Ferredoxin:NADP+ Oxidoreductase Association with Phycocyanin Modulates Its Properties

Anja Korn,Ghada Ajlani,Bernard Lagoutte,Andrew Gall,Pierre Sétif

doi:10.1074/jbc.m109.024638

Abstract

In photosynthetic organisms, ferredoxin:NADP(+) oxidoreductase (FNR) is known to provide NADPH for CO(2) assimilation, but it also utilizes NADPH to provide reduced ferredoxin. The cyanobacterium Synechocystis sp. strain PCC6803 produces two FNR isoforms, a small one (FNR(S)) similar to the one found in plant plastids and a large one (FNR(L)) that is associated with the phycobilisome, a light-harvesting complex. Here we show that a mutant lacking FNR(L) exhibits a higher NADP(+)/NADPH ratio. We also purified to homogeneity a phycobilisome subcomplex comprising FNR(L,) named FNR(L)-PC. The enzymatic activities of FNR(L)-PC were compared with those of FNR(S). During NADPH oxidation, FNR(L)-PC exhibits a 30% decrease in the Michaelis constant K(m)((NADPH)), and a 70% increase in K(m)((ferredoxin)), which is in agreement with its predicted lower activity of ferredoxin reduction. During NADP(+) reduction, the FNR(L)-PC shows a 29/43% decrease in the rate of single electron transfer from reduced ferredoxin in the presence/absence of NADP(+). The increase in K(m)((ferredoxin)) and the rate decrease of single reduction are attributed to steric hindrance by the phycocyanin moiety of FNR(L)-PC. Both isoforms are capable of catalyzing the NADP(+) reduction under multiple turnover conditions. Furthermore, we obtained evidence that, under high ionic strength conditions, electron transfer from reduced ferredoxin is rate limiting during this process. The differences that we observe might not fully explain the in vivo properties of the Synechocystis mutants expressing only one of the isoforms. Therefore, we advocate that FNR localization and/or substrates availability are essential in vivo.

Highlights

Between the one-electron carrier ferredoxin (Fd) and the twoelectron carrier NADPϩ [1,2,3,4,5]: 2 Fdred ϩ NADPϩ ϩ Hϩ % 2 Fdox ϩ NADPH
Quantification of NADPϩ and NADPH in Cell Extracts—The cellular contents of NADPϩ and NADPH were measured in three Synechocystis strains grown under photoautotrophic conditions, the wild type and two mutants containing only one of the ferredoxin:NADP؉ oxidoreductase (FNR) isoforms, i.e. FNRL and FNRS in MI6 and FS1, respectively [24]
From recent data [24], specific roles were proposed for the two Synechocystis FNR isoforms, which seem to parallel the enzymatic selectivity of plant FNR root and leaf isoforms

Summary

Introduction

Between the one-electron carrier ferredoxin (Fd) and the twoelectron carrier NADPϩ [1,2,3,4,5]: 2 Fdred ϩ NADPϩ ϩ Hϩ % 2 Fdox ϩ NADPH. Ternary complexes between the three partners FNR, NADPϩ and Fd have been shown to be involved in NADPϩ-reductase activity [1, 8] This is in line with the fact that fast turnover requires NADPϩ binding before Fdred binding, FAD reduction, and Fdox release [1]. Such ternary complexes may not be required during the NADPH-oxidase catalytic cycle [7, 9], this has yet to be established. FNRS accumulates when photosynthesis is slowed down, i.e. under heterotrophic or starvation conditions [24] These observations support the idea that the two isoforms differ in their NADPϩ-reductase/ NADPH-oxidase activities. This can be regarded as analogous to the leaf and root isoforms of plants

Results

Discussion

Conclusion