Hydride Transfer Step Research Articles

Mechanism and Origins of Stereoselectivity of the Aldol-Tishchenko Reaction of Sulfinimines.

Density functional theory computations have elucidated the mechanism and origins of stereoselectivity in McGlacken’s aldol-Tishchenko reaction for the diastereoselective synthesis of 1,3-amino alcohols using Ellman’s t-butylsulfinimines as chiral auxiliaries. Variations of stereochemical outcome are dependent on the nature of the ketone starting materials used, and the aspects leading to these differences have been rationalized. The intramolecular hydride transfer step is the rate- and stereochemistry-determining step, and all prior steps are reversible.

Computational Study of PmHMGR Thiohemiacetal Breakdown Transition State

3-hydroxyl-3-methylglutaryl CoA reductase (HMGR) catalyzes the rate determining step in the isoprenoid pathway using two NADH molecules. Even though this enzyme is targeted by statins, the atomistic details of the mechanism are still unknown. This objective can be met by combination of computational biophysics and experimental validations through time-resolved Laue crystallography for all steps of the reaction. The catalysis of HMGR can be broken down into three major chemical transformations including two hydride transfers and a thiohemiacetal breakdown step. Moreover, since two cofactor molecules are required, a cofactor exchange process also occurs after the first hydride transfer and before the second hydride transfer. Thiohemiacetal is a known intermediate resulting from the first hydride transfer step. However, the product of the thiohemiacetal breakdown step hypothesized to be mevaldehyde, has not been experimentally validated. This theoretical study explores the atomic details of the enzyme and ligands for the thiohemiacetal breakdown step using the Pseudomonas mevalonii HMGR. Theozymes and QM/MM ONIOM models were used to identify the transition state were performed to reveal the catalytic residues. These models have revealed that the Nδ of His381 is involved in stabilizing the negative charge in the thiolate anion. Ser85 forms a hydrogen bonding network with the first amide of thiohemiacetal ligand, a function performed by His381 in the preceding step first hydride transfer. Transition state force fields (TSFF) using the quantum guided molecular mechanics (Q2MM) program will then be used to perform µsec timescale molecular dynamics simulations. This project focuses on not only developing computational biophysics methods for studying enzyme mechanisms but also uses time-resolved x-ray crystallography to obtain snapshots along the HMGR reaction pathway which involves multiple transformations of the ligand in the same active site, a large flap domain movement, and a cofactor exchange.

Temperature and Solvent Effects on H2 Splitting and Hydricity: Ramifications on CO2 Hydrogenation by a Rhenium Pincer Catalyst.

The catalytic hydrogenation of carbon dioxide holds immense promise for applications in sustainable fuel synthesis and hydrogen storage. Mechanistic studies that connect thermodynamic parameters with the kinetics of catalysis can provide new understanding and guide predictive design of improved catalysts. Reported here are thermochemical and kinetic analyses of a new pincer-ligated rhenium complex (tBuPOCOP)Re(CO)2 (tBuPOCOP = 2,6-bis(di-tert-butylphosphinito)phenyl) that catalyzes CO2 hydrogenation to formate with faster rates at lower temperatures. Because the catalyst follows the prototypical "outer sphere" hydrogenation mechanism, comprehensive studies of temperature and solvent effects on the H2 splitting and hydride transfer steps are expected to be relevant to many other catalysts. Strikingly large entropy associated with cleavage of H2 results in a strong temperature dependence on the concentration of [(tBuPOCOP)Re(CO)2H]- present during catalysis, which is further impacted by changing the solvent from toluene to tetrahydrofuran to acetonitrile. New methods for determining the hydricity of metal hydrides and formate at temperatures other than 298 K are developed, providing insight into how temperature can influence the favorability of hydride transfer during catalysis. These thermochemical insights guided the selection of conditions for CO2 hydrogenation to formate with high activity (up to 364 h-1 at 1 atm or 3330 h-1 at 20 atm of 1:1 H2:CO2). In cases where hydride transfer is the highest individual kinetic barrier, entropic contributions to outer sphere H2 splitting lead to a unique temperature dependence: catalytic activity increases as temperature decreases in tetrahydrofuran (200-fold increase upon cooling from 50 to 0 °C) and toluene (4-fold increase upon cooling from 100 to 50 °C). Ramifications on catalyst structure-function relationships are discussed, including comparisons between "outer sphere" mechanisms and "metal-ligand cooperation" mechanisms.

Structure, dynamics and function of the evolutionarily changing biliverdin reductase B family.

Biliverdin reductase B (BLVRB) family members are general flavin reductases critical in maintaining cellular redox with recent findings revealing that BLVRB alone can dictate cellular fate. However, as opposed to most enzymes, the BLVRB family remains enigmatic with an evolutionarily changing active site and unknown structural and functional consequences. Here, we applied a multi-faceted approach that combines X-ray crystallography, NMR and kinetics methods to elucidate the structural and functional basis of the evolutionarily changing BLVRB active site. Using a panel of three BLVRB isoforms (human, lemur and hyrax) and multiple human BLVRB mutants, our studies reveal a novel evolutionary mechanism where coenzyme 'clamps' formed by arginine side chains at two co-evolving positions within the active site serve to slow coenzyme release (Positions 14 and 78). We find that coenzyme release is further slowed by the weaker binding substrate, resulting in relatively slow turnover numbers. However, different BLVRB active sites imposed by either evolution or mutagenesis exhibit a surprising inverse relationship between coenzyme release and substrate turnover that is independent of the faster chemical step of hydride transfer also measured here. Collectively, our studies have elucidated the role of the evolutionarily changing BLVRB active site that serves to modulate coenzyme release and has revealed that coenzyme release is coupled to substrate turnover.

Unexpected Roles of Triethanolamine in the Photochemical Reduction of CO2 to Formate by Ruthenium Complexes.

A series of 4,4'-dimethyl-2,2'-bipyridyl ruthenium complexes with carbonyl ligands were prepared and studied using a combination of electrochemical and spectroscopic methods with infrared detection to provide structural information on reaction intermediates in the photochemical reduction of CO2 to formate in acetonitrile (CH3CN). An unsaturated 5-coordinate intermediate was characterized, and the hydride-transfer step to CO2 from a singly reduced metal-hydride complex was observed with kinetic resolution. While triethanolamine (TEOA) was expected to act as a proton acceptor to ensure the sacrificial behavior of 1,3-dimethyl-2-phenyl-2,3-dihydro-1H-benzo[d]imidazole as an electron donor, time-resolved infrared measurements revealed that about 90% of the photogenerated one-electron reduced complexes undergo unproductive back electron transfer. Furthermore, TEOA showed the ability to capture CO2 from CH3CN solutions to form a zwitterionic alkylcarbonate adduct and was actively engaged in key catalytic steps such as metal-hydride formation, hydride transfer to CO2 to form the bound formate intermediate, and dissociation of formate ion product. Collectively, the data provide an overview of the transient intermediates of Ru(II) carbonyl complexes and emphasize the importance of considering the participation of TEOA when investigating and proposing catalytic pathways.

Understanding the origin of the enantioselectivity and the mechanism of the asymmetric reduction of ketimine generated from acetophenone with oxazaborolidine catalyst

The enantioselectivity of the oxazaborolidine-catalysed asymmetric reduction reaction of ketimine generated from acetophenone has been investigated by transition state theory at the hydride transfer step using DFT methods at the B3LYP/6-31G(d,p) level of theory. The obtained results indicate that the hydride attack at the Si face is more favourable than the Re one. Analyses of non-covalent interactions and molecular electrostatic potential indicate that the several favourable interactions at the Si hydrogen transfer mode are the origin of the enantioselectivity observed experimentally. Electron Localisation Function topological analysis indicates that the studied hydride transfer step takes place via a non-concerted three-stage mechanism.

Iodoimidazolinium-Catalyzed Reduction of Quinoline by Hantzsch Ester: Halogen Bond or Brønsted Acid Catalysis.

Halogen-bond (XB) catalyzed reactions could also be catalyzed by Brønsted acids, which may complicate the picture of the true activation pathway for these reactions. Herein, we report the first density functional theory study of the mechanistic pathways for the uncatalyzed, iodoimidazolinium halogen bond catalyzed, and a competitive Brønsted acid-catalyzed reduction of quinoline by Hantzsch ester. The uncatalyzed reaction was found to proceed via stepwise pathways. In the lowest energy pathway, proton transfer from Hantzsch ester, a weak Brønsted acid, to quinoline prior to hydride reduction was identified as the key to the lowered energy barriers compared to other reaction pathways. The same reaction steps are involved in the XB-catalyzed pathway, but with substantially lowered reaction barriers, particularly for the hydride-transfer steps. In contrast to the general belief that halogen bond catalysts bind to the electrophile quinoline and activate it by lowering its LUMO energy, we discovered that it is preferable to lower the LUMO energy of quinoline through protonation by Hantzsch ester as a Brønsted acid and stabilize the conjugate anion of Hantzsch ester via halogen bond. Finally, our calculations reveal that the iodoimidazolinium type of catalyst is prone to reduction by Hantzsch ester, generating a Brønsted acid as product. The Brønsted acid catalyzed pathway was calculated to be competitive with the halogen bond catalyzed pathway. Our theoretical findings highlight the need to be cautious when applying iodoimidazolinium catalysts in organocatalysis, and we hope it will aid the design of new halogen bond catalysts that could avoid undesirable Brønsted acid catalysis.

Formate-Catalyzed Selective Reduction of Carbon Dioxide to Formate Products Using Hydrosilanes

Formate salts were found to be active and selective homogeneous catalysts for the hydrosilylation of CO2. Counter cations and solvents strongly affected their catalytic activity. The use of toluene as a solvent rendered the recyclable catalyst system. Lewis basic polar solvents such as N-methylpyrrolidone and dimethyl sulfoxide significantly accelerated the reaction. The turnover number approached up to 1800. Analyses of the Fourier-transform infrared spectroscopy and in situ 1H nuclear magnetic resonance spectra suggested that the catalysis proceeds via electron donation from the solvent to hydrosilane, thereby accelerating the hydride transfer step.

Comparative Computational Approach To Study Enzyme Reactions Using QM and QM-MM Methods.

Choline oxidase catalyzes oxidation of choline into glycine betaine through a two-step reaction pathway employing flavin as the cofactor. On the light of kinetic studies, it is proposed that a hydride ion is transferred from α-carbon of choline/hydrated-betaine aldehyde to the N5 position of flavin in the rate-determining step, which is preceded by deprotonation of hydroxyl group of choline/hydrated-betaine aldehyde to one of the possible basic side chains. Using the crystal structure of glycine betaine–choline oxidase complex, we formulated two computational systems to study the hydride-transfer mechanism including main active-site amino acid side chains, flavin cofactor, and choline as a model system. The first system used pure density functional theory calculations, whereas the second approach used a hybrid ONIOM approach consisting of density functional and molecular mechanics calculations. We were able to formulate in silico model active sites to study the hydride-transfer steps by utilizing noncovalent chemical interactions between choline/betaine aldehyde and active-site amino acid chains using an atomistic approach. We evaluated and compared the geometries and energetics of hydride-transfer process using two different systems. We highlighted chemical interactions and studied the effect of protonation state of an active-site histidine base on the energetics of transfer. Furthermore, we evaluated energetics of the second hydride-transfer process as well as hydration of betaine aldehyde.

Experimental and Computational Studies Delineate the Role of Asparagine 177 in Hydride Transfer for E. coli Thymidylate Synthase.

Thymidylate synthase (TSase), an enzyme responsible for the de novo biosynthesis of 2'-deoxythymidine 5'-monophosphate (thymidylate, dTMP) necessary for DNA synthesis, has been a drug target for decades. TSase is a highly conserved enzyme across species ranging from very primitive organisms to mammals. Among the many conserved active site residues, an asparagine (N177, using Escherichia coli residues numbering) appears to make direct hydrogen bonds with both the C4=O4 carbonyl of the 2'-deoxyuridine 5'-monophosphate (uridylate, dUMP) substrate and its pyrimidine ring's N3. Recent studies have reassessed the TSase catalytic mechanism, focusing on the degree of negative charge accumulation at the O4 carbonyl of the substrate during two critical H-transfers - a proton abstraction and a hydride transfer. To obtain insights into the role of this conserved N177 on the hydride transfer, we examined its aspartic acid (D) and serine (S) mutants - each of which is expected to alter hydrogen bonding and charge stabilization around the C4=O4 carbonyl of the 2'-deoxyuridine 5'-monophosphate (uridylate, dUMP) substrate. Steady-state kinetics, substrate binding order studies and temperature-dependency analysis of intrinsic KIEs for the hydride transfer step of the TSase catalytic cycle suggest the active site of N177D is not precisely organized for that step. A smaller disruption was observed for N177S, which could be rationalized by partial compensation by water molecules and rearrangement of other residues toward preparation of the system for the hydride transfer under study. These experimental findings are qualitatively mirrored by QM/MM computational simulations, thereby shedding light on the sequence and synchronicity of steps in the TSase-catalyzed reaction. This information could potentially inform the design of mechanism-based drugs targeting this enzyme.

Effect of Asp122 Mutation on the Hydride Transfer in E. coli DHFR Demonstrates the Goldilocks of Enzyme Flexibility

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) in the presence of NADPH. The key hydride transfer step in the reaction is facilitated by a combination of enzyme active site preorganization and correlated protein motions in the Michaelis-Menten (E:NADPH:DHF) complex. The present theoretical study employs mutagenesis to examine the relation between structural and functional properties of the enzyme. We mutate Asp122 in Escherichia coli DHFR, which is a conserved amino acid in the DHFR family. The consequent effect of the mutation on enzyme catalysis is examined from an energetic, structural and short-time dynamic perspective. Our investigations suggest that the structural and short-time dynamic perturbations caused by Asp122X mutations (X = Asn, Ser, Ala) are along the reaction coordinate and lower the rate of hydride transfer. Importantly, analysis of the correlated and principle component motions in the enzyme suggest that the mutation alters the coupled motions that are present in the wild-type enzyme. In the case of D122N and D122S, the mutations inhibit coupled motion, whereas in the case of D122A, the mutation enhances coupled motion, although all mutations result in similar rate reduction. These results emphasize a Goldilocks principle of enzyme flexibility, that is, enzymes should neither be too rigid nor too flexible.

Structurally Flexible Oxocarbenium/Borohydride Ion Pair: Dynamics of Hydride Transfer on the Background of Conformational Roaming.

We apply Born-Oppenheimer molecular dynamics to the practically significant [dioxane-H(+)-acetone][(C6F5)3B-H(-)] and [Et2O-H(+)-OCPr2][(C6F5)3B-H(-)] ion pair intermediates. Dynamics of hydride transfer in cation/anion ion pair takes place on the background of large-amplitude configurational changes. Geometry of oxocarbenium/borohydride ion pairs is flexible, meaning that we uncover significant actual structural disorder at a finite temperature. Therefore, although the starting structure can be fairly close to the configurational area of the hydride transfer transition state (TS) and despite a low potential energy barrier (ca. 1.5 kcal/mol, according to the literature), already at T ≈ 325 K the system can remain ignorant of the TS region and move round and about ("roam") in the configurational space for a period of time in the range between 10 and 100 ps. This indicates structural flexibility of oxocarbenium/borohydride ion pair on apparently a flat potential energy "landscape" of cation/anion interaction, and this has not been taken into consideration by the free energy estimations in static considerations made thus far. The difference between the dynamics-based representation of the system versus the static representation amounts to the difference between quasi-bimolecular versus unimolecular descriptions of the hydride transfer step.

On the role of oxocarbenium ions formed in Brønsted acidic condition on γ-Al2O3 surface in the ring-opening of γ-valerolactone

The ring-opening and decarboxylation reaction of γ-valerolactone (GVL), yielding a mixture of butene isomers, constitutes an important transformative step for the upgradation of biomass-derived precursors into high value fuels and chemicals. In this study, density functional theory (DFT) simulations were utilized to gain insights into the ring-opening of GVL in presence of Brønsted acid promoted γ-Al2O3 catalyst. DFT simulations suggested towards the formation of stable oxocarbenium ion of GVL on the γ- Al2O3 surface via a proton transfer to the carbonyl oxygen in presence of the Brønsted acid. The surface oxygen atoms of γ-Al2O3 were observed to show high affinity for H-abstraction from the oxocarbenium ion to yield the carbenium ion intermediate, which subsequently gave the product butene via a combination of hydride transfer and decarboxylation steps. Higher reactivity of the Brønsted acid promoted γ-Al2O3 surface, was attributed to the direct ring-opening (Ea = 13 kJ/mol) of the surface adsorbed oxocarbenium ion of GVL, leading to the formation of the γ-carbenium ion, which was unlikely to form with the adsorbed GVL structure. In addition, H-abstraction by the surface oxygen from the C5 of adsorbed oxocarbenium ion (Ea = 13 kJ/mol) was calculated to be of lower activation barrier as compared to the H-abstraction from C5 of the adsorbed GVL (Ea = 40 kJ/mol), which may also lead to facile ring-opening in presence of a Brønsted acid on γ-Al2O3.

Dynamic Characteristics of Guanosine-5'-monophosphate Reductase Complexes Revealed by High-Resolution 31P Field-Cycling NMR Relaxometry.

The ability of enzymes to modulate the dynamics of bound substrates and cofactors is a critical feature of catalysis, but the role of dynamics has largely been approached from the perspective of the protein. Here, we use an underappreciated NMR technique, subtesla high-resolution field-cycling 31P NMR relaxometry, to interrogate the dynamics of enzyme bound substrates and cofactors in guanosine-5'-monophosphate reductase (GMPR). These experiments reveal distinct binding modes and dynamic profiles associated with the 31P nuclei in the Michaelis complexes for the deamination and hydride transfer steps of the catalytic cycle. Importantly, the substrate is constrained and the cofactor is more dynamic in the deamination complex E·GMP·NADP+, whereas the substrate is more dynamic and the cofactor is constrained in the hydride transfer complex E·IMP·NADP+. The presence of D2O perturbed the relaxation of the 31P nuclei in E·IMP·NADP+ but not in E·GMP·NADP+, providing further evidence of distinct binding modes with different dynamic properties. dIMP and dGMP are poor substrates, and the dynamics of the cofactor complexes of dGMP/dIMP are disregulated relative to GMP/IMP. The substrate 2'-OH interacts with Asp219, and mutation of Asp219 to Ala decreases the value of Vmax by a factor of 30. Counterintuitively, loss of Asp219 makes both substrates and cofactors less dynamic. These observations suggest that the interactions between the substrate 2'-OH and Asp219 coordinate the dynamic properties of the Michaelis complexes, and these dynamics are important for progression through the catalytic cycle.

Revised Theoretical Model on Enantiocontrol in Phosphoric Acid Catalyzed H-Transfer Hydrogenation of Quinoline

The enantioselective H-transfer hydrogenation of quinoline by Hantzsch ester is a relevant example of Brønsted acid catalyzed cascade reactions, with phosphoric acid being a privileged catalyst. The generally accepted mechanism points out the hydride transfer step as the rate- and stereodetermining step, however computations based on these models do not totally fit with experimental observations. We hereby present a computational study that enlightens the stereochemical outcome and quantitatively reproduces the experimental enantiomeric excesses in a series of H-transfer hydrogenations. Our calculations suggest that the high stereocontrol usually attained with BINOL-derived phosphoric acids results mostly from the steric constraints generated by an aryl substituent of the catalyst, which hinders the access of the Hantzsch ester to the catalytic site and enforces approach through a specific way. It relies on a new model involving the preferential assembly of one of the stereomeric complexes formed by the chiral phosphoric acid and the two reaction partners. The stereodetermining step thus occurs prior to the H-transfer step.

Temperature dependence of dynamic, tunnelling and kinetic isotope effects in formate dehydrogenase.

The origin of the catalytic power of enzymes has been a question of debate for a long time. In this regard, the possible contribution of protein dynamics in enzymatic catalysis has become one of the most controversial topics. In the present work, the hydride transfer step in the formate dehydrogenase (FDH EC 1.2.1.2) enzyme is studied by means of molecular dynamic (MD) simulations with quantum mechanics/molecular mechanics (QM/MM) potentials in order to explore any correlation between dynamics, tunnelling effects and the rate constant. The temperature dependence of the kinetic isotope effects (KIEs), which is one of the few tests that can be studied by experiments and simulations to shed light on this debate, has been computed and the results have been compared with previous experimental data. The classical mechanical free energy barrier and the number of recrossing trajectories seem to be temperature-independent while the quantum vibrational corrections and the tunnelling effects are slightly temperature-dependent over the interval of 5-45 °C. The computed primary KIEs are in very good agreement with previous experimental data, being almost temperature-independent within the standard deviations. The modest dependence on the temperature is due to just the quantum vibrational correction contribution. These results, together with the analysis of the evolution of the collective variables such as the electrostatic potential or the electric field created by the protein on the key atoms involved in the reaction, confirm that while the protein is well preorganised, some changes take place along the reaction that favour the hydride transfer and the product release. Coordinates defining these movements are, in fact, part of the real reaction coordinate.

Understanding the Relationship Between Kinetics and Thermodynamics in CO2Hydrogenation Catalysis

Catalysts that are able to reduce carbon dioxide under mild conditions are highly sought after for storage of renewable energy in the form of a chemical fuel. This study describes a systematic kinetic and thermodynamic study of a series of cobalt and rhodium bis(diphosphine) complexes that are capable of hydrogenating carbon dioxide to formate under ambient temperature and pressure. Catalytic CO2 hydrogenation was studied under 1.8 and 20 atm of pressure (1:1 CO2/H2) at room temperature in tetrahydrofuran with turnover frequencies (TOF) ranging from 20 to 74 000 h–1. The catalysis was followed by 1H and 31P NMR spectroscopy in real time under all conditions to yield information about the rate-determining step. The cobalt catalysts displayed a rate-determining step of hydride transfer to CO2, while the hydrogen addition and/or deprotonation steps were rate limiting for the rhodium catalysts. Thermodynamic analysis of the complexes provided information on the driving force for each step of catalysis in term...

The role of the Met20 loop in the hydride transfer in Escherichia coli dihydrofolate reductase

A key question concerning the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) is whether the Met20 loop is dynamically coupled to the chemical step during catalysis. A more basic, yet unanswered question is whether the Met20 loop adopts a closed conformation during the chemical hydride transfer step. To examine the most likely conformation of the Met20 loop during the chemical step, we studied the hydride transfer in wild type (WT) ecDHFR using hybrid quantum mechanics-molecular mechanics free energy simulations with the Met20 loop in a closed and disordered conformation. Additionally, we investigated three mutant forms (I14X; X = Val, Ala, Gly) of the enzyme that have increased active site flexibility and donor-acceptor distance dynamics in closed and disordered Met20 loop states. We found that the conformation of the Met20 loop has a dramatic effect on the ordering of active site hydration, although the Met20 loop conformation only has a moderate effect on the hydride transfer rate and donor-acceptor distance dynamics. Finally, we evaluated the pKa of the substrate N5 position in closed and disordered Met20 loop states and found a strong correlation between N5 basicity and the conformation of the Met20 loop.

Unsymmetrical Iron P-NH-P' Catalysts for the Asymmetric Pressure Hydrogenation of Aryl Ketones.

The reductive amination of α-dialkylphosphine acetaldehydes with enantiopure β-aminophosphines is a new, versatile route to unsymmetrical tridentate (pincer) ligands P-NH-P'. Four new ligands PR2 CH2 CH2 NHCHR'CHR''PPh2 (R=iPr, Cy, R'=Ph, CH(CH3 )2 , R''=Ph, H) prepared in this way are used to make the iron(II) complexes mer-FeCl2 (CO)(P-NH-P') and mer-FeCl(H)(CO)(P-NH-P'). The hydride complex with the rigid ligand with R'=R''=Ph is an efficient and highly enantioselective homogeneous asymmetric pressure hydrogenation (APH) catalyst. Prochiral aryl ketones are reduced under mild conditions (THF, 0.1 mol % catalyst, 1 mol % KOtBu, 5-10 bar, 50 °C) to the (S)-alcohols, usually in enantiomeric excess (ee) greater than 90 %. DFT calculations provided transition-state structures for the enantiodetermining hydride-transfer step.

Interaction and electron transfer between ferredoxin-NADP+ oxidoreductase and its partners: structural, functional, and physiological implications.

Ferredoxin-NADP+ reductase (FNR) catalyzes the last step of linear electron transfer in photosynthetic light reactions. The FAD cofactor of FNR accepts two electrons from two independent reduced ferredoxin molecules (Fd) in two sequential steps, first producing neutral semiquinone and then the fully anionic reduced, or hydroquinone, form of the enzyme (FNRhq). FNRhq transfers then both electrons in a single hydride transfer step to NADP+. We are presenting the recent progress in studies focusing on Fd:FNR interaction and subsequent electron transfer processes as well as on interaction of FNR with NADP+/H followed by hydride transfer, both from the structural and functional point of views. We also present the current knowledge about the physiological role(s) of various FNR isoforms present in the chloroplasts of higher plants and the functional impact of subchloroplastic location of FNR. Moreover, open questions and current challenges about the structure, function, and physiology of FNR are discussed.