Open questions in ferredoxin‐NADP+ reductase catalytic mechanism

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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.

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