TAILIEUCHUNG - Báo cáo khóa học: Isolation and biochemical characterization of two soluble iron(III) reductases from Paracoccus denitrificans

Two soluble enzymes (FerA and FerB) catalyzing the reduction of a number of iron(III) complexes by NADH, were purified to near homogeneity from the aerobically grown iron-limited culture ofParacoccus denitrificansusing a combination of anion-exchange chromatography (Seph-arose Q), chromatofocusing (Mono P), and gel permeation chromatography (Superose 12). FerA is a monomer with a molecular mass of 19 kDa, whereas FerB exhibited a molecular mass of about 55 kDa and consists of probably two identical subunits. . | Eur. J. Biochem. 271 553-562 2004 FEBS 2004 doi Isolation and biochemical characterization of two soluble iron III reductases from Paracoccus denitrificans Jiri Mazoch1 Radek Tesarik2 Vojtech Sedlacek1 Igor Kucera1 and Jaroslav Turanek2 1 Department of Biochemistry Faculty of Science Masaryk University Brno Czech Republic department of Immunology Veterinary Research Institute Brno Czech Republic Two soluble enzymes FerA and FerB catalyzing the reduction of a number of iron III complexes by NADH were purified to near homogeneity from the aerobically grown iron-limited culture of Paracoccus denitrificans using a combination of anion-exchange chromatography Sepharose Q chromatofocusing Mono P and gel permeation chromatography Superose 12 . FerA is a monomer with a molecular mass of 19 kDa whereas FerB exhibited a molecular mass of about 55 kDa and consists of probably two identical subunits. FerA can be classified as an NADH flavin oxidoreductase with a sequential reaction mechanism. It requires the addition of FMN or riboflavin for activity on Fe III substrates. In these reactions the apparent substrate specificity of FerA seems to stem exclusively from different chemical reactivities of Fe III compounds with the free reduced flavin produced by the enzyme. Observations on reducibility of Fe III chelated by vicinal dihydroxy ligands support the view that FerA takes part in releasing iron from the catechol type siderophores synthesized by P. denitrficans. Contrary to FerA the purified FerB contains a noncovalently bound redox-active FAD coenzyme can utilize NADPH in place of NADH does not reduce free FMN al an apprecatble aate and gives a pingpong type kinetic pattern with NADH and Fe III -nitrilo-triacetate as substrates. FerB is able to reduce chromate in agreement with the fact that its N-terminus bears a homology to the previously described chromate reductase from Pseudomonas putida. Besides this it also readily reduces quinones .

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