Influences of the Hydrophobicity of the Heme－binding Pocket on the Properties and Functions of Cytochrome b5 Mutants

The mutation sites of the four mutants F35Y, P40V, V45E and V45Y of cytochrome b5 are located at the edge of the heme-binding pocket. The solvent accessible areas of the “pocket inte-rior“ of the four mutants and the wild-type cytochrome b5 have been calculated based on their crystal structures at high resolu-tion. The change in the hydrophobicity of the heme-binding pocket resulting from the mutation can be quantitatively de-scribed using the difference of the solvent accessible area of the “pocket interior“ of each mutant from that of the wild-type cy-tochrome b5. The influences of the hydrophobicity of the heme-binding pocket on the protein stability and redox potential are discussed.     

Proline-40 is Essential to Maintaining Cytochrome b_5''''s Stability and Its Electron Transfer with Cytochrome c

ＩｎｔｒｏｄｕｃｔｉｏｎＣｙｔｏｃｈｒｏｍｅｂ5(Ｃｙｔｂ5)ｉｓａｍｅｍｂｒａｎｅ ｂｏｕｎｄｐｒｏ ｔｅｉｎ .Ｉｔｃａｎｂｅｐｒｏｔｅｏｌｙｚｅｄｔｏｙｉｅｌｄａｓｏｌｕｂｌｅ ,ｈｙｄｒｏｐｈｉｌｉｃｄｏｍａｉｎｃｏｎｔａｉｎｉｎｇａｎｏｎ ｃｏｖａｌｅｎｔｌｙｂｏｕｎｄｈｅｍｅｇｒｏｕｐ .Ｃｙｔｂ5ｉｓｉｎｖｏｌｖｅｄｉｎｅｌｅｃｔｒｏｎｔｒａｎｓｆｅｒｗｉｔｈａｖａｒｉｅｔｙｏｆｐｒｏｔｅｉｎｓ,ｓｕｃｈａｓｃｙｔｏｃｈｒｏｍｅｃ (Ｃｙｔｃ) ,1 3 ｍｅｔｍｙｏ ｇｌｏｂｉｎ ,2 ｍｅｔｈｅｍｏｇｌｏ…     

The CO Photodissociation and Recombination Dynamics of the W172Y/F282T Ligand Channel Mutant of Rhodobacter sphaeroides aa3 Cytochrome c Oxidase

In the ligand channel of the cytochrome c oxidase from Rhodobacter sphaeroides (Rs aa₃) W172 and F282 have been proposed to generate a constriction that may slow ligand access to and from the active site. To explore this issue, the tryptophan and phenylalanine residues in Rs aa₃ were mutated to the less bulky tyrosine and threonine residues, respectively, which occupy these sites in Thermus thermophilus (Tt) ba₃ cytochrome oxidase. The CO photolysis and recombination dynamics of the reduced wild‐type Rs aa₃ and the W172Y/F282T mutant were investigated using time‐resolved optical absorption spectroscopy. The spectral changes associated with the multiple processes are attributed to different conformers. The major CO recombination process (44 μs) in the W172Y/F282T mutant is ~500 times faster than the predominant CO recombination process in the wild‐type enzyme (~23 ms). Classical dynamic simulations of the wild‐type enzyme and double mutant showed significant structural changes at the active site in the mutant, including movement of the heme a₃ ring‐D propionate toward Cu_B and reduced binuclear center cavity volume. These structural changes effectively close the ligand exit pathway from the binuclear center, providing a basis for the faster CO recombination in the double mutant.     

Regioselective Hydroxylation of C12–C15 Fatty Acids with Fluorinated Substituents by Cytochrome P450 BM3

We demonstrate herein that wild‐type cytochrome P450 BM3 can recognize non‐natural substrates, such as fluorinated C₁₂–C₁₅ chain‐length fatty acids, and show better catalysis for their efficient conversion. Although the binding affinities for fluorinated substrates in the P450 BM3 pocket are marginally lower than those for non‐fluorinated substrates, spin‐shift measurements suggest that fluoro substituents at the ω‐position can facilitate rearrangement of the dynamic structure of the bulk‐water network within the hydrophobic pocket through a micro desolvation process to expel the water ligand of the heme iron that is present in the resting state. A lowering of the Michaelis–Menten constant (K_m), however, indicates that fluorinated fatty acids are indeed better substrates compared with their non‐fluorinated counterparts. An enhancement of the turnover frequencies (k_cat) for electron transfer from NADPH to the heme iron and for C? H bond oxidation by compound I (Cpd I) to yield the product suggests that the activation energies associated with going from the enzyme–substrate (ES state) to the corresponding transition state (ES^≠ state) are significantly lowered for both steps in the case of the fluorinated substrates. Delicate control of the regioselectivity by the fluorinated terminal methyl groups of the C₁₂–C₁₅ fatty acids has been noted. Despite the fact that residues Arg47/Tyr51/Ser72 exert significant control over the hydroxylation of the subterminal carbon atoms toward the hydrocarbon tail, the fluorine substituent(s) at the ω‐position affects the regioselective hydroxylation. For substrate hydroxylation, we have found that fluorinated lauric acids probably give a better structural fit for the heme pocket than fluorinated pentadecanoic acid, even though pentadecanoic acid is by far the best substrate among the reported fatty acids. Interestingly, 12‐fluorododecanoic acid, with only one fluorine atom at the terminal methyl group, exhibits a comparable turnover frequency to that of pentadecanoic acid. Thus, fluorination of the terminal methyl group introduces additional interactions of the substrate within the hydrophobic pocket, which influence the electron transfers for both dioxygen activation and the controlled oxidation of aliphatics mediated by high‐valent oxoferryl species.     

Folding behaviors of apocytochrome b5 and its mutants: Insights from high temperature molecular dynamics simulations

Apocytochrome b₅ (apocyt b₅) with heme removal from the heme-binding core 1, and its mutants with amino acid replaced in the hydrophobic core 2, namely apocyt b₅ Y7P, P81A and H15R/S20E, have been subjected to molecular dynamics (MD) simulation at high temperature (500 K) for elucidating their folding behaviors. The early events upon thermal induced unfolding were found to be in good agreement with available experimental results, and the lowest stability of Y7P was predicted among the four apoproteins. The influences of these key residues on protein folding behavior were compared directly at an atomic level. At the same time, the influences of non-native interactions of hydrogen bonds and salt-bridges on protein stabilities were analyzed in detail. The insights from current MD simulations are valuable for understanding the apoprotein folding and the holoprotein formation in terms of heme proteins.     

Targeting the Substrate Binding Site of E. coli Nitrile Reductase QueF by Modeling,Substrate and Enzyme Engineering

Nitrile reductase QueF catalyzes the reduction of 2‐amino‐5‐cyanopyrrolo[2,3‐d]pyrimidin‐4‐one (preQ₀) to 2‐amino‐5‐aminomethylpyrrolo[2,3‐d]pyrimidin‐4‐one (preQ₁) in the biosynthetic pathway of the hypermodified nucleoside queuosine. It is the only enzyme known to catalyze a reduction of a nitrile to its corresponding primary amine and could therefore expand the toolbox of biocatalytic reactions of nitriles. To evaluate this new oxidoreductase for application in biocatalytic reactions, investigation of its substrate scope is prerequisite. We report here an investigation of the active site binding properties and the substrate scope of nitrile reductase QueF from Escherichia coli. Screenings with simple nitrile structures revealed high substrate specificity. Consequently, binding interactions of the substrate to the active site were identified based on a new homology model of E. coli QueF and modeled complex structures of the natural and non‐natural substrates. Various structural analogues of the natural substrate preQ₀ were synthesized and screened with wild‐type QueF from E. coli and several active site mutants. Two amino acid residues Cys190 and Asp197 were shown to play an essential role in the catalytic mechanism. Three non‐natural substrates were identified and compared to the natural substrate regarding their specific activities by using wild‐type and mutant nitrile reductase.     

神经红蛋白突变体(F28Y,F106Y)的构建、 表达与表征

构建了神经红蛋白F28Y, F106Y的两种突变体, 并进行了表达、 纯化和谱学表征. 电喷雾质谱表明突变体蛋白的分子量与理论值一致. 氧化型和还原型F28Y及F106Y的紫外 可见吸收光谱与野生型相似, 仅氧化型F28Y的Soret 带有2 nm的蓝移, 说明这两种突变体蛋白仍保持六配位形式. F28Y的荧光最大发射峰明显红移(340 nm→347 nm), 表明其荧光基团更加暴露于极性环境中. 圆二色光谱表明, 突变体蛋白的α 螺旋含量降低且F28Y产生了β 折叠, 这是由于F28相对于F106则位于疏水腔外部且更加接近于溶剂表面所致. 热稳定性顺序为NGB>F28Y>F106Y, F106Y最不稳定, 是因为其与血红素间存在着较强的疏水作用, 突变使F106与血红素间的作用力减弱, 从而导致血红素在热变性条件下更容易从蛋白中解离出来.     

Mimicking Tyrosine Phosphorylation in Human Cytochrome c by the Evolved tRNA Synthetase Technique

Phosphorylation of tyrosine 48 of cytochrome c is related to a wide range of human diseases due to the pleiotropic role of the heme‐protein in cell life and death. However, the structural conformation and physicochemical properties of phosphorylated cytochrome c are difficult to study as its yield from cell extracts is very low and its kinase remains unknown. Herein, we report a high‐yielding synthesis of a close mimic of phosphorylated cytochrome c, developed by optimization of the synthesis of the non‐canonical amino acid p‐carboxymethyl‐L ‐phenylalanine (pCMF) and its efficient site‐specific incorporation at position 48. It is noteworthy that the Y48pCMF mutation significantly destabilizes the Fe?Met bond in the ferric form of cytochrome c, thereby lowering the pK_a value for the alkaline transition of the heme‐protein. This finding reveals the differential ability of the phosphomimic protein to drive certain events. This modified cytochrome c might be an important tool to investigate the role of the natural protein following phosphorylation.     

Oxygen‐binding Protein Fiber and Microgel: Supramolecular Myoglobin–Poly(acrylate) Conjugates

A supramolecular conjugate of myoglobin (Mb) and water‐soluble poly(acrylate), (PA_5k and PA_25k, where 5k and 25k represent the molecular weight of the polymers, respectively), is constructed on the basis of a noncovalent heme‐heme pocket interaction. The modified heme with an amino group linked to the terminus of one of the heme‐propionates is coupled to the side‐chain carboxyl groups of poly(acrylate) activated by N‐hydroxysuccinimide. The ratios of the heme‐modified monomer unit and the unmodified monomer unit (m:n) in the polymer chains of Heme‐PA_5k and Heme‐PA_25k were determined to be 4.5:95.5 and 3.1:96.9, respectively. Subsequent addition of apoMb to the conjugated polymers provides Mb‐connected fibrous nanostructures confirmed by atomic force microscopy. A mixture of the heme‐modified polymer and dimeric apomyoglobin as a cross‐linker forms a microgel in which the reconstituted myoglobin retains its native exogenous ligand binding activity.     

Crystal Structures of Bacterial (6‐4) Photolyase Mutants with Impaired DNA Repair Activity

PhrB from Agrobacterium fabrum is the first prokaryotic photolyase which repairs (6‐4) UV DNA photoproducts. The protein harbors three cofactors: the enzymatically active FAD chromophore, a second chromophore, 6,7‐dimethyl‐8‐ribityllumazine (DMRL) and a cubane‐type Fe‐S cluster. Tyr424 of PhrB is part of the DNA‐binding site and could provide an electron link to the Fe‐S cluster. The PhrB_Y424F mutant showed reduced binding of lesion DNA and loss of DNA repair. The mutant PhrB_I51W is characterized by the loss of the DMRL chromophore, reduced photoreduction and reduced DNA repair capacity. We have determined the crystal structures of both mutants and found that both mutations only affect local protein environments, whereas the overall fold remained unchanged. The crystal structure of PhrB_Y424F revealed a water network extending to His366, which are part of the lesion‐binding site. The crystal structure of PhrB_I51W shows how the bulky Trp leads to structural rearrangements in the DMRL chromophore pocket. Spectral characterizations of PhrB_I51W suggest that DMRL serves as an antenna chromophore for photoreduction and DNA repair in the wild type. The energy transfer from DMRL to FAD could represent a phylogenetically ancient process.     

Stabilization of the Oxidation State +IV in Siloxide‐Supported Terbium Compounds

The synthesis of lanthanides other than cerium in the oxidation state +IV has remained a desirable but unmet target until recently, when two examples of Tb^IV with saturated coordination spheres were isolated. Here we report the third example of an isolated molecular complex of terbium(IV), where the supporting siloxide ligands do not saturate the coordination sphere. The fully characterized six‐coordinate complex [Tb^IV(OSiPh₃)₄(MeCN)₂], 2 ‐Tb^Ph, shows high stability and the labile MeCN ligands can be replaced by phosphinoxide ligands. Computational studies suggest that the stability is due to a strong π(O?Tb) interaction which is stronger than in the previously reported Tb^IV complexes. Cyclic‐voltammetry experiments demonstrate that non‐binding counterions contribute to the stability of Tb^IV in solution by destabilizing the +III oxidation state, while alkali ions promote Tb^IV/Tb^III electron transfer.     

Additive Effect of Mutations Affecting the Rate of Phylloquinone Reoxidation and Directionality of Electron Transfer within Photosystem I†

Optical pump‐probe spectroscopy in the nanosecond–microsecond timescale has been used to study the electron transfer reactions taking place within the Photosystem I reaction center of intact Chlamydomonas reinhardtii cells. The biphasic kinetics of phylloquinone (PhQ) reoxidation were investigated in double mutants that combine a mutation (PsaA‐Y696F) near the primary acceptor chlorophyll, ec3_A, with those near PhQ_A (PsaA‐S692A, PsaA‐W697F). The PsaA‐S692A and PsaA‐W697F mutations selectively lengthened the 200 ns lifetime component observed in the wild‐type (WT). The ?20 ns component was unaltered in the single mutant, both in terms of lifetime and relative amplitude. However, both double mutants possessed a ?20 ns component (PhQ_B^? reoxidation) with increased amplitude compared with the WT and the individual PhQ_A mutants. The component assigned to PhQ_A^? reoxidation was slowed, like the individual PhQ_A mutants, and of lower amplitude, as observed in the single ec3_A mutant. Hence, the effects of these mutations are almost entirely additive, providing strong support for the previously proposed bidirectional electron transfer model, which attributes the ?20 and ?200 ns phases to reoxidation of PhQ_B or PhQ_A, respectively. Moreover, in all the mutants investigated, it was also possible to observe an intermediate (～180 ns) component, as previously reported for mutants of the PhQ_A binding pocket (Biochim. Biophys. Acta [2006] 1757 , 1529–1538), which we have tentatively attributed to forward electron transfer between the iron–sulfur clusters F _X and F _A/B .     

Chemo‐ and Regioselective Dihydroxylation of Benzene to Hydroquinone Enabled by Engineered Cytochrome P450 Monooxygenase

Hydroquinone (HQ) is produced commercially from benzene by multi‐step Hock‐type processes with equivalent amounts of acetone as side‐product. We describe an efficient biocatalytic alternative using the cytochrome P450‐BM3 monooxygenase. Since the wildtype enzyme does not accept benzene, a semi‐rational protein engineering strategy was developed. Highly active mutants were obtained which transform benzene in a one‐pot sequence first into phenol and then regioselectively into HQ without any overoxidation. A computational study shows that the chemoselective oxidation of phenol by the P450‐BM3 variant A82F/A328F leads to the regioselective formation of an epoxide intermediate at the C3=C4 double bond, which departs from the binding pocket and then undergoes fragmentation in aqueous medium with exclusive formation of HQ. As a practical application, an E. coli designer cell system was constructed, which enables the cascade transformation of benzene into the natural product arbutin, which has anti‐inflammatory and anti‐bacterial activities.     

Impact of proximal and distal pocket site-directed mutations on the ferric/ferrous heme redox potential of yeast cytochrome c peroxidase

Cytochrome c peroxidase (CCP) contains a five-coordinate heme active site. The reduction potential for the ferric to ferrous couple in CCP is anomalously low and pH dependent (Eo?=?~?180?mV vs. S.H.E. at pH 7). The contribution of the protein environment to the tuning of the redox potential of this couple is evaluated using site-directed mutants of several amino acid residues in the environment of the heme. These include proximal pocket mutation of residues Asp-235, Trp-191, Phe-202, and His-175, distal pocket mutation of residues Trp-51, His-52, and Arg-48; and a heme edge mutation of Ala-147. Where unknown, the structural changes resulting from the amino acid substitution have been studied by X-ray crystallography. In most cases, ostensibly polar or charged residues are replaced by large hydrophobic groups or alternatively by Ala or Gly. These latter have been shown to generate large, solvent-filled cavities. Reduction potentials are measured as a function of pH by spectroelectrochemistry. Starting with the X-ray-derived structures of CCP and the mutants, or with predicted structures generated by molecular dynamics (MD), predictions of redox potential changes are modeled using the protein dipoles Langevin dipoles (PDLD) method. These calculations serve to model an electrostatic assessment of the redox potential change with simplified assumptions about heme iron chemistry, with the balance of the experimentally observed shifts in redox potential being thence attributed to changes in the ligand set and heme coordination chemistry, and/or other changes in the structure not directly evident in the X-ray structures (e.g., ionization states, specific roles played by solvent species, or conformationally flexible portions of the protein). Agreement between theory and experiment is good for all mutant proteins with the exception of the mutation Arg 48 to Ala, and His 52 to Ala. In the former case, the influence of phosphate buffer is adduced to account for the discrepancy, with evidence for phosphate binding in the distal pocket, and measurements made in a bis?Ctris propane/2-(N-morpholino)ethanesulfonic acid buffer system agree well with theory. For the latter case, an unknown structural element relevant to His-52 and/or solvent influence in the mutant akin to anion binding in the distal pocket (though lacking proof that it is, and in this case lacking a phosphate effect) manifests in this mutant. The use of exogenous (sixth) ligands in dissecting the contributions to control of redox potential is also explored as a pathway for model building.     

Kinetic Isotope Effects and Hydrogen/Deuterium Exchange Reveal Large Conformational Changes During the Catalysis of the Clostridium acetobutylicum Spore Photoproduct Lyase

Spore photoproduct lyase (SPL) catalyzes the direct reversal of a thymine dimer 5‐thyminyl‐5,6‐dihydrothymine (i.e. the spore photoproduct (SP)) to two thymine residues in germinating endospores. Previous studies suggest that SPL from the bacterium Bacillus subtilis (Bs) harbors an unprecedented radical‐transfer pathway starting with cysteine 141 proceeding through tyrosine 99. However, in SPL from the bacterium Clostridium acetobutylicum (Ca), the cysteine (at position 74) and the tyrosine are located on the opposite sides of a substrate‐binding pocket that has to collapse to bring the two residues into proximity, enabling the C→Y radical passage as implied in SPL_(Bs). To test this hypothesis, we adopted hydrogen/deuterium exchange mass spectrometry (HDX‐MS) to show that C74_(Ca) is located at a highly flexible region. The repair of dinucleotide SP TpT by SPL_(Ca) is eight‐fold to 10‐fold slower than that by SPL_(Bs); the process also generates a large portion of the aborted product TpTSO₂^?. SPL_(Ca) exhibits apparent (^DV) kinetic isotope effects (KIEs) of ~6 and abnormally large competitive (^DV/K) KIEs (~20), both of which are much larger than the KIEs observed for SPL_(Bs). All these observations indicate that SPL_(Ca) possesses a flexible active site and readily undergoes conformational changes during catalysis.     

Structure‐Guided Minimalist Redesign of the L‐Fuculose‐1‐Phosphate Aldolase Active Site: Expedient Synthesis of Novel Polyhydroxylated Pyrrolizidines and their Inhibitory Properties Against Glycosidases and Intestinal Disaccharidases

A minimalist active site redesign of the L ‐fuculose‐1‐phosphate aldolase from E. coli FucA was envisaged, to extend its tolerance towards bulky and conformationally restricted N‐Cbz‐amino aldehyde acceptor substrates (Cbz=benzyloxycarbonyl). Various mutants at the active site of the FucA wild type were obtained and screened with seven sterically demanding N‐Cbz‐amino aldehydes including N‐Cbz‐prolinal derivatives. FucA F131A showed an aldol activity of 62 μmol h^?1 mg^?1 with (R)‐N‐Cbz‐prolinal, whereas no detectable activity was observed with the FucA wild type. For the other substrates, the F131A mutant gave aldol activities from 4 to about 25 times higher than those observed with the FucA wild type. With regard to the stereochemistry of the reactions, the (R)‐amino aldehydes gave exclusively the anti configured aldol adducts whereas their S counterparts gave variable ratios of anti/syn diastereoisomers. Interestingly, the F131A mutant was highly stereoselective both with (R)‐ and with (S)‐N‐Cbz‐prolinal, exclusively producing the anti and syn aldol adducts, respectively. Molecular models suggest that this improved activity towards bulky and more rigid substrates, such as N‐Cbz‐prolinal, could arise from a better fit of the substrate into the hydrophobic pocket created by the F131A mutation, due to an additional π–cation interaction with the residue K205′ and to efficient contact between the substrate and the mechanistically important Y113′ and Y209′ residues. An expedient synthesis of novel polyhydroxylated pyrrolizidines related to the hyacinthacine and alexine types was accomplished through aldol additions of dihydroxyacetone phosphate (DHAP) to hydroxyprolinal derivatives with the hyperactive FucA F131A as catalyst. The iminocyclitols obtained were fully characterised and found to be moderate to weak inhibitors (relative to 1,4‐dideoxy‐1,4‐imino‐L ‐arabinitol (LAB) and 1,4‐dideoxy‐1,4‐imino‐D ‐arabinitol (DAB)) against glycosidases and rat intestinal saccharidases.     

Origins of the Intermediate Spectral Form in M100 Mutants of Photoactive Yellow Protein

Numerous single‐site mutants of photoactive yellow protein (PYP) from Halorhodospira halophila and as well as PYP homologs from other species exhibit a shoulder on the short wavelength side of the absorbance maximum in their dark‐adapted states. The structural basis for the occurrence of this shoulder, called the “intermediate spectral form,” has only been investigated in detail for the Y42F mutation. Here we explore the structural basis for occurrence of the intermediate spectral form in a M121E derivative of a circularly permuted H. halophila PYP (M121E‐cPYP). The M121 site in M121E‐cPYP corresponds to the M100 site in wild‐type H. halophila PYP. High‐resolution NMR measurements with a salt‐tolerant cryoprobe enabled identification of those residues directly affected by increasing concentrations of ammonium chloride, a salt that greatly enhances the fraction of the intermediate spectra form. Residues in the surface loop containing the M121E (M100E) mutation were found to be affected by ammonium chloride as well as a discrete set of residues that link this surface loop to the buried hydroxyl group of the chromophore via a hydrogen bond network. Localized changes in the conformational dynamics of a surface loop can thereby produce structural rearrangements near the buried hydroxyl group chromophore while leaving the large majority of residues in the protein unaffected.     

Binding structures of tri‐N‐acetyl‐β‐glucosamine in hen egg white lysozyme using molecular dynamics with a polarizable force field

Lysozyme is a well‐studied enzyme that hydrolyzes the β‐(1,4)‐glycosidic linkage of N‐acetyl‐β‐glucosamine (NAG)_n oligomers. The active site of hen egg‐white lysozyme (HEWL) is believed to consist of six subsites, A‐F that can accommodate six sugar residues. We present studies exploring the use of polarizable force fields in conjunction with all‐atom molecular dynamics (MD) simulations to analyze binding structures of complexes of lysozyme and NAG trisaccharide, (NAG)₃. MD trajectories are applied to analyze structures and conformation of the complex as well as protein–ligand interactions, including the hydrogen‐bonding network in the binding pocket. Two binding modes (ABC and BCD) of (NAG)₃ are investigated independently based on a fixed‐charge model and a polarizable model. We also apply molecular mechanics with generalized born and surface area (MM‐GBSA) methods based on MD using both nonpolarizable and polarizable force fields to compute binding free energies. We also study the correlation between root‐mean‐squared deviation and binding free energies of the wildtype and W62Y mutant; we find that for this prototypical system, approaches using the MD trajectories coupled with implicit solvent models are equivalent for polarizable and fixed‐charge models. © 2012 Wiley Periodicals, Inc.     

Enhancement of Photophysical and Photosensitizing Properties of Flavin Adenine Dinucleotide by Mutagenesis of the C‐Terminal Extension of a Bacterial Flavodoxin Reductase

The role of the mobile C‐terminal extension present in Rhodobacter capsulatus ferredoxin–NADP(H) reductase (RcFPR) was evaluated using steady‐state and dynamic spectroscopies for both intrinsic Trp and FAD in a series of mutants in the absence of NADP(H). Deletion of the six C‐terminal amino acids beyond Ala266 was combined with the replacement A266Y to emulate the structure of plastidic reductases. Our results show that these modifications of the wild‐type RcFPR produce subtle global conformational changes, but strongly reduce the local rigidity of the FAD‐binding pocket, exposing the isoalloxazine ring to the solvent. Thus, the ultrafast charge‐transfer quenching of ¹FAD* by the conserved Tyr66 residue was absent in the mutant series, producing enhancement of the excited singlet‐ and triplet‐state properties of FAD. This work highlights the delicate balance of the specific interactions between FAD and the surrounding amino acids, and how the functionality and/or photostability of redox flavoproteins can be modified.