Purification of a flavoprotein having NADPH-cytochrome c reductase and transhydrogenase activities from Nitrobacter winogradskyi and its molecular and enzymatic properties |
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Authors: | T Kurokawa Y Fukumori T Yamanaka |
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Institution: | (1) Department of Life Science, Faculty of Science, Tokyo Institute of Technology, O-okayama, Meguro-ku, 152 Tokyo, Japan |
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Abstract: | A flavoenzyme which showed NADPH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase EC 1.6.2.4) and transhydrogenase (NADPH-NAD+ oxidoreductase, EC 1.6.1.1) activities was purified to an electrophoretically homogeneous state from Nitrobacter winogradskyi. The reductase was a flavoprotein which contained one FAD per molecule but no FMN. The oxidized form of the enzyme showed absorption maxima at 272, 375 and 459 nm with a shoulder at 490 nm, its molecular weight was estimated to be 36,000 by SDS polyacrylamide gel electrophoresis, and the enzyme seemed to exist as a dimer in aqueous solution. The enzyme catalyzed reduction of cytochrome c, DCIP and benzylviologen by NADPH, oxidation of NADPH with menadione and duroquinone, and showed transhydrogenase activity. NADH was less effective than NADPH as the electron donor in the reactions catalyzed by the enzyme. The NADPH-reduction catalyzed by the enzyme of N. winogradskyi cytochrome c-550 and horse cytochrome c was stimulated by spinach ferredoxin. The enzyme reduced NADP+ with reduced spinach ferredoxin and benzylviologen radical.Abbreviations DCIP
dichlorophenolindophenol
- Tris
trishydroxy-methylaminomethane
- Mops
3-(N-morpholino) propanesulfonic acid
- SDS
sodium dodecylsufate |
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Keywords: | Nitrobacter winogradskyi Chemolithotroph NAD(P)+ reductase NADPH-cytochrome c reductase Transhydrogenase |
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