Methyl Viologen-linked Activity Research Articles

Chemical modification studies of tryptophan, arginine and lysine residues in maize chloroplast ferredoxin:sulfite oxidoreductase

The ferredoxin-dependent sulfite reductase from maize was treated, in separate experiments, with three different covalent modifiers of specific amino acid side chains. Treatment with the tryptophan-modifying reagent, N-bromosuccinimide (NBS), resulted in a loss of enzymatic activity with both the physiological donor for the enzyme, reduced ferredoxin, and with reduced methyl viologen, a non-physiological electron donor. Formation of the 1:1 ferredoxin/sulfite reductase complex prior to treating the enzyme with NBS completely protected the enzyme against the loss of both activities. Neither the secondary structure, nor the oxidation-reduction midpoint potential (Em) values of the siroheme and [4Fe-4S] cluster prosthetic groups of sulfite reductase, nor the binding affinity of the enzyme for ferredoxin were affected by NBS treatment. Treatment of sulfite reductase with the lysine-modifying reagent, N-acetylsuccinimide, inhibited the ferredoxin-linked activity of the enzyme without inhibiting the methyl viologen-linked activity. Complex formation with ferredoxin protects the enzyme against the inhibition of ferredoxin-linked activity produced by treatment with N-acetylsuccinimide. Treatment of sulfite reductase with N-acetylsuccinimide also decreased the binding affinity of the enzyme for ferredoxin. Treatment of sulfite reductase with the arginine-modifying reagent, phenylglyoxal, inhibited both the ferredoxin-linked and methyl viologen-linked activities of the enzyme but had a significantly greater effect on the ferredoxin-dependent activity than on the reduced methyl viologen-linked activity. The effects of these three inhibitory treatments are consistent with a possible role for a tryptophan residue the catalytic mechanism of sulfite reductase and for lysine and arginine residues at the ferredoxin-binding site of the enzyme.

Isolation and partial characterization of homogeneous ferredoxin-sulfite reductase from a red alga, Porphyra yezoensis.

Ferredoxin-dependent sulfite reductase (Fd-SiR) (EC 1.8.7.1) was purified about 1136-fold, with a yield of 11%, from fresh thalli of Porphyra yezoensis by a procedure involving ammonium sulfate precipitation, DEAE-cellulose chromatography, Buty 1-Toyopearl chromatography, Sephadex G-100 gel filtration and ferredoxin-Sepharose affinity chromatography. The purified enzyme was apparently homogeneous, as judged on polyacrylamide disc gel electrophoresis, with a specific activity of 100 units/mg of protein. The molecular weight of the enzyme was estimated to be 70 kilodaltons by gel filtration. On subunit analysis by SDS-PAGE, a single band corresponding to molecular weight of 65 kilodaltons appeared. The purified enzyme (Fd-SiR) showed 5-times higher ferredoxin-dependent activity than methyl viologen-linked activity. In the oxidized form, the enzyme exhibited absorption maxima at 278, 390 (Soret band), 586 (a band) and 714 (CT band) nm, indicating that siroheme is involved in the catalysis of sulfite reductio...

Spinach ferredoxin-nitrite reductase: characterization of catalytic activity and interaction of the enzyme with substrates.

The steady-state kinetic parameters of the enzymatic reduction of nitrite by spinach ferredoxin-nitrite reductase [EC 1.7.7.1] were measured under anaerobic conditions. The maximum velocity of ferredoxin-linked activity was essentially the same as for the methyl viologen-linked activity of the enzyme. The initial velocity patterns of the oxidation of reduced ferredoxin suggested a sequential reaction scheme by which nitrite and reduced ferredoxin bind to the free enzyme. The binding of nitrite and ferredoxin to the enzyme was also investigated by different spectra produced by the complex formed by the enzyme with the substrates. Nitrite and ferredoxin each gave a 1: 1 complex with the enzyme. The dissociation constant (Kd) of the enzyme-nitrite complex agreed well with the Km value for the ferredoxin-linked activity, whereas the Kd of the enzyme-ferredoxin complex differed from the Km value for the enzyme activity. It was concluded that our preparation of spinach ferredoxin-nitrite reductase differs from both the complex (Mr = 85,000) and the modified (Mr = 61,000) forms of the enzyme reported by Hirasawa et al. [J. Biol. Chem. 262, 12428-12433 (1987)].

Some properties of ferredoxin-nitrite reductase from green shoots of bean and an immunological comparison with nitrite reductase from roots and etiolated shoots

Ferredoxin-nitrite reductase (EC 1.7.7.1), an enzyme which catalyzes the 6-electron reduction of nitrite to ammonia, has been isolated from green shoots of bean ( Phaseolus angularis). The isolated enzyme (GR-NiR), having a molecular mass of 68 000, showed 1.4 times higher ferredoxin-dependent activity than methyl viologen-linked activity. The enzyme was homogeneous by polyacrylamide gel electrophoresis (PAGE). In the oxidized form, the enzyme had absorption maxima at 275, 393 (Soret band), 535 and 571 (α band) nm, indicating that siroheme is involved in the catalysis of nitrite reduction. The absorbance ratios, A 393 : A 275 and A 571 : A 393 were 0.26 and 0.32, respectively. Antibody against the isolated enzyme was raised in rabbits. Analysis of the antiserum by immunodiffusion and immunoelectrophoresis suggested that it was a specific antiserum against GR-NiR. Using the antiserum, immunodiffusion and immunoprecipitation procedures were employed to compare the immunological similarity of NiR from green shoots, etiolated shoots and roots of bean. These tests revealed that the three forms of assimilatory NiR have antigenic determinants in common.

Purification and properties of ferredoxin—nitrate reductase from the cyanobacterium Plectonema boryanum

Assimilatory ferrodoxin—nitrate reductase has been purified to homogeneity from the cyanobacterium Plectonema boryanum. The enzyme was solubilized by sonication and, following heat treatment, ammonium sulfate precipitation and chromatography on DEAE-Toyopearl, Sephadex G-150 and hydroxyapatite, was purified 19000-fold with a yield of 15%. The purified enzyme had a specific activity of 305 and 1020 μmol NO 2 − formed/min per mg protein for ferrodoxin-linked and methyl viologen-linked nitrate reductase activities, respectively. The molecular weights measured by sodium dodecyl sulfate gel electrophoresis and sedimentation equilibrium were 83 000 and 85 000, respectively, indicating that the enzyme consists of a single polypeptide chain. The purified nitrate reductase contained 0.95 atom of molybdenum, and four iron and four acid-labile sulfur atoms per molecule ( M r 85 000). The spectrum of the enzyme resembled that of iron-sulfur proteins. The CD and MCD spectra suggested the presence of an iron-sulfur cluster in the molecule. Some similarities of amino-acid composition were observed between the cyanobacterial nitrate reductase, NAD(P)H-nitrate reductase and the catalytic subunit of the dissimilatory nitrate reductase from Escherichia coli. The stoichiometry of the nitrate reductase reaction confirmed a two-electron reduction of nitrate to nitrite by reduced ferredoxin or reduced methyl viologen. The K m values of the native enzyme was 38 μM for reduced ferredoxin and 2500 μM for reduced methyl viologen. Dithionite converted the native enzyme into a modified form which had no ferredoxin-linked nitrate reductase activity but retained methyl viologen-linked activity with a pH optimum at 10.2.

Two hydrogenases with distinct electrn-carrier specificity and subunit composition in Methanobacterium formicicum

Two hydrogenases, differing in their specificity for the electron carrier F 420, were present in cell-free extracts of Methanobacterium formicicum. The two hydrogenases were resolved either by gel filtration, ion-exchange chromatography, ro by electrophoresis in polyacrylamide gel. The larger hydrogenase (designated the F 420-reactive hydrogenase) showed factor F 420-linked and methyl viologen-linked activities; two forms ( M r 600 000 and 380 000) were obtained from M. formicicum strain MF. The smaller enzyme (designated F 420-non-reactive hydrogenase; M r 70 000 from M. formicicum strain MF) showed only methyl viologen-linked activity. The F 420-reactive hydrogenase contained subunits of M r 42 600, 34 000 and 23 500. The F 420-non-reactive hydrogenase contained subunits of M r 48 000 and 38 000. The subunit patterns and the results of peptide mapping indicate that F 420-reactive and F 420-non-reactive hydrogenases are distinct enzymes. The F 420-reactive hydrogenase was more stable under aerobic conditions, and both hydrogenases could be purified in air. The aerobically prepared enzymes must be incubated with a reducing agent to become active. After activation, the F 420-reactive hydrogenase became unstable at 25–30°C but was stable at 0–5°C. Besides the major F 420-non-reactive hydrogenase, a minor F 420-non-reactive hydrogenase ( M r 145 000) was also observed in extracts of M. formicicum. The hydrogenases of M. formicicum strains MF and JF were very similar. In M. bryantii, several hydrogenase species with different electrophoretic mobilities were observed, but their properties different from those of the M. formicicum enzymes.

Further characterization of ferredoxin nitrite reductase and the relationship between the enzyme and methyl viologen-dependent nitrite reductase.

Ferredoxin-dependent nitrite reductase of spinach has been further characterized and the relationship between this enzyme and methyl viologen-dependent nitrite reductase studied. Purified ferredoxin nitrite reductase, having a molecular weight of 86, 000, showed 2.5 times higher ferredoxin-dependent activity than methyl viologen-linked activity. Besides 4 mol of labile sulfide the enzyme contained about 2 mol of siroheme per mol. When dithionite, methyl viologen and nitrite were added, ESR signals of a heme nitrosyl complex at g=2.14, 2.07 and 2.02 were observed. Moreover, hyperfine splitting of the signal due to 14N nuclear spin was also observed at 2.033, 2.023 and 2.013. The sole addition of hydroxylamine to the ferric enzyme also caused the same but much less intense signals with the hyperfine splitting. On treatment of the ferredoxin nitrite reductase (native enzyme) with DEAE-Sephadex A-50 chromatography, a modified nitrite reductase having a molecular weight of 61, 000 and a protein fraction having an apparent molecular weight of 24, 000 were separated. The modified enzyme contained about one mol of siroheme and 4 mol of labile sulfide per mol and showed essentially the same heme ESR signals as the native enzyme. Contrary to the native enzyme, this modified enzyme accepted electrons more efficiently from methyl viologen than ferredoxin and the reduction of nitrite to ammonia catalyzed by the modified enzyme was not stoichiometric. The observed nitrite to ammonia ratio was 1 to less than 0.6. Cyanide at concentrations between 0.02 to 0.2 mM inhibited the activity of the native enzyme almost completely but the modified enzyme was inhibited only partially. From the results obtained, it is suggested that the native ferredoxin-linked nitrite reductase consists of two components (or subunits) and removal of the light component results in formation of a modified enzyme with increased relative affinity to methyl viologen.