Ferredoxin-NADP+ reductase -: Kinetics of electron transfer, transient intermediates, and catalytic activities studied by flash-absorption spectroscopy with isolated photosystem I and ferredoxin

The electron transfer cascade from photosystem I to NADP(+) was studied at physiological pH by flash-absorption spectroscopy in a Synechocystis PCC6803 reconstituted system comprised of purified photosystem I, ferredoxin, and ferredoxin-NADP(+) reductase. Experiments were conducted with a 34-kDa ferredoxin-NADP(+) reductase homologous to the chloroplast enzyme and a 38-kDa N-terminal extended form. Small differences in kinetic and catalytic properties were found for these two forms, although the largest one has a 3-fold decreased affinity for ferredoxin. The dissociation rate of reduced ferredoxin from photosystem I (800 s(-1)) and the redox potential of the first reduction of ferredoxin-NADP(+) reductase (-380 mV) were determined. In the absence of NADP(+), differential absorption spectra support the existence of a high affinity complex between oxidized ferredoxin and semireduced ferredoxin-NADP(+) reductase. An effective rate of 140-170 s(-1) was also measured for the second reduction of ferredoxin-NADP(+) reductase, this process having a rate constant similar to that of the first reduction. In the presence of NADP(+), the second-order rate constant for the first reduction of ferredoxin-NADP(+) reductase was 20% slower than in its absence, in line with the existence of ternary complexes ( ferredoxin-NADP(+) reductase)-NADP(+)-ferredoxin. A single catalytic turnover was monitored, with 50% NADP(+) being reduced in 8-10 ms using 1.6 mu M photosystem I. In conditions of multiple turnover, we determined initial rates of 360-410 electrons per s and per ferredoxin-NADP(+) reductase for the reoxidation of 3.5 mu M photoreduced ferredoxin. Identical rates were found with photosystem I lacking the PsaE subunit and wild type photosystem I. This suggests that, in contrast with previous proposals, the PsaE subunit is not involved in NADP(+) photoreduction.