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J. Biol. Chem., Vol. 280, Issue 28, 25960-25972, July 15, 2005

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Ferredoxin-NADP⁺ Reductase

KINETICS OF ELECTRON TRANSFER, TRANSIENT INTERMEDIATES, AND CATALYTIC ACTIVITIES STUDIED BY FLASH-ABSORPTION SPECTROSCOPY WITH ISOLATED PHOTOSYSTEM I AND FERREDOXIN^*

Nicolas Cassan, Bernard Lagoutte, and Pierre Sétif

From the Service de Bioénergétique and CNRS URA 2096, Département de Biologie Joliot Curie, CEA Saclay, 91191 Gif sur Yvette, France

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 µM 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 µM 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.

Received for publication, April 6, 2005 , and in revised form, May 11, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: CEA Saclay, DBJC/SBE, 91191 Gif sur Yvette, France. Tel.: 33-169-08-98-77; Fax: 33-169-08-87-17; E-mail; pierre.setif{at}cea.fr.

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