determined by Sephadex G-100column (1.0 by 100 cm) chromatography by the method ofAndrews (1). The subunit molecular weight was determinedby sodium dodecyl sulfate-PAGE by the method of Weberand Osborn (23).
Protein content. The protein content was determined bythe method of Lowry et al. (5), with crystalline bovine serumalbumin as the reference protein.
RESULTS
Properties of sulfur:ferric ion oxidoreductase. Sulfur:ferricion oxidoreductase was purified from iron-grown T. fer-rooxidans AP19-3 as described in Materials and Methods.Results of a typical purification are summarized in Table 1.The procedure gave 154-fold purification over the crude cellextract. The purified enzyme was homogeneous by thecriterion of PAGE and showed a specific activity of 1,543.2U/mg of protein (Fig. 1; Table 1). The apparent molecularweight of sulfur:ferric ion oxidoreductase was 46,000 bySephadex G-100 column chromatography (Fig. 2). Only oneband was observed at a molecular weight of 23,000 on the gelfrom sodium dodecyl sulfate-PAGE (Fig. 3), indicating thatthe enzyme was composed of two subunits of the samemolecular weight. The enzyme had an isoelectric point at pH4.6 (data not shown).The properties of sulfur:ferric ion oxidoreductase (except
molecular weight and subunit structure) were examined withan enzyme solution at the stage of Mono Q column chronma-tography because the hornogeneous enzyme at the stage ofPAGE-gel cut (specific activity of 1543.2 U/mg of protein)
Sulfur: ferric ionoxidoreductase
+ - Dye
FIG. 1. PAGE of a purified sulfur:ferric ion oxidoreductase. Discgel electrophoresis was done in 7.5% polyacrylamide gel at pH 9.4 at3 mA per tube for 1.5 h. Purified sulfur:ferric ion oxidoreductase (3,ug of protein) was placed on the gel.
Elution volume (ml)FIG. 2. Determination of molecular weight of sulfur:ferric ion
oxidoreductase by Sephadex G-100 gel filtration. Elution was donewith 0.1 M sodium phosphate buffer, pH 7.5. Abbreviations: A,phosphorylase b (94,000 molecular weight); B, albumin (67,000); C,sulfur:ferric ion oxidoreductase (46,000); D, ovalbumin (43,000); E,carbonic anhydrase (30,000); F, trypsin inhibitor (20,100); G, a-lactalbumin (14,400).
could not be obtained in sufficient quantities. Enzyme solu-tion at the stage of Mono Q column chromatography (spe-cific activity, 1019.8 U/mg of protein) showed one main bandand three faint bands on the gel from PAGE, and the activitywas observed only in the main band.Under anaerobic conditions in the presence of Fe3" at pH
6.5, the enzyme reduced 4 mol of Fe3+ with 1 mol ofelemental sulfur to give 4 mol of Fe2' and 1 mol of sulfite(Fig. 4). The results indicate that sulfur:ferric ion oxidore-ductase catalyzed the reaction of equation 1 proposed for thesulfur oxidation in T. ferrooxidans AP19-3 (13) (S0 + 4Fe3++ 3H20 -O H2SO3 + 4Fe2+ + 4H+). From the results, weconcluded that sulfur:ferric ion oxidoreductase correspondsto an FIR system (13).
ABCDFG
(0~
Q,.0
A
I 3L0
va,LI
0_
0.-cL -
en EN _~
0
0LI)
G
0G25 0.50 0.75Rf
FIG. 3. Determination of molecular weight of sulfur:ferric ionoxidoreductase by SDS-PAGE. SDS-PAGE was done in a 10%polyacrylamide gel at 8 mA per tube. Purified sulfur:ferric ionoxidoreductase (3 p.g of protein) was placed on the gel (E). Abbre-viations: A, phosphorylase b (94,000 molecular weight); B, albumin(67,000); C, ovalbumin (43,000); D, carbonic anhydrase (30,000); E,sulfur:ferric ion oxidoreductase (23,000); F, trypsin inhibitor(20,100); G, at-lactalbumin (14,400).
E
0E
MIV10Go
03
0
0.1
0
EI
0
10
La
Time (m;n)
FIG. 4. Production of ferrous ion and sulfite by anaerobic oxida-tion of elemental sulfur by sulfur:ferric ion oxidoreductase. Thecomposition of the reaction mixture and the method for analysis aredescribed in the text. Sulfur:ferric ion oxidoreductase purified at thestage of Mono Q column chromatography (30 ,ug of protein) wasused in the reaction. Symbols: 0, production of Fe2"; U, productionof sulfite.
Under aerobic conditions at pH 6.5, sulfite, but not Fe2+,was produced during the oxidation of elemental sulfur bythis enzyme because any Fe2+ produced was rapidly reoxid-ized by molecular oxygen chemically (Fig. 5). After 20 min,thiosulfate appeared in the reaction mixture because anexcess of elemental sulfur in the reaction mixture reactedchemically with sulfite produced by sulfur:ferric ion oxido-
E
CEc
a,
130
0.Na,
f4Ld
Time (min)FIG. 5. Ferrous ion, sulfite, and thiosulfate formation by the
aerobic oxidation of elemental sulfur by sulfur:ferric ion oxidore-ductase. The composition of the reaction mixture and the methodfor analysis are described in the text. Enzyme purified at the stage ofMono Q column chromatography (30 ,ug of protein) was added to thereaction mixture. Symbols: A, production of Fe2" by native en-zyme; *, production of sulfite by native enzyme; 0, production ofsulfite by enzyme boiled for 15 min; *, production of thiosulfate bynative enzyme; O, production of thiosulfate by enzyme boiled for 15min.
SULFUR:FERRIC ION OXIDOREDUCTASE IN T. FERROOXIDANS
5
C0 E9o f_
-0~0
a E0
fN 0
c
4
3
2
1 2 3 4 5
Sulfur: ferric ion ox;doreductase
(pgl ml )
FIG. 6. Aerobic production of ferrous ion by sulfur:ferric ionoxidoreductase. The reaction mixture contained 4.0 ml of 0.1 Msodium phosphate buffer (pH 6.5), 0 to 20 j±mol of enzyme purifiedat the stage of Mono Q column chromatography, 20 xg of GSH(adjusted to pH 6.5 with diluted NaOH), 100 mg of SI, 0.2 mg ofbovine serum albumin, 0.5 ,umol of Fe3", and 1 ,umol of o-phenanthroline. Total volume was 5.0 ml. Symbols: 0, completereaction mixture; A, without Fe3+; *, without o-phenanthroline.
reductase. Suzuki and Silver (21) also observed thiosulfateproduced chemically during the oxidation of elemental sulfurby a partially purified sulfur:oxygen oxidoreductase. Sincesulfur:ferric ion oxidoreductase did not decompose thiosulf-ate, the thiosulfate accumulated in the reaction mixture. Thepossibility that Fe3+ serves as an electron acceptor underaerobic conditions was ascertained by adding o-phen-anthroline to the reaction mixture to trap Fe2+ producedduring the sulfur oxidation (Fig. 6). Ferrous ion was not
C
0.
E
0EV.-
as
V
.00.0-
14)
0.2
0.1
0
observed in those reaction mixtures without Fe3" and o-phenanthroline.The activity was completely destroyed by heating the
enzyme solution in a water bath at 69°C for 10 min. Theenzyme was unstable, especially when it was diluted mark-edly, so bovine serum albumin was incorporated in thereaction mixture at a concentration of 40 ,ug/ml. The activitywas absolutely dependent on GSH (Fig. 7). The enzyme hada pH optimum at 6.5, and no activities were observed belowpH 4.5 or above pH 8.5 (Fig. 8).
Localization of sulfur:ferric ion oxidoreductase in T. fer-rooxidans AP19-3 cells. Intact T. ferrooxidans AP19-3 cells(100 mg of protein) were suspended in 2 ml of 50 mM Tris-10mM sodium EDTA (pH 7.5), with or without 20% sucrose,and incubated for 4 h at 30°C. The cell suspensions wererapidly poured into 100 ml of ice-cooled water, gently stirredfor 30 min, and then centrifuged at 10,000 x g for 15 min.The supernatant solutions were concentrated and assayedfor sulfur:ferric ion oxidoreductase and for acid phospha-tase. Both activities were found in the osmotic shock fluidsobtained by treating the cells with 20% sucrose, but couldnot be detected in the control fluids obtained by treating thecells in the same manner without 20% sucrose (Fig. 9). Theresults strongly suggest that sulfur:ferric ion oxidoreductasewas present in the periplasmic space of the strain.The following results also support the presence of
sulfur:ferric ion oxidoreductase in the periplasmic space ofthe strain. Treatment of intact cells with 1 mg/ml each ofpepsin, protease, pronase E, zymolyase, chitinase, lyso-zyme, amylase, lipase, phospholipase C, or phospholipaseA2 for 1 h did not affect the activity of either the FIR system(sulfur:ferric ion oxidoreductase) or the iron-oxidizing sys-tem (data not shown), indicating that the enzyme was notpresent on the outer surface of the outer membrane. Intactcells were treated with or without 1% phenol for 10 min andwashed three times with 0.1 M 1-alanine-SO42- buffer (pH3.0) to remove the phenol, and then the activities of iron
1001
501401,
to
GD
'aGa:
0
20 40 60 80
GSH added (yjmol )FIG. 7. Effect of GSH on the activity of sulfur:ferric ion oxido-
reductase. The activity was determined by measuring sulfite aero-
bically as described in the text. Enzyme purified at the stage ofMono Q column chromatography (30 ,ug of protein) was used in thereactions.
4
pHFIG. 8. Effect of pH on the activity of sulfur:ferric ion oxidore-
ductase. The activity was determined by measuring sulfite producedunder aerobic conditions as described in the text. The buffer used inthe pH curve was 0.1 M sodium phosphate buffer. Enzyme purifiedat the stage of Mono Q column chromatography (30 ,ug of protein)was used in the reactions.
T;ime(min) Time ( m;n)FIG. 9. Activities of sulfur:ferric ion oxidoreductase (A) and
acid phosphatase (B) in osmotic shock fluid. Osmotic shock fluidwas prepared by treating intact cells with 50 mM Tris-10 mmsodium EIDTA (pH 7.5) with (0) or without (U) 20% sucrose by themethod described in the text. The activities of sulfur:ferric ionoxidoreductase and acid phosphatase were measured. The proteinconcentration in the assays was 0.094 mg/ml.
oxidase, the FIR system, and cytochrome c oxidase weredetermined. The FIR system (sulfur:ferric ion oxidoreduc-tase) of phenol-treated cells was completely destroyed. Incontrast, the activities of iron oxidase and cytochrome coxidase remained at 38 and 65%, respectively, after thephenol treatment (data not shown). The iron-oxidizing sys-tem and cytochrome c oxidase were considered present inthe inner membrane of T. ferrooxidans, and a copper-containing protein which stimulates both activities seemedto be loosely bound to the outer side of inner membrane (17,18). Since the FIR system was destroyed competely beforethe activities of iron oxidase and cytochrome c oxidase werelost, the FIR system seems to be located outside of theiron-oxidizing system.
DISCUSSION
Intact cells of T. ferrooxidans AP19-3 possess an FIRsystem that catalyzes the reduction of Fe3+ with elementalsulfur under anaerobic conditions (13). The rate and amountof Fe3+ reduction in T. thiooxidans were very much lowerthan those of T. ferrooxidans (13), suggesting that the FIRsystem does not operate in the sulfur oxidation of T.thiooxidans. In the present work, sulfur:ferric ion oxidore-ductase of T. ferrooxidans AP19-3 was purified to anelectrophoretically homogeneous state, and the purified ho-mogeneous protein (specific activity, 1,543.2 U/mg of pro-
tein) could catalyze the reduction of Fe3+ by elementalsulfur. It was distinguished from the sulfur:oxygen oxidore-ductase of T. thiooxidans (20) and T. thioparus (21) by thefact that the former utilizes Fe3+, but the latter utilizesmolecular oxygen, as an electron acceptor of elementalsulfur. Both enzymes absolutely required GSH in the reac-
tion, suggesting that GSH is commonly utilized in an earlystep of sulfur oxidation reaction by a mechanism that isunknown.
Stoichiometric studies of anaerobic sulfur oxidation bythis enzyme showed that it catalyzed equation 1 proposedfor sulfur oxidation in T. ferrooxidans AP19-3 (13), indicat-ing that sulfur:ferric ion oxidoreductase corresponds to theFIR system. Under aerobic conditions, sulfite but not Fe2+was produced during the oxidation of elemental sulfur bythis enzyme because any Fe2+ produced was rapidly reoxid-ized chemically by molecular oxygen. The possibility thatFe3+ serves as an electron acceptor under aerobic conditionswas ascertained by adding o-phenanthroline to the reactionmixture to chelate Fe2+ produced by the sulfur oxidation.However, the amount of Fe2` produced under these condi-tions was low because the amount of sQluble Fe3+ availableas an electron acceptor was low at pH 6.5. Thus, underaerobic conditions at pH 6.5, both Fe3+ and molecularoxygen could be utilized as electron acceptors. However,Fe3+ seems to be a physiological electron acceptor in sulfuroxidation under these conditions since elemental sulfur wasoxidized by the intact cells to produce a large amount ofsulfite or an intermediate of sulfur oxidation in the reactionmixture (16).From the results obtained in this and previous work (13,
15, 16, 17), we propose the mechanism of sulfur oxidation inT. ferrooxidans AP19-3 to be that shown in Fig. 10. At thefirst step of sulfur oxidation, solid elemental sulfur may passthrough the outer membrane by a mechanism that remains tobe established. In the periplasmic space, sulfur:ferric ionoxidoreductase oxidizes elemental sulfur with 4 mol of Fe3+to give 1 mol of sulfite and 4 mol of Fe2 . Under physiolog-ical conditions the 4 mol of Fe2" thus produced is rapidlyoxidized by the iron-oxidizing system of the organism to give4 mol of Fe3+, and the Fe3+ can be utilized as an electronacceptor of elemental sulfur to complete a cyclical process.Under anaerobic conditions, the 4 mol of Fe2+ produced bysulfur:ferric ion oxidoreductase is leaked from the cells intothe medium (13). Sulfite produced by the enzyme may beoxidized chemically with Fe3+ to give additional Fe2+ (13).The proposed mechanism of sulfur oxidation in T. fer-rooxidans AP19-3 explains why sulfur-grown T. fer-rooxidans so often also has comparatively high iron-oxidizing activity (15).
Recently, Hooper and Dispirito (4) proposed that thegeneration of a proton gradient involves extracytoplasmicoxidation of a substrate in bacteria that grow on simplereductants. This can reasonably explain the generation ofcellular energy by chemolithotrophic bacteria via achemiosmotic coupling mechanism (6, 7, 8, 9). Their propo-sition suggests that the periplasmic environment ofchemolithotrophs must be protected from nonphysiologicalinvasion of protons from the exterior and leakage of protonsto the outside of the cells to maintain a proton gradientproduced by the oxidation of a simple substrate. The outermembrane or lipopolysaccharide layer of gram-negativechemolithotrophs may play an important role in retaining aproton gradient. Sulfur:ferric ion oxidoreductase waspresent in the periplasmic space of this strain. When elemen-tal sulfur is oxidized by this enzyme, six protons are pro-duced in the periplasmic space, which can give the cells aproton gradient. In this way, the proposition by Hooper andDispirito (4) on the generation of cellular energy inchemolithotrophic bacteria supports the sulfur oxidationmechanism shown in Fig. 10. No activity was observed atpH 4.5, but 36.1 and 53.7% of the activity was observed at
SULFUR:FERRIC ION OXIDOREDUCTASE IN T. FERROOXIDANS
Sulfur: ferric - ion coxidoreductase( Ferric-ion reducing system)
45 \4~4Fee;- 6Fe' 3 H20
2hmil r tronioxidisystem
3F2Fe 2*32z*6____ 2 * 2~~~~~ A Fe31 02 6H
SO4 2Fe
Chemical reaction
Otr Peptidog lycan ne4embrane MembraneFIG. 10. Proposed mechanism of sulfur oxidation of T. ferrooxidans AP19-3.
pH 5.5, and 6.0, respectively, suggesting that the periplasmicpH of this strain is above pH 4.5.
Corbett and Ingledew (2) also observed the reduction ofFe3+ by elemental sulfur in iron-grown T. ferrooxidansNCIB 8455. They showed that the reduction of Fe3+ byelemental sulfur with intact cells of T. ferrooxidans NCIB8455 was inhibited 50% by approximately 20 ,uM 2-heptyl-4-hydroxyquinoline N-oxide (HOQNO), which is a specificinhibitor of electron transfer in the cytochrome b-cl segmentof respiratory chains, and suggested that site 2 energyconservation in electron transfer reactions between So andeither Fe3+ or 02 occurs in T. ferrooxidans. Whether thecytochrome b-cl segment is present in the purified sulfur:fer-ric ion oxidoreductase (apparent molecular weight, 46,000)was not determined. However, the results obtained with apartially purified enzyme solution at the stage of Mono Qcolumn chromatography strongly suggest that cytochromeb-cl was not involved in reduction of Fe3" by elementalsulfur because the activity of sulfur:ferric ion oxidoreduc-tase was not inhibited by 40 ,uM HOQNO, but slightlystimulated (data not shown). The fact that sulfur:ferric ionoxidoreductase was easily solubilized in osmotic shock fluidwithout detergents also supports the view that cytochromeb-cl is not involved in the reduction of Fe3+ by elementalsulfur.
Isolation of an enzyme that plays a crucial role in sulfuroxidation of T. ferrooxidans to an electrophoretically homo-geneous state will open the way to make a geneticallyengineered microorganism that can be used for bacterialleaching of sulfide ores.
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15. Sugio, T., W. Mizunashi, T. Tano, and K. Imai. 1986. Produc-tion of ferrous ions as intermediates during aerobic sulfuroxidation in Thiobacillus ferrooxidans. Agric. Biol. Chem.50:2755-2761.
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