Effects of Amino and Thiol Group Reagents on the Ferredoxin:NADP⁺ Oxidoreductase Catalysed Reduction of Dibromothymoquinone

Abstract

Effects of selective reagents of amino groups (fluorescamine, Fc) and thiol [5,5'-dithio-bis(2-nitrobenzoic) acid, DTNB] groups on the diaphorase activity of spinach ferredoxin:NADP + oxidoreductase (FNR, E.C 1.18.1.2) in the presence of dibromothymoquinone (DBMIB) as an electron acceptor were studied. The incubation of FNR with 250 µM Fc in the time range from 0 to 120 min led to the gradual decrease of FNR activity according to biphasic kinetics. At the initial phase the activity (defined as the rate of NADPH oxidation) decreased about 4-time faster than at the subsequent second slower phase. Incubation of FNR simultaneously with Fc and DBMIB for more than 20 min caused restoration of the activity to about 80 % of the control. The inhibitory effect of Fc on the FNR-catalysed DBMIB reduction had non-competitive character. Incubation of FNR with DTNB led also to a gradual decrease of the enzyme activity, which reached about 45 % of the control after 2 h of incubation. Thus neither amino nor thiol groups in the FNR molecule are involved directly in the DBMIB reduction. However, the presence of DBMIB in the incubation medium influenced the inhibitory pattern of Fc and DTNB, and this suggests that DBMIB modified the conformational state of the FNR molecule.

Effect of cadmium on ferredoxin:NADP+ oxidoreductase activity

“…That means that the metal ion does not react with any amino acid residues that are involved directly in the quinone binding (or reduction) site, but could impact by sterical handicap or induction of conformational changes. This result confirms previously published data which indicating that -SH groups are not involved directly in the DBMIB binding or reduction [20,22]. Our unpublished data show, that also 5,5 0 -dithiobis(2-nitrobenzoic acid), thiol reactant [48], brought about comparable inhibition of DBMIB reduction (about 50%).…”

“…We suggest that Cd induced conformational changes within the enzyme molecule which facilitated formation of adducts between certain thiol groups in FNR and ME and this caused only the decrease of the DBMIB reduction. Such interpretation coincides with our previously published data indicating that -SH groups in FNR are only indirectly involved in the DBMIB dependent enzyme activity [20,22]. The formed adducts, if any, did not affect FNR activity in the presence of FeCy.…”

“…Probably short incubation caused preferential Cd 2þ interaction with some of surface-located ligands, such as prevalent acidic amino acid residues, and this may led to formation of a more favourable conformational state for the enzyme activity. Similar effect was observed in the sample treated with 5,5 0 -dithiobis(2-nitrobenzoic acid) [22]. Moreover, analysis of the enzyme activity in the presence of various concentration of NADPH (Figs.…”

“…The earlier data, indicating that monoclonal antibodies against FNR inhibit the dichlorophenol indophenol reduction but have no effect on the enzyme activity in the presence of Fd and cytochrome c as an electron acceptor system [21], are consistent with this proposal. The three substrate binding sites have further been supported by the studies on respondents of the enzyme to selective inhibitors [20,22]. Our preliminary experimental data suggest that these sites are also responded differently to the action of cadmium.…”