Characterization of the flavoprotein moieties of NADPH-sulfite reductase from Salmonella typhimurium and Escherichia coli. Physicochemical and catalytic properties, amino acid sequence deduced from DNA sequence of cysJ, and comparison with NADPH-cytochrome P-450 reductase.
NADPH-sulfite reductase flavoprotein (SiR-FP) was purified from a Salmonella typhimurium cysG strain that does not synthesize the hemoprotein component of the sulfite reductase holoenzyme. cysJ, which codes for SiR-FP, was cloned from S. typhimurium LT7 and Escherichia coli B, and both genes were sequenced. Physicochemical analyses and deduced amino acid sequences indicate that SiR-FP is an octamer of identical 66-kDa peptides and contains 4 FAD and 4 FMN per octamer. Potentiometric titrations of SiR holoenzyme, SiR-FP, and FMN-depleted SiR-FP yielded the following redox potentials for the prosthetic groups at pH 7.7: E'1 (FMNH./FMN) = -152 mV; E'2 (FMNH2/FMNH.) = -327 mV; E'3 (FADH./FAD) = -382 mV; E'4 (FADH2/FADH.) = -322 mV. Microcoulometric titration of SiR-FP at 25 degrees C yielded data which were in full agreement with these potentials. Spectroscopic and catalytic studies of native SiR-FP and of SiR-FP depleted of FMN support the following electron flow sequence: NADPH----FAD----FMN. FMN can then contribute electrons to the hemoprotein component of sulfite reductase, as well as to cytochrome c and various diaphorase acceptors. The FMN is postulated to cycle between the FMNH2 and FMNH. oxidation states during catalysis; in this sense SiR-FP shares a catalytic mechanism with NADPH-cytochrome P-450 oxidoreductase. SiR-FP domains involved in binding FMN, FAD, and NADPH are proposed from amino acid sequence homologies with Desulfovibrio vulgaris flavodoxin (Dubourdieu, M., and Fox, J.L. (1977) J. Biol. Chem. 252, 1453-1463) and spinach ferredoxin-NADP+ oxidoreductase (Karplus, P.A., Walsh, K.A., and Herriott, J. R. (1984) Biochemistry 23, 6576-6583). Comparison of the deduced amino acid sequences of SiR-FP and NADPH-cytochrome P-450 oxidoreductase (Porter, T. D., and Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U. S.A. 82, 973-977) also showed identities that suggest these two proteins are descended from a common precursor, which contained binding regions for both FMN and FAD.
Ostrowski
J
J
Howard Hughes Medical Institute Laboratory, Duke University Medical Center, Durham, North Carolina 27710.
Barber
M J
MJ
Rueger
D C
DC
Miller
B E
BE
Siegel
L M
LM
Kredich
N M
NM
D000595:Amino Acid Sequence; D001483:Base Sequence; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005420:Flavoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005796:Genes; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000378:metabolism*; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D010957:Plasmids; D015183:Restriction Mapping; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013053:Spectrophotometry; D050864:Sulfite Reductase (NADPH)
D005420:Flavoproteins; D010446:Peptide Fragments; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors; D050864:Sulfite Reductase (NADPH); C494314:sulfite reductase (NADPH), E coli