Thermophilic iron reductases from thermus scotoductus
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A thermophilic bacterium, Thermus scotoductus, was previously isolated form hot, alkaline groundwater sampled in a South African gold mine. Thermus scotoductus, was grown microaerophically / anaerobically with Fe(III) citrate or KNO3 serving as the final electron acceptor and acetate as the potential electron donor. Subcellular fractionation indicated that the ferric reductase activity was located in both the soluble (cytoplasmic) and the membrane fraction. The major part of the activity present in the membrane fraction was found to be associated with the outer membrane. Both the soluble and membrane-associated ferric reductases were purified. Purification of the soluble ferric reductase included ion exchange and hydrophobic interaction chromatography resulting in a 9.8-fold purification of the main active fraction and a specific activity of 61.8 U/mg with a final yield of 22.6%. The protein was purified to homogeneity as indicated by SDS-PAGE and 2 D-PAGE with a relative molecular mass of 39 000. Isoelectric focusing was performed on the purified protein that displayed a pI of 5.6 - 5.8. The purified enzyme was analyzed for ferric reductase activity by a zymogram. The KCl-extracted ferric reductase activity from the membrane fraction was isolated by using hydrophobic interaction chromatography. The final purification protocol resulted in a 4.3-fold purification of the main active fraction and a specific activity of 17.1 U/mg with a final yield of 11.8%. A main protein band with relative molecular mass of 49 000 was detected on SDS-PAGE. The purified enzyme was analyzed for ferric reductase activity by a zymogram. The soluble ferric reductase exhibited an optimum temperature of 60 – 65 ºC and an optimum pH of 6. The enzyme was extremely stable yielding half-lives of 16 h at 50 ºC, 14 h at 70 ºC and 4 h at 90 ºC. The heavy metal AgCl significantly inhibited the ferric 126 reductase activity. The Km value for Fe(III)-NTA was estimated to be approximately 6.1 mM. The membrane-associated ferric reductase appeared to by extremely thermostable, yielding an astonishing half-live of 81 h at 70 ºC. The half-lives for 50 ºC and 90 ºC were estimated to be 62 h and 4 h, respectively. The enzyme exhibited an optimum temperature of 70 – 75 ºC and an optimum pH of 5. With NADH as the electron donor, the Km value for Fe(III)-NTA was estimated to be approximately 2.6 mM. Of the 13 metals tested, only AgCl, Hg2(NO3)2 and Pb(NO3)2 had a significant inhibitory effect on the activity. Both ferric reductases were stimulated upon incubation with EDTA although higher concentrations of EDTA slightly inactivated the membrane-associated ferric reductase. PMSF had only slight effect on both ferric reductases. Water-soluble carbodiimide resulted in the inactivation of the soluble and membrane-associated ferric reductase after 125 min and 100 min, respectively. Both ferric reductases were affected by high concentrations of urea up to 6 M; minor enzyme activity was restored upon 6-fold dilution. The membrane-associated and soluble ferric reductases differ favourably from their mesophilic counterparts in some characteristics. The aim of this study, namely the isolation and characterization of the thermophilic ferric reductases from Thermus scotoductus and to compare it with that of hyperthermophilic and mesophilic species, was successfully completed.