Nuclear magnetic resonance studies of the old yellow enzyme. 2. 13C NMR of the enzyme recombined with 13C-labeled flavin mononucleotides

The apoenzyme of NADPH oxidoreductase, 'old yellow enzyme', was reconstituted with selectively 13C-enriched flavin mononucleotides and investigated by 13C NMR spectroscopy. The 13C NMR results confirm the results obtained by 15N NMR spectroscopy and yield additional information about the coenzyme-apoenzyme interaction. A strong deshielding of the C(2) and C(4) atoms of enzyme-bound FMN both in the oxidized and reduced state is observed, which is supposed to be induced by hydrogen-bond formation between the protein and the two carbonyl groups at C(2) and C(4) of the isoalloxazine ring system. The chemical shifts of all 13C resonances of the flavin in the two-electron-reduced state indicate that the N(5) atom is sp3-hybridized. From 31P NMR measurements it is concluded that the FMN phosphate group is not accessible to bulk solvent. The unusual 31P chemical shift of FMN in old yellow enzyme seems to indicate a different binding mode of the FMN phosphate group in this enzyme as compared to the flavodoxins. The 13C and 15N NMR data on the old-yellow-enzyme--phenolate complexes show that the atoms of the phenolate are more deshielded whereas the atoms of the enzyme-bound isoalloxazine ring are more shielded upon complexation. A non-linear correlation exists between the chemical shifts of the N(5) and the N(10) atoms and the pKa value of the phenolate derivative bound to the protein. Since the chemical shifts of N(5), N(10) and C(4a) are influenced most on complexation it is suggested that the phenolate is bound near the pyrazine ring of the isoalloxazine system. 15N NMR studies on the complex between FMN and 2-aminobenzoic acid indicate that the structure of this complex differs from that of the old-yellow-enzyme--phenolate complexes.     

Crystal structure of pentaerythritol tetranitrate reductase: "flipped" binding geometries for steroid substrates in different redox states of the enzyme.

Pentaerythritol tetranitrate reductase (PETN reductase) degrades high explosive molecules including nitrate esters, nitroaromatics and cyclic triazine compounds. The enzyme also binds a variety of cyclic enones, including steroids; some steroids act as substrates whilst others are inhibitors. Understanding the basis of reactivity with cyclic enones requires structural information for the enzyme and key complexes formed with steroid substrates and inhibitors. The crystal structure of oxidised and reduced PETN reductase at 1.5 A resolution establishes a close structural similarity to the beta/alpha-barrel flavoenzyme, old yellow enzyme. In complexes of oxidised PETN reductase with progesterone (an inhibitor), 1,4-androstadiene-3,17-dione and prednisone (both substrates) the steroids are stacked over the si-face of the flavin in an orientation different from that reported for old yellow enzyme. The specifically reducible 1,2 unsaturated bonds in 1,4-androstadiene-3,17-dione and prednisone are not optimally aligned with the flavin N5 in oxidised enzyme complexes. These structures suggest either relative "flipping" or shifting of the steroid with respect to the flavin when bound in different redox forms of the enzyme. Deuterium transfer from nicotinamide coenzyme to 1,4-androstadiene-3,17-dione via the enzyme bound FMN indicates 1alpha addition at the steroid C2 atom. These studies rule out lateral motion of the steroid and indicate that the steroid orientation is "flipped" in different redox states of the enzyme.     

Structure of the sodium borohydride-reduced N-(cyclopropyl)glycine adduct of the flavoenzyme monomeric sarcosine oxidase

Monomeric sarcosine oxidase (MSOX) is a flavoprotein that contains covalently bound FAD [8a-(S-cysteinyl)FAD] and catalyzes the oxidation of sarcosine (N-methylglycine) and other secondary amino acids, such as l-proline. Our previous studies showed that N-(cyclopropyl)glycine (CPG) acts as a mechanism-based inactivator of MSOX [Zhao, G., et al. (2000) Biochemistry 39, 14341-14347]. The reaction results in the formation of a modified reduced flavin that can be further reduced and stabilized by treatment with sodium borohydride. The borohydride-reduced CPG-modified enzyme exhibits a mass increase of 63 +/- 2 Da as compared with native MSOX. The crystal structure of the modified enzyme, solved at 1.85 A resolution, shows that FAD is the only site of modification. The modified FAD contains a fused five-membered ring, linking the C(4a) and N(5) atoms of the flavin ring, with an additional oxygen atom bound to the carbon atom attached to N(5) and a tetrahedral carbon atom at flavin C(4) with a hydroxyl group attached to C(4). On the basis of the crystal structure of the borohydride-stabilized adduct, we conclude that the labile CPG-modified flavin is a 4a,5-dihydroflavin derivative with a substituent derived from the cleavage of the cyclopropyl ring in CPG. The results are consistent with CPG-mediated inactivation in a reaction initiated by single electron transfer from the amine function in CPG to FAD in MSOX, followed by collapse of the radical pair to yield a covalently modified 4a,5-dihydroflavin.     

Structural basis of free reduced flavin generation by flavin reductase from Thermus thermophilus HB8

Free reduced flavins are involved in a variety of biological functions. They are generated from NAD(P)H by flavin reductase via co-factor flavin bound to the enzyme. Although recent findings on the structure and function of flavin reductase provide new information about co-factor FAD and substrate NAD, there have been no reports on the substrate flavin binding site. Here we report the structure of TTHA0420 from Thermus thermophilus HB8, which belongs to flavin reductase, and describe the dual binding mode of the substrate and co-factor flavins. We also report that TTHA0420 has not only the flavin reductase motif GDH but also a specific motif YGG in C terminus as well as Phe-41 and Arg-11, which are conserved in its subclass. From the structure, these motifs are important for the substrate flavin binding. On the contrary, the C terminus is stacked on the NADH binding site, apparently to block NADH binding to the active site. To identify the function of the C-terminal region, we designed and expressed a mutant TTHA0420 enzyme in which the C-terminal five residues were deleted (TTHA0420-ΔC5). Notably, the activity of TTHA0420-ΔC5 was about 10 times higher than that of the wild-type enzyme at 20-40 °C. Our findings suggest that the C-terminal region of TTHA0420 may regulate the alternative binding of NADH and substrate flavin to the enzyme.     

A rate-limiting conformational change of the flavin in p-hydroxybenzoate hydroxylase is necessary for ligand exchange and catalysis: studies with 8-mercapto- and 8-hydroxy-flavins

The FAD of p-hydroxybenzoate hydroxylase (PHBH) is known to exist in two conformations. The FAD must be in the in-position for hydroxylation of p-hydroxybenzoate (pOHB), whereas the out-position is essential for reduction of the flavin by NADPH. In these investigations, we have used 8-mercapto-FAD and 8-hydroxy-FAD to probe the movement of the flavin in catalysis. Under the conditions employed, 8-mercapto-FAD (pK(a) = 3.8) and 8-hydroxy-FAD (pK(a) = 4.8) are mainly anionic. The spectral characteristics of the anionic forms of these flavins are very sensitive to their environment, making them sensitive probes for detecting movement of the flavin during catalysis. With these flavin analogues, the enzyme hydroxylates pOHB efficiently, but at a rate much slower than that of enzyme with FAD. Reaction of oxygen with reduced forms of these modified enzymes in the absence of substrate appears to proceed through the formation of the flavin-C4a-hydroperoxide intermediate, as with normal enzyme, but the decay of this intermediate is so fast compared to its formation that very little accumulates during the reaction. However, after elimination of H2O2 from the flavin-C4a-hydroperoxide, a perturbed oxidized enzyme spectrum is observed (Eox*), and this converts slowly to the spectrum of the resting oxidized form of the enzyme (Eox). In the presence of pOHB, PHBH reconstituted with 8-mercapto-FAD also shows the additional oxidized intermediate (Eox*) after the usual oxygenated C4a-intermediates have formed and decayed in the course of the hydroxylation reaction. This Eox* to Eox step is postulated to be due to flavin movement. Furthermore, binding of pOHB to resting (Eox) follows a three-step equilibrium mechanism that is also consistent with flavin movement being the rate-limiting step. The rate for the slowest step during pOHB binding is similar to that observed for the conversion of Eox* to Eox during the oxygen reaction in the absence or presence of substrate. Steady-state kinetic analysis of PHBH substituted with 8-mercapto-FAD demonstrated that the apparent k(cat) is also similar to the rate of Eox* conversion to Eox. Presumably, the protein environment surrounding the flavin in Eox* differs slightly from that of the final resting form of the enzyme (Eox).     

Active site residues critical for flavin binding and 5,6-dimethylbenzimidazole biosynthesis in the flavin destructase enzyme BluB

The "flavin destructase" enzyme BluB catalyzes the unprecedented conversion of flavin mononucleotide (FMN) to 5,6-dimethylbenzimidazole (DMB), a component of vitamin B(12). Because of its unusual chemistry, the mechanism of this transformation has remained elusive. This study reports the identification of 12 mutant forms of BluB that have severely reduced catalytic function, though most retain the ability to bind flavin. The "flavin destructase" BluB is an unusual enzyme that fragments the flavin cofactor FMNH(2) in the presence of oxygen to produce 5,6-dimethylbenzimidazole (DMB), the lower axial ligand of vitamin B(12) (cobalamin). Despite the similarities in sequence and structure between BluB and the nitroreductase and flavin oxidoreductase enzyme families, BluB is the only enzyme known to fragment a flavin isoalloxazine ring. To explore the catalytic residues involved in this unusual reaction, mutants of BluB impaired in DMB biosynthesis were identified in a genetic screen in the bacterium Sinorhizobium meliloti. Of the 16 unique point mutations identified in the screen, the majority were located in conserved residues in the active site or in the unique "lid" domain proposed to shield the active site from solvent. Steady-state enzyme assays of 12 purified mutant proteins showed a significant reduction in DMB synthesis in all of the mutants, with eight completely defective in DMB production. Ten of these mutants have weaker binding affinities for both oxidized and reduced FMN, though only two have a significant effect on complex stability. These results implicate several conserved residues in BluB's unique ability to fragment FMNH(2) and demonstrate the sensitivity of BluB's active site to structural perturbations. This work lays the foundation for mechanistic studies of this enzyme and further advances our understanding of the structure-function relationship of BluB.     

Unusual folded conformation of nicotinamide adenine dinucleotide bound to flavin reductase P.

The 2.1 A resolution crystal structure of flavin reductase P with the inhibitor nicotinamide adenine dinucleotide (NAD) bound in the active site has been determined. NAD adopts a novel, folded conformation in which the nicotinamide and adenine rings stack in parallel with an inter-ring distance of 3.6 A. The pyrophosphate binds next to the flavin cofactor isoalloxazine, while the stacked nicotinamide/adenine moiety faces away from the flavin. The observed NAD conformation is quite different from the extended conformations observed in other enzyme/NAD(P) structures; however, it resembles the conformation proposed for NAD in solution. The flavin reductase P/NAD structure provides new information about the conformational diversity of NAD, which is important for understanding catalysis. This structure offers the first crystallographic evidence of a folded NAD with ring stacking, and it is the first enzyme structure containing an FMN cofactor interacting with NAD(P). Analysis of the structure suggests a possible dynamic mechanism underlying NADPH substrate specificity and product release that involves unfolding and folding of NADP(H).     

Solvent accessibility to flavin in oxynitrilase

Oxynitrilase containing 2-thioFAD [C(2) = S] in place of FAD exhibits catalytic activity similar to that of native enzyme. Reaction of methyl methanethiolsulfonate with 2-thioFAD bound to oxynitrilase results in the formation of the corresponding flavin disulfide [C(2)-SSCH3]. Normal flavin [C(2) = O] is formed by reacting 2-thioFAD oxynitrilase with m-chloroperoxybenzoate or H2O2. Both reactions proceed via a spectrally detectable flavin 2-S-oxide intermediate [C(2) = S+-O-], but sizable amounts of this intermediate accumulate only in the m-chloroperoxybenzoate reaction (about 40%). While similar reactions have been reported with free 2-thioflavin, kinetic and other data indicate that the oxynitrilase reactions occur with intact enzyme. This shows that the 2-position of the pyrimidine ring in the bound coenzyme is accessible to solvent. The data are consistent with previous studies on the reaction of peroxides with oxynitrilase-bound 5-deazaFAD which show that the pyrimidine ring is accessible at position 4. Analogous studies indicate that the pyrimidine ring is buried in the case of flavin bound to lactate oxidase, since the data indicate that both positions 2 and 4 are inaccessible to solvent.     

Effect of flavin structure and redox state on catalysis by and flavin-pterin energy transfer in Escherichia coli DNA photolyase

5-DeazaFAD bound to a hydrophobic site in apophotolyase and formed a stable reconstituted enzyme, similar to that observed with FAD. Although stoichiometric incorporation was observed, the flavin ring modification in 1-deazaFAD interfered with normal binding, decreased protein stability, and prevented formation of a stable flavin radical, unlike that observed with FAD. The results suggest that an important hydrogen bond is formed between the protein and N (1) in FAD, but not N (5), and that there is sufficient space at the normal flavin binding site near N (5) to accommodate an additional hydrogen but not near N (1). Catalytic activity was observed with enzyme containing 5-deazaFADH2 (42% of native enzyme) or 1-deazaFADH2 (11% of native enzyme) as its only chromophore, but no activity was observed with the corresponding oxidized flavins, similar to that observed with FAD and consistent with a mechanism where dimer cleavage is initiated by electron donation from excited reduced flavin to substrate. The protein environment in photolyase selectively enhanced photochemical reactivity in the fully reduced state, as evidenced by comparison with results obtained in model studies with the corresponding free flavins. Phosphorescence was observed with free or photolyase-bound 5-deazaFADH2, providing the first example of a flavin that exhibits phosphorescence in the fully reduced state. Formation of an enzyme-substrate complex resulted in a nearly identical extent of quenching of 5-deazaFADH2 phosphorescence (85.1%) and fluorescence (87.5%). The data are consistent with a mechanism involving exclusive reaction of substrate with the excited singlet state of 5-deazaFADH2, analogous to that proposed for FADH2 in native enzyme. Direct evidence for singlet-singlet energy transfer from enzyme-bound 5-deazaFADH2 to 5,10-CH(+)-H4folate was provided by the fact that pterin fluorescence was observed upon excitation of 5-deazaFADH2, accompanied by a decrease in 5-deazaFADH2 fluorescence. On the other hand, the fluorescence of enzyme-bound pterin was quenched by 5-deazaFADox, consistent with energy transfer from pterin to 5-deazaFADox. In each case, the spectral properties of the chromophores were consistent with the observed direction of energy transfer and indicated that transfer in the opposite direction was energetically unlikely. Unlike 5-deazaFAD, energy transfer from pterin to FAD is energetically feasible with FADH2 or FADox. The results indicate that the direction of flavin-pterin energy transfer at the active site of photolyase can be manipulated by changes in the flavin ring or redox state which alter the energy level of the flavin singlet.     

Crystal structure of NAD(P)H:flavin oxidoreductase from Escherichia coli.

Flavin reductases use flavins as substrates and are distinct from flavoenzymes which have tightly bound flavins. The reduced flavin can serve to reduce ferric complexes and iron proteins. In Escherichia coli, reactivation of ribonucleotide reductase is achieved by reduced flavins produced by flavin reductase. The crystal structure of E. coli flavin reductase reveals that the enzyme structure is similar to the structures of the ferredoxin reductase family of flavoproteins despite very low sequence similarities. The main difference between flavin reductase and structurally related flavoproteins is that there is no binding site for the AMP moiety of FAD. The direction of the helix in the flavin binding domain, corresponding to the phosphate binding helix in the flavoproteins, is also slightly different and less suitable for phosphate binding. Interactions for flavin substrates are instead provided by a hydrophobic isoalloxazine binding site that also contains a serine and a threonine, which form hydrogen bonds to the isoalloxazine of bound riboflavin in a substrate complex.     

Nuclear magnetic resonance studies of the old yellow enzyme. 1. 15N NMR of the enzyme recombined with 15N-labeled flavin mononucleotides

The apoenzyme of NADPH oxidoreductase, 'old yellow enzyme', was reconstituted with specifically 15N-labeled flavin mononucleotide and investigated by 15N NMR spectroscopy in the oxidized and reduced state. The results indicate that in the oxidized state a hydrogen bond is formed between the N(5) atom and the apoprotein. In addition, hydrogen bonds exist between the N(1) and N(3) atoms of FMN and the apoprotein. The resonance position of N(10) indicates that this atom is somewhat sp3-hybridized, i.e. lifted out of the molecular plane of the isoalloxazine ring system. In the reduced state the N(1) atom is negatively charged and the N(3) atom forms a hydrogen bond with the apoprotein. The N(10) atom in protein-bound FMN exhibits about the same hybridization state as in free anionic reduced FMN, i.e. it is located in the plane of the isoalloxazine ring. The chemical shift of the N(5) resonance indicates that this atom is almost completely sp3-hybridized. This interpretation can also be derived from the 15N(5)-1H coupling constant. Among the flavoproteins thus far studied by NMR techniques, old yellow enzyme is the only protein that shows a conformation of the reduced prosthetic group with the N(5) atom lifted out of the molecular plane. The isoelectric focussing properties of old yellow enzyme and a new easy method for the preparation of the apoprotein are also reported.     

Structural basis for the inactivation of Thermus thermophilus proline dehydrogenase by N-propargylglycine

The flavoenzyme proline dehydrogenase catalyzes the first step of proline catabolism, the oxidation of proline to pyrroline-5-carboxylate. Here we report the first crystal structure of an irreversibly inactivated proline dehydrogenase. The 1.9 A resolution structure of Thermus thermophilus proline dehydrogenase inactivated by the mechanism-based inhibitor N-propargylglycine shows that N5 of the flavin cofactor is covalently connected to the -amino group of Lys99 via a three-carbon linkage, consistent with the mass spectral analysis of the inactivated enzyme. The isoalloxazine ring has a butterfly angle of 25 degrees , which suggests that the flavin cofactor is reduced. Two mechanisms can account for these observations. In both, N-propargylglycine is oxidized to N-propargyliminoglycine. In one mechanism, this alpha,beta-unsaturated iminium compound is attacked by the N5 atom of the now reduced flavin to produce a 1,4-addition product. Schiff base formation between Lys99 and the imine of the 1,4-addition product releases glycine and links the enzyme to the modified flavin. In the second mechanism, hydrolysis of N-propargyliminoglycine yields propynal and glycine. A 1,4-addition reaction with propynal coupled with Schiff base formation between Lys99 and the carbonyl group tethers the enzyme to the flavin via a three-carbon chain. The presumed nonenzymatic hydrolysis of N-propargyliminoglycine and the subsequent rebinding of propynal to the enzyme make the latter mechanism less likely.     

Excited flavin and pterin coenzyme molecules in evolution

Excited flavin and pterin molecules are active in intermolecular energy transfer and in photocatalysis of redox reactions resulting in conservation of free energy. Flavin-containing pigments produced in models of the prebiotic environment are capable of converting photon energy into the energy of phosphoanhydride bonds of ATP. However, during evolution photochemical reactions involving excited FMN or FAD molecules failed to become participants of bioenergy transfer systems, but they appear in enzymes responsible for repair of UV-damaged DNA (DNA photolyases) and also in receptors of blue and UV-A light regulating vital functions of organisms. The families of these photoproteins (DNA-photolyases and cryptochromes, LOV-domain- and BLUF-domain-containing proteins) are different in the structure and in mechanisms of the photoprocesses. The excited flavin molecules are involved in photochemical processes in reaction centers of these photoproteins. In DNA photolyases and cryptochromes the excitation energy on the reaction center flavin is supplied from an antenna molecule that is bound with the same polypeptide. The role of antenna is played by MTHF or by 8-HDF in some DNA photolyases, i.e. also by molecules with known coenzyme functions in biocatalysis. Differences in the structure of chromophore-binding domains suggest an independent origin of the photoprotein families. The analysis of structure and properties of coenzyme molecules reveals some specific features that were significant in evolution for their being selected as chromophores in these proteins.     

Evidence for a novel mechanism of time-resolved flavin fluorescence depolarization in glutathione reductase

Time-resolved flavin fluorescence anisotropy studies on glutathione reductase (GR) have revealed a remarkable new phenomenon: wild-type GR displays a rapid process of fluorescence depolarization, that is absent in mutant enzymes lacking a nearby tyrosine residue that blocks the NADPH-binding cleft. Fluorescence lifetime data, however, have shown a more rigid active-site structure for wild-type GR than for the tyrosine mutants. These results suggest that the rapid depolarization in wild-type GR originates from an interaction with the flavin-shielding tyrosine, and not from restricted reorientational motion of the flavin. A novel mechanism of fluorescence depolarization is proposed that involves a transient charge-transfer complex between the tyrosine and the light-excited flavin, with a concomitant change in the direction of the emission dipole moment of the flavin. This interaction is likely to result from side-chain relaxation of the tyrosine in the minor fraction of enzyme molecules in which this residue is in an unsuitable position for immediate fluorescence quenching at the moment of excitation. Support for this mechanism is provided by binding studies with NADP+ and 2'P-5'ADP-ribose that can intercalate between the flavin and tyrosine and/or block the latter. Fluorescence depolarization analyses as a function of temperature and viscosity confirm the dynamic nature of the process. A comparison with fluorescence depolarization effects in a related flavoenzyme indicates that this mechanism of flavin fluorescence depolarization is more generally applicable.     

Thioredoxin reductase from Escherichia coli: evidence of restriction to a single conformation upon formation of a crosslink between engineered cysteines.

Thioredoxin reductase is a flavoprotein which catalyzes the reduction of the small protein thioredoxin by NADPH. It contains a redox active disulfide and an FAD in each subunit of its dimeric structure. Each subunit is further divided into two domains, the FAD and the pyridine nucleotide binding domains. The orientation of the two domains determined from the crystal structure and the flow of electrons determined from mechanistic studies suggest that thioredoxin reductase requires a large conformational change to carry out catalysis (Williams CH Jr, 1995, FASEB J 9:1267-1276). The constituent amino acids of an ion pair, E48/R130, between the FAD and pyridine nucleotide binding domains, were mutagenized to cysteines to form E48C,R130C (CC mutant). Formation of a stable bridge between these cysteines was expected to restrict the enzyme largely in the conformation observed in the crystal structure. Crosslinking with the bifunctional reagent N,N,1,2 phenylenedimaleimide, spanning 4-9 A, resulted in a >95 % decrease in thioredoxin reductase and transhydrogenase activity. SDS-PAGE confirmed that the crosslink in the CC-mutant was intramolecular. Dithionite titration showed an uptake of electrons as in wild-type enzyme, but anaerobic reduction of the flavin with NADPH was found to be impaired. This indicates that the crosslinked enzyme is in the conformation where the flavin and the active site disulfide are in close proximity but the flavin and pyridinium rings are too far apart for effective electron transfer. The evidence is consistent with the hypothesis that thioredoxin reductase requires a conformational change to complete catalysis.     

Structural and biochemical characterization of the prenylated flavin mononucleotide-dependent indole-3-carboxylic acid decarboxylase

The ubiquitous UbiD family of reversible decarboxylases is implicated in a wide range of microbial processes and depends on the prenylated flavin mononucleotide cofactor for catalysis. However, only a handful of UbiD family members have been characterized in detail, and comparison between these has suggested considerable variability in enzyme dynamics and mechanism linked to substrate specificity. In this study, we provide structural and biochemical insights into the indole-3-carboxylic acid decarboxylase, representing an UbiD enzyme activity distinct from those previously studied. Structural insights from crystal structure determination combined with small-angle X-ray scattering measurements reveal that the enzyme likely undergoes an open-closed transition as a consequence of domain motion, an event that is likely coupled to catalysis. We also demonstrate that the indole-3-carboxylic acid decarboxylase can be coupled with carboxylic acid reductase to produce indole-3-carboxyaldehyde from indole + CO₂ under ambient conditions. These insights provide further evidence for a common mode of action in the widespread UbiD enzyme family.     

Dual role of NADP(H) in the reaction of a flavin dependent N-hydroxylating monooxygenase

Aspergillus fumigatus siderophore A (Af SidA) is a flavin-dependent monooxygenase that catalyzes the hydroxylation of ornithine, producing N(5)-hydroxyornithine. This is the first step in the biosynthesis of hydroxamate-containing siderophores in A. fumigatus. Af SidA is essential for virulence, validating this enzyme as a drug target. Af SidA can accept reducing equivalents from either NADPH or NADH and displays similar kinetic parameters when using either coenzyme. When the enzyme is reduced with NADPH and reacted with molecular oxygen, a C4a-hydroperoxyflavin intermediate is observed. When the enzyme is reduced with NADH, the intermediate is 2-fold less stable. Steady-state kinetic isotope effect values of 3 and 2 were determined for NADPH and NADH, respectively. The difference in the isotope effect values is due to differences in the rate of flavin reduction by these coenzymes. A difference in the binding mode between these coenzymes was observed by monitoring flavin fluorescence. Limited proteolysis studies show that NADP(+), and not NAD(+), protects Af SidA from proteolysis, suggesting that it induces conformational changes upon binding. Together, these results are consistent with NADPH having a role in flavin reduction and in the modulation of conformational changes, which positions NADP(+) to also play a role in stabilization of the C4a-hydroperoxyflavin.     

Cryptochrome blue light photoreceptors are activated through interconversion of flavin redox states

Cryptochromes are blue light-sensing photoreceptors found in plants, animals, and humans. They are known to play key roles in the regulation of the circadian clock and in development. However, despite striking structural similarities to photolyase DNA repair enzymes, cryptochromes do not repair double-stranded DNA, and their mechanism of action is unknown. Recently, a blue light-dependent intramolecular electron transfer to the excited state flavin was characterized and proposed as the primary mechanism of light activation. The resulting formation of a stable neutral flavin semiquinone intermediate enables the photoreceptor to absorb green/yellow light (500-630 nm) in addition to blue light in vitro. Here, we demonstrate that Arabidopsis cryptochrome activation by blue light can be inhibited by green light in vivo consistent with a change of the cofactor redox state. We further characterize light-dependent changes in the cryptochrome1 (cry1) protein in living cells, which match photoreduction of the purified cry1 in vitro. These experiments were performed using fluorescence absorption/emission and EPR on whole cells and thereby represent one of the few examples of the active state of a known photoreceptor being monitored in vivo. These results indicate that cry1 activation via blue light initiates formation of a flavosemiquinone signaling state that can be converted by green light to an inactive form. In summary, cryptochrome activation via flavin photoreduction is a reversible mechanism novel to blue light photoreceptors. This photocycle may have adaptive significance for sensing the quality of the light environment in multiple organisms.     

The dissociation of flavin coenzymes from trypsin-solubilized NADPH/Cytochrome c (P-450) reductase of pig-liver microsomes.

The change in fluorescence emission at 520 nm after excitation at 365 nm was used to investigate the effect of pH and ionic strength on the dissociation of flavin cofactors from microsomal NADPH/cytochrome c (P-450) reductase. In the unmodified enzyme both the FAD and FMN moieties appeared to dissociate at a similar rate and followed first-order kinetics. The rate constant for the dissociation was increased by low pH and high ionic strength, particularly in the range pH 4.4-3.8 (0.02 M acetate buffer) where the rate constants increased 80-fold. Modification of the enzyme by treatment with p-chloromercuribenzoate enhanced the rate of flavin dissociation and, in the region of pH 4, resulted in a biphasic increase in fluorescence consistent with two simultaneous parallel first-order dissociations. It was concluded that p-chloromercuribenzoate treatment modified the protein so that the two flavin cofactors dissociated at different rates. Using the measured rate constants for the dissociations, and the known variation in fluorescence of flavin nucleotides with pH, an analogue computer simulation of the dissociation as well as a manual curve-fitting procedure showed that the biphasic response could be explained as a simultaneous rapid dissociation of FAD and a slower loss of FMN from the protein.