Abstract
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 microM photosystem I. In conditions of multiple turnover, we determined initial rates of 360-410 electrons per s and per ferredox-in-NADP+ reductase for the reoxidation of 3.5 microM 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.
Highlights
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
The substrate NADPϩ was used at a concentration of 1 mM, which is saturating for its binding to FNR and its catalytic reduction, as dissociation and Michaelis constants were found previously to lie in the range of 1–20 M [40, 55]
Photoactivation of FNR reduction and catalysis has been studied in the absence or presence of the substrate NADPϩ, with a special emphasis upon intermediate states
Summary
Biological Materials—All experiments were performed with PSI monomers from Synechocystis 6803 and recombinant forms of Fd/FNR from Synechocystis 6803. The absorption changes arising from P700 oxidation were recorded between 740 and 950 nm, with the P700ϩ spectrum exhibiting its usual shape with a broad maximum around 800 – 810 nm (data not shown) It appears that using a coefficient of 6500 MϪ1 cmϪ1 at 820 nm in previous publications was leading to an 11% overestimation of the Synechocystis 6803 PSI concentration. 2 and 4 –7), the flash-induced absorption changes of a control cuvette containing wild type PSI, Fd, in some cases NADPϩ, but no FNR, were subtracted from a similar sample containing FNR. This subtraction procedure is both useful and necessary for the following reasons. Numerical simulations were performed using the commercial software Mathcad (version 8.0; MathSoft)
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