Studies of interaction of homo-dimeric ferredoxin-NAD(P)+ oxidoreductases of Bacillus subtilis and Rhodopseudomonas palustris, that are closely related to thioredoxin reductases in amino acid sequence, with ferredoxins and pyridine nucleotide coenzymes.
Ferredoxin-NADP(+) oxidoreductases (FNRs) of Bacillus subtilis (YumC) and Rhodopseudomonas palustris CGA009 (RPA3954) belong to a novel homo-dimeric type of FNR with high amino acid sequence homology to NADPH-thioredoxin reductases. These FNRs were purified from expression constructs in Escherichia coli cells, and their steady-state reactions with [2Fe-2S] type ferredoxins (Fds) from spinach and R. palustris, [4Fe-4S] type Fd from B. subtilis, NAD(P)(+)/NAD(P)H and ferricyanide were studied. From the K(m) and k(cat) values for the diaphorase activity with ferricyanide, it is demonstrated that both FNRs are far more specific for NADPH than for NADH. The UV-visible spectral changes induced by NADP(+) and B. subtilis Fd indicated that both FNRs form a ternary complex with NADP(+) and Fd, and that each of the two ligands decreases the affinities of the others. The steady-state kinetics of NADPH-cytochrome c reduction activity of YumC is consistent with formation of a ternary complex of NADPH and Fd during catalysis. These results indicate that despite their low sequence homology to other FNRs, these enzymes possess high FNR activity but with measurable differences in affinity for different types of Fds as compared to other more conventional FNRs.
Seo
Daisuke
D
Division of Material Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192, Japan. dseo@cacheibm.s.kanazawa-u.ac.jp
Okabe
Seisuke
S
Yanase
Mitsuhiro
M
Kataoka
Kunishige
K
Sakurai
Takeshi
T
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005288:Ferredoxins / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D011725:Pyridines / Q000378:metabolism*; D012241:Rhodopseudomonas / Q000201:enzymology*; D016415:Sequence Alignment; D018724:Spinacia oleracea; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D005288:Ferredoxins; D010940:Plant Proteins; D011725:Pyridines; D009243:NAD; D009249:NADP; D045304:Cytochromes c; D005287:Ferredoxin-NADP Reductase; D013880:Thioredoxin-Disulfide Reductase