Pre-steady-state kinetic studies of redox reactions catalysed by Bacillus subtilis ferredoxin-NADP+ oxidoreductase with NADP+/NADPH and ferredoxin.

Ferredoxin-NADP + oxidoreductase ([EC1.18.1.2], FNR) from Bacillus subtilis ( Bs FNR) is a homodimeric flavoprotein sharing structural homology with bacterial NADPH-thioredoxin reductase. Pre-steady-state kinetics of the reactions of Bs FNR with NADP + , NADPH, NADPD (deuterated form) and B. subtilis ferredoxin ( Bs Fd) using stopped-flow spectrophotometry were studied. Mixing Bs FNR with NADP + and NADPH yielded two types of charge-transfer (CT) complexes, oxidized FNR (FNR ox )-NADPH and reduced FNR (FNR red )-NADP + , both having CT absorption bands centered at approximately 600 nm. After mixing Bs FNR ox with about a 10-fold molar excess of NADPH (forward reaction), Bs FNR was almost completely reduced at equilibrium. When Bs FNR red was mixed with NADP + , the amount of Bs FNR ox increased with increasing NADP + concentration, but Bs FNR red remained as the major species at equilibrium even with about 50-fold molar excess NADP + . In both directions, the hydride-transfer was the rate-determining step, where the forward direction rate constant (~ 500 s − 1 ) was much higher than the reverse one (< 10 s − 1 ). Mixing Bs Fd red with Bs FNR ox induced rapid formation of a neutral semiquinone form. This process was almost completed within 1 ms. Subsequently the neutral semiquinone form was reduced to the hydroquinone form with an apparent rate constant of 50 to 70 s − 1 at 10 °C, which increased as Bs Fd red increased from 40 to 120 μM. The reduction rate of Bs FNR ox by Bs Fd red was markedly decreased by premixing Bs FNR ox with Bs Fd ox , indicating that the dissociation of Bs Fd ox from Bs FNR sq is rate-limiting in the reaction. The characteristics of the Bs FNR reactions with NADP + /NADPH were compared with those of other types of FNRs. [ABSTRACT FROM AUTHOR]