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. and J. Grzyb, M. Bojko, S. Więckowski.
The effects of phenylmercuric acetate (PMA) and apoferredoxin (apoFd) on the diaphorase activity of spinach ferredoxin:NADP+ oxidoreductase (FNR) in the presence of dibromothymoquinone (DBMIB) or cytochrome c (Cyt c) were studied. PMA inhibited effectively (I50 = < 5 μM) ferredoxin-dependent Cyt c reduction but did not affect evidently the enzyme activity in the presence of DBMIB as an electron acceptor. ApoFd caused also inhibition of Cyt c reduction but slightly stimulated, like ferredoxin, DBMIB reduction. We confirm a hypothesis according to which three binding sites for substrates [NADP(H), Fd-Cyt c, quinone/dichlorophenol indophenol] occur within the molecule of isolated FNR. and M. Bojko, S. Więckowski.