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.
Hirasawa
Masakazu
M
Department of Chemistry and Biochemistry and Center for Biotechnology and Genomics, Texas Tech University, Lubbock, TX 79409-1061, USA.
Nakayama
Masato
M
Kim
Sung-Kun
SK
Hase
Toshiharu
T
Knaff
David B
DB
D000107:Acetylation; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D001974:Bromosuccinimide / Q000494:pharmacology; D002736:Chloroplasts / Q000201:enzymology*; D017124:Conserved Sequence; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010658:Phenylglyoxal / Q000494:pharmacology; D011485:Protein Binding; D016415:Sequence Alignment; D013057:Spectrum Analysis; D013388:Succinimides / Q000737:chemistry / Q000494:pharmacology; D050889:Sulfite Reductase (Ferredoxin) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*; D003313:Zea mays / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics
D013388:Succinimides; D014364:Tryptophan; D001120:Arginine; D050889:Sulfite Reductase (Ferredoxin); D008239:Lysine; D001974:Bromosuccinimide; D010658:Phenylglyoxal