Open questions in ferredoxin‐NADP⁺ reductase catalytic mechanism

Abstract

Ferredoxin (flavodoxin)-NADP(H) reductases (FNR) are ubiquitous flavoenzymes that deliver NADPH or low potential one-electron donors (ferredoxin, flavodoxin) to redox-based metabolisms in plastids, mitochondria and bacteria. The plant-type reductase is also the basic prototype for one of the major families of flavin-containing electron transferases that display common functional and structural properties. Many aspects of FNR biochemistry have been extensively characterized in recent years using a combination of site-directed mutagenesis, steady-state and transient kinetic experiments, spectroscopy and X-ray crystallography. Despite these considerable advances, various key features in the enzymology of these important reductases remain yet to be explained in molecular terms. This article reviews the current status of these open questions. Measurements of electron transfer rates and binding equilibria indicate that NADP(H) and ferredoxin interactions with FNR result in a reciprocal decrease of affinity, and that this induced-fit step is a mandatory requisite for catalytic turnover. However, the expected conformational movements are not apparent in the reported atomic structures of these flavoenzymes in the free state or in complex with their substrates. The overall reaction catalysed by FNR is freely reversible, but the pathways leading to NADP + or ferredoxin reduction proceed through entirely different kinetic mechanisms. Also, the reductases isolated from various sources undergo inactivating denaturation on exposure to NADPH and other electron donors that reduce the FAD prosthetic group, a phenomenon that might have profound consequences for FNR function in vivo. The mechanisms underlying this reductive inhibition are so far unknown. Finally, we provide here a rationale to interpret FNR evolution in terms of catalytic efficiency. Using the formalism of the Albery-Knowles theory, we identified which parameter(s) have to be modified to make these reductases even more proficient under a variety of conditions, natural or artificial. Flavoenzymes with FNR activity catalyse a number of reactions with potential importance for biotechnological processes, so that modification of their catalytic competence is relevant on both scientific and technical grounds.

Cores and pH-dependent Dynamics of Ferredoxin-NADP+ Reductase Revealed by Hydrogen/Deuterium Exchange

“…The higher dynamic motion in the NADP ϩ -flexible subdomain at pD r 8.0 than at pD r 6.0 is most likely to facilitate rapid sampling in the conformational space for selecting the most suitable conformation for enhancing the binding affinity for NADP ϩ , especially in the first binding step of the adenosine portion of NADP ϩ in the bipartite NADP ϩ binding mode (20,35). The fact that the presence of NADP ϩ at the active site is a prerequisite for the catalysis of FNR (35) can explain why the NADP ϩ -flexible subdomain exhibits the higher motional behavior than other regions of FNR at pD r 8.0.…”

Transgenic Tobacco Plants Overexpressing Chloroplastic Ferredoxin-NADP(H) Reductase Display Normal Rates of Photosynthesis and Increased Tolerance to Oxidative Stress

“…The net CO 2 uptake rate at saturating CO 2 internal concentration (A max ) and the initial slope at low Ci were derived from Ci response curves by fitting experimental data to the equation of a nonrectangular hyperbola as described (Hajirezaei et al, 2002). reaction (Bruns and Karplus, 1995;Carrillo and Ceccarelli, 2003). Whereas many functional properties of FNR have been thoroughly characterized (Carrillo and Ceccarelli, 2003;Ceccarelli et al, 2004), until recently, little was known on the actual contribution of this flavoenzyme to electron transport and photosynthesis in planta.…”

“…reaction (Bruns and Karplus, 1995;Carrillo and Ceccarelli, 2003). Whereas many functional properties of FNR have been thoroughly characterized (Carrillo and Ceccarelli, 2003;Ceccarelli et al, 2004), until recently, little was known on the actual contribution of this flavoenzyme to electron transport and photosynthesis in planta. The combined use of FNR antisense (Hajirezaei et al, 2002) and overexpressing transgenic plants (this work) revealed somehow paradoxical behavior.…”

“…Although FNR is highly specific for NADP(H) and a very poor NAD(H)-dependent oxidoreductase, it can derive electrons to a wide variety of acceptors of appropriate redox potential but very different structures, mediating the so-called diaphorase reaction (Carrillo and Ceccarelli, 2003). Expression of pea FNR in tobacco chloroplasts led to a concomitant increase in diaphorase activity (Fig.…”

Characterization of SpnQ from the Spinosyn Biosynthetic Pathway of Saccharopolyspora spinosa: Mechanistic and Evolutionary Implications for C-3 Deoxygenation in Deoxysugar Biosynthesis

“…13 In the SpnQ case, a similar electron relay path as in the E 1 -E 3 reaction may be operative (see Scheme 2, path A). It should be noted that when E 1 was assayed in the presence of its natural substrate (10), ferredoxin/ferredoxin reductase, and NADPH, it was five-fold less effective at C-3 deoxygenation than when assayed using E 3 under standard conditions (18% versus 100% conversion after 1.5 h at 25 °C).…”