title: Ferredoxin-NADP+ reductase. Kinetics of electron transfer, transient intermediates, and catalytic activities studied by flash-absorption spectroscopy with isolated photosystem I and ferredoxin.
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.
authors.affiliation: Service de Bioénergétique and CNRS URA 2096, Département de Biologie Joliot Curie, CEA Saclay, 91191 Gif sur Yvette, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Cassan
authors.forename: Bernard
authors.initials: B
authors.lastname: Lagoutte
authors.forename: Pierre
authors.initials: P
authors.lastname: Sétif
mesh_terms: D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D004583:Electrons; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000502:physiology*; D005288:Ferredoxins / Q000737:chemistry*; D017353:Gene Deletion; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D009249:NADP; D010100:Oxygen / Q000737:chemistry; D045331:Photosystem I Protein Complex / Q000737:chemistry*; D011485:Protein Binding; D013053:Spectrophotometry; D046939:Synechocystis / Q000378:metabolism*; D013997:Time Factors
chemical_list: D005288:Ferredoxins; D045331:Photosystem I Protein Complex; C079741:photosystem I, psaE subunit; D009249:NADP; D005287:Ferredoxin-NADP Reductase; D010100:Oxygen