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

Abstract

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.

NMR Study of the Electron Transfer Complex of Plant Ferredoxin and Sulfite Reductase

“…Thus, one would expect that at least two positively charged surfaces on SiR operate as the counterparts of the Fd acidic patches when the complex is formed. This was experimentally suggested by chemical modification of lysine and arginine residues of SiR that caused an inhibitory effect on Fd-dependent activity of SiR, but not on redox dye-dependent activity (10).…”

“…The results of gel filtration chromatography (7) and spectral perturbation experiments (10) showed that Fd and SiR form a complex of high affinity at a lower ionic strength but such molecular interaction does not occur at a higher ionic strength, indicative of the major contribution of electrostatic force to stabilizing the complex. Previous experiments, using site-specific mutants of root-type Fd isoform from maize, demonstrated that negatively charged residues of Fd participate in this electrostatic interaction (7).…”

“…A recent study on chemical modifications of lysine and arginine residues of maize SiR showed that some basic residues are also involved in SiR binding to Fd (10).…”

Kinetic, Thermodynamic and Structural Studies of Native and N-Bromosuccinimide-Modified Mushroom Tyrosinase

“…Chemical modification of some residues such as tryptophan, lysine, and arginine in sulfite reductase, respectively by NBS, N-acetylsuccinimide, and phenylglyoxal revealed the inhibitory effect of these three modifiers and delineated the importance role of these residues in the catalytic mechanism of enzyme (30). In a previous study, Histidine, arginine, tryptophan, and lysine residues of Aspergillus niger lipase were chemically modified by using bromoacetic acid (BrAc), 2, 3-butanedione (BD), NBS, and metanal, respectively, and the results suggested that these residues are essential for enzyme activity and might be involved in the catalytic site of the enzyme (31).…”

Curcumin Cross-links Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Polypeptides and Potentiates CFTR Channel Activity by Distinct Mechanisms

“…The inability of reducing agents like dithiothreitol and ␤-mercaptoethanol to disrupt curcumin-induced CFTR oligomers, as well as the ability of curcumin to cross-link a cysteine-free CFTR construct (data not shown), indicate that curcumin cross-links CFTR without reacting with cysteine sulfhydryl groups. We attempted to determine which other amino acid residues could be involved in the curcumin-induced CFTR cross-linking by pretreating CFTR-containing microsomes with nucleophilic amino acid modifiers like N-acetylsuccinimide (lysine modifier), phenylglyoxal (arginine modifier) (43), and DEPC (histidine modifier) (44) before curcumin treatment. None of these modifiers prevented CFTR cross-linking (data not shown) indicating either that curcumin interacts with CFTR via a different reaction than the Michael addition or that the modifiers were unable to block all reactive groups.…”